RNAi技术干扰喉癌细胞Egf17表达抑制侵袭转移和血管生成的体内外实验研究
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摘要
第一部分Egfl7在喉癌组织中的表达测定及临床意义
目的:探讨Egfl7(Egf-like domain 7)在喉鳞状细胞癌(laryngealsquamous cell carcinoma,LSCC)组织中的表达及意义。
方法:采用RT-PCR和Western-blot方法检测33例新鲜喉癌组织和配对的癌旁非癌喉组织(non-carcinoma laryngeal tissues,NCLT)中Egfl7mRNA和Egfl7蛋白的表达;采用免疫组化方法检测116例喉癌组织中Egfl7蛋白的表达;采用Cox多因素回归法,结合临床随访资料和临床病理资料分析,研究Egfl7的表达与临床病理特征和预后的关系。
结果:
1.33例新鲜喉癌标本中,LSCC组织中Egfl7 mRNA的阳性表达率87.9%(29/33)显著高于NCLT的阳性表达率33.3%(11/33)(P＜0.01)。LSCC组织中Egfl7 mRNA的平均表达水平也明显高于NCLT(1.42±0.21/0.86±0.11,P=0.008)。同一批样本中LSCC中Egfl7蛋白的阳性表达率90.9%(30/33)显著高于NCLT阳性表达率27.3%(9/33)(P＜0.01)。Egfl7蛋白在LSCC组织中的平均表达水平也明显高于NCLT组织(0.97±0.21/0.41±0.13,P=0.001)。且Egfl7mRNA和Egfl7蛋白表达具有显著相关性(R=0.786,P＜0.01)。Egfl7mRNA和Egfl7蛋白的表达水平与LSCC的临床分期、肿瘤的直径大小及有无淋巴结转移等临床病理特征明显相关(P＜0.05)。Egfl7mRNA和Egfl7蛋白的表达水平和MVD呈正相关性(R=0.842,P=0.231)。
2.Egfl7蛋白阳性表达绝大多数位于细胞浆内。在116例LSCC组织中Egfl7蛋白阳性表达率为81.03%(94/116),Egfl7蛋白阴性表达率为18.97%(22/116)。
3.Egfl7蛋白表达与LSCC的临床分期(P=0.003)、肿瘤的直径大小(P=0.001)及有无淋巴结转移(P=0.002)明显相关。Egfl7蛋白在喉癌组织中的表达水平与患者性别、年龄、癌灶原发部位无关(P＞0.05)。116例喉癌中Egfl7~(++/+++)组67例,Egfl7~(-/+)组49例,患者平均生存时间为36.9月,中位生存时间为43.2月。116例中55例在5年内死亡,在死亡病例中40例为Egfl7~(++/+++)组,15例为Egfl7~(-/+)组,两组之间具有显著性差异(P=0.007),Egfl7~(++/+++)组喉癌患者手术后生存率低于Egfl7~(-/+)组。采用Cox多因素回归分析,结果发现Egfl7蛋白表达(RR,1.74;P=0.012),淋巴结转移(RR,1.52;P=0.015)是喉鳞癌预后的独立预测因子。
结论:
1.Egfl7可能参与了喉癌的发生发展;
2.Egfl7蛋白可能成为预测喉癌的不良预后的肿瘤标志物。
第二部分RNAi干扰Egfl7对喉癌细胞肿瘤生物学行为影响的体内外研究
目的:探讨靶向沉默Egfl7的表达对LSCC侵袭转移的作用机制。
方法:构建pSUPER.Retro.neo逆转录病毒小RNA干扰质粒,转染PT67包装细胞,获得病毒颗粒并瞬时二重感染Hep-2喉癌细胞系,干预沉默Hep-2中Egfl7的表达,体外采用划痕实验、Transwell侵袭实验、MTT和流式细胞仪检测等方法,研究喉癌细胞株Hep-2分泌Egfl7调控喉癌细胞分化增殖、喉癌侵袭运动的分子机制。体内采用裸鼠成瘤实验研究体内抑制Egfl7的表达对喉癌细胞成瘤能力的影响。
结果:
1.本研究成功构建了靶向Egfl7的siRNA质粒和对照组表达质粒。
2.获得了稳定表达序列1和序列C的Hep-2细胞系,分别命名为Hep-2~(Egfl7RNAi+)和Hep-2~(Egfl7RNAi-)。
3.Western blot结果显示:Hep-2~(Egfl7RNAi+)中Egfl7的表达被明显抑制。
4.体外实验中抑制Egfl7的表达可以抑制喉癌细胞的侵袭迁移。
①划痕实验:Hep-2~(Egfl7RNAi+)较Hep-2~(Egfl7RNAi-)组,Hep-2细胞迁移能力明显下降,24小时划痕愈合率分别为62%和77%,差异具有显著性意义(P＜0.05)。
②Transwell侵袭实验:Hep-2~(Egfl7RNAi+)较Hep-2~(Egfl7RNAi-)细胞侵袭能力明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为63±11和129±13,差异具有显著性(P＜0.05)。
5.体外实验中抑制Egfl7的表达不影响喉癌细胞的增殖和凋亡。
①MTT法比较Hep-2~(Egfl7RNAi+)和Hep-2~(Egfl7RNAi-)的增殖能力,两组细胞增殖能力差异不具有显著性意义(P＞0.05)。
②AnnexinⅤ和PI双染法结合流式细胞仪技术检测Hep-2~(Egfl7RNAi+)和Hep-2~(Egfl7RNAi-)。的凋亡情况,两组细胞的凋亡差异不具有显著性意义(P＞0.05)。
6.体内裸鼠成瘤实验结果:体内抑制Egfl7的表达可以抑制喉癌细胞的成瘤能力。
比较Mice~(Egfl7RNAi+)和Mice~(Egfl7RNAi-)两组裸鼠模型中Hep-2原发瘤的大小:皮下种植45天后Hep-2原发瘤的大小(cm~3)分别为3.01±0.22和3.45±0.23,差异具有显著性意义(P＜0.05)。
结论:
1.Egfl7在喉癌侵袭转移中发挥关键性作用;
2.Egfl7可能成为抗喉癌侵袭转移的基因治疗新靶点。
第三部分Egfl7调控喉癌新生血管生成的体内外研究
目的:探讨靶向沉默Egfl7的表达对LSCC血管生成的作用机制。
方法:干预沉默人微血管内皮细胞系(HMEC-1)中Egfl7表达,体外采用划痕实验、Transwell侵袭实验、二维和三维血管成管实验、MTT和流式细胞仪等方法检测,研究喉癌细胞株Hep-2分泌Egfl7调控喉癌细胞分化增殖、喉癌侵袭运动和喉癌血管成管的分子机制,体内采用三维共培养的细胞接种裸鼠成瘤实验验证Egfl7调控喉癌血管成管的分子机制导致肿瘤生物学行为的改变。
结果:
1.逆转录病毒介导的小RNA干扰特异性沉默HMEC-1人微血管内皮细胞系中Egfl7的表达。感染HMEC-1的效率为80%～90%。Western blot检测结果:实验组HMEC-1~(Egfl7RNAi+)较对照组HMEC-1~(Egfl7RNAi-)Egfl7蛋白表达明显下降,分别为0.36±0.07和0.86±0.02(P＜0.05),这提示成功地抑制了HMEC-1细胞中Egfl7的表达。
2.体外实验中抑制Egfl7的表达可以抑制人微血管内皮细胞(HMEC-1)的迁移。
①划痕实验:HMEC-1~(Egfl7RNAi+)较对照组HMEC-1~(Egfl7RNAi-)的迁移能力明显下降,24小时划痕愈合率分别为69%和92%,差异具有显著性(P＜0.05)。
②Transwell侵袭实验:HMEC-1~(Egfl7RNAi+)较对照组HMEC-1~(Egfl7RNAi-)细胞侵袭能力明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为49±6和70±3,差异具有显著性意义(P＜0.05)。
3.体外实验中抑制Egfl7的表达不影响人微血管内皮细胞(HMEC-1)的增殖和凋亡。
①MTT法比较HMEC-1~(Egfl7RNAi+)和HMEC-1~(Egfl7RNAi-)的增殖能力,两组细胞增殖能力差异不具有显著性意义(P＞0.05)。
②AnnexinⅤ和PI双染法结合流式细胞仪技术检测HMEC-1~(Egfl7RNAi+)较对照组HMEC-1~(Egfl7RNAi-)的凋亡情况,两组细胞的凋亡差异不具有显著性意义(P＞0.05)。
4.体外实验中抑制Egfl7的表达可以抑制人微血管内皮细胞(HMEC-1)的血管生成。
①二维血管新生模型结果:实验组HMEC-1~(Egfl7RNAi+)内皮细胞不能在Matrigel上形成网状结构;对照组成管指数为2397±726,实验组成管指数为489±215,两组差异具有显著性(P＜0.05)。
②三维血管新生模型结果:对照组HMEC-1~(Egfl7RNAi-)内皮细胞在三维环境中增殖并且形成三维管状结构,而实验组HMEC-1~(Egfl7RNAi+)在三维环境中仅仅发生增殖而很少形成三维管状结构,对照组和实验组中每10个微载体成管数分别为45±9和9±7,差异具有显著性意义(P＜0.05)。
5.体内实验中抑制Egfl7的表达可以抑制VEGF诱导的血管生成。
①比较Mice~(Egfl7RNAi+)和Mice~(Egfl7RNAi-)两组裸鼠模型Hep-2原发瘤中的微血管密度(MVD),Mice~(Egfl7RNAi+)和Mice~(Egfl7RNAi-)两组裸鼠模型Hep-2原发瘤中每高倍镜视野平均MVD值分别为29±2和37±3,差异具有显著性(P＜0.05)。
②实验组和对照组的包装细胞种植于裸鼠皮下并成瘤,同时将混合了VEGF的Matrigel种植于裸鼠皮下做为血管新生的诱生物,实验组Matrigel中的新生血管少于对照组,实验组和对照组Matrigel中新生血管总长度分别为23634±6421μm和32145±4314μm,差异具有显著性意义(P＜0.05)。
结论:
1.Egfl7可能通过调控血管成管分子机制参与了喉癌的侵袭转移;
2.Egfl7可能成为抗喉癌血管生成的基因治疗新靶点。
PartyⅠThe study on clinical significance of the expression of Egfl7 in laryngeal squamous cell carcinoma
Objective:To investigate the expression of Egfl7 in laryngeal squamous cell carcinoma(LSCC)and its significance.
Methods:The expression of Egfl7 mRNA and protein were detected by RT-PCR and Western-blot in 33 fresh cases laryngeal cancer tissue; The expression of Egfl7 protein in 116 cases laryngeal carcinoma were detected by immunohistochemical methods.To research the expression of Egfl7 relation to clinicopathologic characteristics and prognosis,the multi-factor COX and the combination of clinical pathology and clinical follow-up data were analysis.
Results:
1.The expression of Egfl7 mRNA positive rate is significantly difference between 33 cases fresh laryngeal squamous cell carcinoma(LSCC) tissues 87.9%(29/33) to non-carcinoma laryngeal tissues(NCLT) 33.3%(11/33)(P＜0.01).The expression average level of Egfl7 mRNA in LSCC was significantly higher than NCLT(1.42±0.21/ 0.86±0.11,P=0.008).The expression of Egfl7 protein positive rate is significantly difference between 33 cases fresh laryngeal squamous cell carcinoma(LSCC) tissues 90.9%(30/33) to non-carcinoma laryngeal tissues(NCLT) 27.3%(9/33)(P＜0.01).The expression average level of Egfl7 protein in LSCC was significantly higher than NCLT(0.97±0.21/ 0.41±0.13,P=0.001).Furthermore,the expression of Egfl7 mRNA between protein is significant positive correlation(R=0.786,P＜0.01). Egfl7 mRNA and protein expression were significantly correlated with LSCC clinical stage,tumor size and the presence of lymph node metastasis(P＜0.05).The expression of Egfl7 mRNA and protein is significant positive correlation with MVD in 33 cases LSCC(R=0.842, P=0.231).
2.The positive immue staining of Egfl7 protein expression is majority located in the cytoplasm.Among 116 cases LSCC tissues,the expression of Egfl7 protein positive rate is significantly difference between the positive expression 81.03%(94/116) and negative expression rate 18.97%(22/116).
3.The expression Egfl7 protein in 116 cases LSCC is significantly related the clinical stage(P=0.003),tumor size in diameter(P=0.001) and lymph node metastasis(P=0.002).However,the expression Egfl7 protein in LSCC is unrelated sex,age and to the primary site(P＞0.05). Among 116 patients with LSCC,67 in Egfl7~(++/+++) group,49 in Egfl7~(-/+) group,the average survival time was 36.9 months,median survival time was 43.2 months and 55cases of them died in five years.Of which 55 died cases,40 cases was in Egfl7~(++/+++) group,15 in Egfl7~(-/+) group.Egfl7 high-expression group post-operative survival rate is significant difference lower than Egfl7 low expression group(P=0.007).Cox analysis result is show that Egfl7 expression(RR,1.74;P=0.012),lymph node metastases(RR,1.52;P=0.015) is an independent predictor of the prognosis laryngeal squamous cell carcinoma.
Conclusion:
1.Egfl7 may have been involved in the occurrence and development of LSCC.
2.Egfl7 might be a tumor marker as a predict prognosis of laryngeal cancer.
PartyⅡStudy on the effect of biological behavior of laryngeal cancer to interfere with RNAi target to Egfl7 in vivo and in vitro
Objective:To investiga the mechanisms of the expression of Egfl7 in LSCC invasion and metastasis by interfence targeting Egfl7 silence.
Methods:PT67 packaging cells were transferred construction of retroviral pSUPER.Retro.neo small RNA interference plasmid,the virus particles and instantaneous access to double infection Hep-2 laryngeal cancer cell lines,the intervention of silence Egfl7 in Hep-2 expression in vitro Use the scratch test,Transwell invasion experiments,MTT and flow cytometry and other methods to study the laryngeal cancer cell line Hep-2 secretion Egfl7 control the proliferation of laryngeal cancer cells, laryngeal attack the molecular mechanism of movement.To examine the role of the expression of Egfl7 in laryngeal cancer cells is effected the ability of development of LSCC,Hep-2 cell lines were by RNAi interfered and were carry out a subcutaneous injection into node mice body in vivo.
Results:
1.In our present study,targeting of Egfl7siRNA plasmid and the control group expression vector were successfully constructed.
2.We obtained the stability of the expressed sequence 1 and C sequences of Hep-2 cell line,and named Hep-2~(Egfl7RNAi+)and Hep.2~(Egfl7RNAi-).
3.The expression of Egfl7 protein in Hep-2~(Egfl7RNAi+) cell lines was significantly inhibited by Western blot detection.
4.The expression of Egfl7 is inhibited in laryngeal cancer cells can lead to inhibition the invasion of migration is decreased in vitro.
①There is significant difference between Hep-2~(Egfl7RNAi+) (62%)and Hep-2~(Egfl7RNAi-)group(77%)(P＜0.05) by wound healing assay.
②There is significant difference Hep-2~(Egfl7RNAi+)(63±11) than Hep-2~(Egfl7RNAi-)cell invasion(129±13)(P＜0.05) by Transwell invasion experiment:
5.The expression of Egfl7 is inhibited in laryngeal cancer cells without affected cell proliferation and apoptosis.
①There is no significant difference between Hep-2~(Egfl7RNAi+) and Hep-2~(Egfl7RNAi-) proliferation by MTT detection method(P＞0.05).
②AnnexinⅤand PI double staining combined with flow cytometry detection technology show that in Hep-2~(Egfl7RNAi+) and Hep-2~(Egfl7RNAi-) groups apoptosis difference is not significant(P＞0.05).
6.To inhibit the expression of Egfl7 in laryngeal cancer cells can inhibit the ability of tumor development.The size of Mice~(Egfl7RNAi+)group is smaller(3.01±0.22) compare to Mice~(Egfl7RNAi-) group(3.45±0.23) after 45 days by subcutaneous injection,(P＜0.05)
Conclusion:
1.Egfl7 may be play a key role in LSCC invasion and metastasis.
2.Egfl7 would to be a new target for gene therapy in LSCC invasion and metastasis.
PartyⅢStudy on the effect of angiogenesis of laryngeal cancer to interfere with RNAi target to Egfl7 in vivo and in vitro
Objective:To investiga the mechanism of the expression of Egfl7 in LSCC angiogenesis by interfering targeted Egfl7 silence.
Methods:Silent targert to Egfl7 expression in human microvascular endothelial cells(HMEC-1) was interfered by silencing specific Retrovirus-mediated small interfering RNA;The molecular mechanism that secreted Egfl7 in Hep-2 regulate the cell differentiation,proliferation, invasion and angiogenesis of LSCC was studied by using scratch test, Transwell invasion test,two and three-dimensional vasicular tubulogenesis test,MTT and flow cytometry assay and other detection methods in vitro;The mechanisms which Egfl7 regulate vasicular tubulogenesis leading to tumor biological behavior change in LSCC was confirmed by using three-dimensional co-culture of tumor cells into nude mice experiments in vivo.
Results:
1.The HMEC-1 infection efficiency is about to 80%to 90%.The expression of Egfl7 in HMEC-1 lines were silencing specific Retrovirus-mediated small interfering RNA.The expression of Egfl7 protein is significantly decreased in HMEC-1~(Egfl7RNAi+)(0.36±0.07) group than that in the control HMEC-1~(Egfl7RNAi-Egfl7)(0.86±0.02) group by Western blot decation(P＜0.05).
2.The expression of Egfl7 is inhibited in HMEC-1 can lead to inhibition the invasion of migration is decreased in vitro.
①There is significant difference between HMEC-1~(Egfl7RNAi+) (69%)and HMEC-1~(Egfl7RNAi-)group(92%)(P＜0.05) by wound healing assay.
②There is significant difference in HMEC-1~(Egfl7RNAi+) invasion(49±6) compared with HMEC-1~(Egfl7RNAi-) invasion(70±3)(P＜0.05) by Transwell invasion experiment.
3.The expression of Egfl7 is inhibited in laryngeal cancer cells cannot be effected cell proliferation and apoptosis.
①There is no significant difference between HMEC-1~(Egfl7RNAi+) and HMEC-1~(Egfl7RNAi-) proliferation by MTT detection method(P＞0.05).
②AnnexinⅤand PI double staining combined with flow cytometry detection technology show that apoptosis in HMEC-1~(Egfl7RNAi+) and HMEC-1~(Egfl7RNAi-) groups is not significant(P＞0.05).
4.Egfl7 inhibition in vitro can inhibit the expression of HMEC-1 angiogenesis.
①The HMEC-1~(Egfl7RNAi+) endothelial cells can not form a mesh structure on Matrigel.There is a significant difference between the two groups(2397±726 vs 489±215)(P＜0.05).
②The control group HMEC-1~(Egfl7RNAi-)endothelial cell proliferation in the three-dimensional environment and the formation of threedimensional structure of the tube,while the HMEC-1~(Egfl7RNAi+)group in the three-dimensional environment and the proliferation took place only a few three-dimensional structure of the tube,In the control group and experimental group of 10 micro-carrier into a pipe a few were 45±9 and 9±7,with the difference was significant(P＜0.05).
5.In vivo inhibition Egfl7 can inhibit the expression of VEGF-induced angiogenesis.
①To compare Mice~(Egfl7RNAi+) with Mice~(Egfl7RNAi-) two nude models the primary tumor microvessel density(MVD),There is a significant difference MVD values between the two groups(29±2 vs 37±3)(P＜0.05).
②Matrigel cultivation of VEGF in nude mice as a bio-induced angiogenesis,the experimental group in the Matrigel neovascularization was less than the control group,experimental and control groups of and, with the difference was significant(P＜0.05).There is a significant difference in Matrigel neovascularization of the total length between the two groups(23634±6421μm vs 32145±4314μm)(P＜0.05).
Conclusion:
1.Egfl7 may be involved in angiogenesis of LSCC invasion and metastasis;
2.Egfl7 could be as a key anti-angiogenesis target for LSCC gene therapy.
目的:探讨Egfl7(Egf-like domain 7)在喉鳞状细胞癌(laryngealsquamous cell carcinoma,LSCC)组织中的表达及意义。
方法:采用RT-PCR和Western-blot方法检测33例新鲜喉癌组织和配对的癌旁非癌喉组织(non-carcinoma laryngeal tissues,NCLT)中Egfl7mRNA和Egfl7蛋白的表达;采用免疫组化方法检测116例喉癌组织中Egfl7蛋白的表达;采用Cox多因素回归法,结合临床随访资料和临床病理资料分析,研究Egfl7的表达与临床病理特征和预后的关系。
结果:
1.33例新鲜喉癌标本中,LSCC组织中Egfl7 mRNA的阳性表达率87.9%(29/33)显著高于NCLT的阳性表达率33.3%(11/33)(P＜0.01)。LSCC组织中Egfl7 mRNA的平均表达水平也明显高于NCLT(1.42±0.21/0.86±0.11,P=0.008)。同一批样本中LSCC中Egfl7蛋白的阳性表达率90.9%(30/33)显著高于NCLT阳性表达率27.3%(9/33)(P＜0.01)。Egfl7蛋白在LSCC组织中的平均表达水平也明显高于NCLT组织(0.97±0.21/0.41±0.13,P=0.001)。且Egfl7mRNA和Egfl7蛋白表达具有显著相关性(R=0.786,P＜0.01)。Egfl7mRNA和Egfl7蛋白的表达水平与LSCC的临床分期、肿瘤的直径大小及有无淋巴结转移等临床病理特征明显相关(P＜0.05)。Egfl7mRNA和Egfl7蛋白的表达水平和MVD呈正相关性(R=0.842,P=0.231)。
2.Egfl7蛋白阳性表达绝大多数位于细胞浆内。在116例LSCC组织中Egfl7蛋白阳性表达率为81.03%(94/116),Egfl7蛋白阴性表达率为18.97%(22/116)。
3.Egfl7蛋白表达与LSCC的临床分期(P=0.003)、肿瘤的直径大小(P=0.001)及有无淋巴结转移(P=0.002)明显相关。Egfl7蛋白在喉癌组织中的表达水平与患者性别、年龄、癌灶原发部位无关(P＞0.05)。116例喉癌中Egfl7~(++/+++)组67例,Egfl7~(-/+)组49例,患者平均生存时间为36.9月,中位生存时间为43.2月。116例中55例在5年内死亡,在死亡病例中40例为Egfl7~(++/+++)组,15例为Egfl7~(-/+)组,两组之间具有显著性差异(P=0.007),Egfl7~(++/+++)组喉癌患者手术后生存率低于Egfl7~(-/+)组。采用Cox多因素回归分析,结果发现Egfl7蛋白表达(RR,1.74;P=0.012),淋巴结转移(RR,1.52;P=0.015)是喉鳞癌预后的独立预测因子。
结论:
1.Egfl7可能参与了喉癌的发生发展;
2.Egfl7蛋白可能成为预测喉癌的不良预后的肿瘤标志物。
第二部分RNAi干扰Egfl7对喉癌细胞肿瘤生物学行为影响的体内外研究
目的:探讨靶向沉默Egfl7的表达对LSCC侵袭转移的作用机制。
方法:构建pSUPER.Retro.neo逆转录病毒小RNA干扰质粒,转染PT67包装细胞,获得病毒颗粒并瞬时二重感染Hep-2喉癌细胞系,干预沉默Hep-2中Egfl7的表达,体外采用划痕实验、Transwell侵袭实验、MTT和流式细胞仪检测等方法,研究喉癌细胞株Hep-2分泌Egfl7调控喉癌细胞分化增殖、喉癌侵袭运动的分子机制。体内采用裸鼠成瘤实验研究体内抑制Egfl7的表达对喉癌细胞成瘤能力的影响。
结果:
1.本研究成功构建了靶向Egfl7的siRNA质粒和对照组表达质粒。
2.获得了稳定表达序列1和序列C的Hep-2细胞系,分别命名为Hep-2~(Egfl7RNAi+)和Hep-2~(Egfl7RNAi-)。
3.Western blot结果显示:Hep-2~(Egfl7RNAi+)中Egfl7的表达被明显抑制。
4.体外实验中抑制Egfl7的表达可以抑制喉癌细胞的侵袭迁移。
①划痕实验:Hep-2~(Egfl7RNAi+)较Hep-2~(Egfl7RNAi-)组,Hep-2细胞迁移能力明显下降,24小时划痕愈合率分别为62%和77%,差异具有显著性意义(P＜0.05)。
②Transwell侵袭实验:Hep-2~(Egfl7RNAi+)较Hep-2~(Egfl7RNAi-)细胞侵袭能力明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为63±11和129±13,差异具有显著性(P＜0.05)。
5.体外实验中抑制Egfl7的表达不影响喉癌细胞的增殖和凋亡。
①MTT法比较Hep-2~(Egfl7RNAi+)和Hep-2~(Egfl7RNAi-)的增殖能力,两组细胞增殖能力差异不具有显著性意义(P＞0.05)。
②AnnexinⅤ和PI双染法结合流式细胞仪技术检测Hep-2~(Egfl7RNAi+)和Hep-2~(Egfl7RNAi-)。的凋亡情况,两组细胞的凋亡差异不具有显著性意义(P＞0.05)。
6.体内裸鼠成瘤实验结果:体内抑制Egfl7的表达可以抑制喉癌细胞的成瘤能力。
比较Mice~(Egfl7RNAi+)和Mice~(Egfl7RNAi-)两组裸鼠模型中Hep-2原发瘤的大小:皮下种植45天后Hep-2原发瘤的大小(cm~3)分别为3.01±0.22和3.45±0.23,差异具有显著性意义(P＜0.05)。
结论:
1.Egfl7在喉癌侵袭转移中发挥关键性作用;
2.Egfl7可能成为抗喉癌侵袭转移的基因治疗新靶点。
第三部分Egfl7调控喉癌新生血管生成的体内外研究
目的:探讨靶向沉默Egfl7的表达对LSCC血管生成的作用机制。
方法:干预沉默人微血管内皮细胞系(HMEC-1)中Egfl7表达,体外采用划痕实验、Transwell侵袭实验、二维和三维血管成管实验、MTT和流式细胞仪等方法检测,研究喉癌细胞株Hep-2分泌Egfl7调控喉癌细胞分化增殖、喉癌侵袭运动和喉癌血管成管的分子机制,体内采用三维共培养的细胞接种裸鼠成瘤实验验证Egfl7调控喉癌血管成管的分子机制导致肿瘤生物学行为的改变。
结果:
1.逆转录病毒介导的小RNA干扰特异性沉默HMEC-1人微血管内皮细胞系中Egfl7的表达。感染HMEC-1的效率为80%～90%。Western blot检测结果:实验组HMEC-1~(Egfl7RNAi+)较对照组HMEC-1~(Egfl7RNAi-)Egfl7蛋白表达明显下降,分别为0.36±0.07和0.86±0.02(P＜0.05),这提示成功地抑制了HMEC-1细胞中Egfl7的表达。
2.体外实验中抑制Egfl7的表达可以抑制人微血管内皮细胞(HMEC-1)的迁移。
①划痕实验:HMEC-1~(Egfl7RNAi+)较对照组HMEC-1~(Egfl7RNAi-)的迁移能力明显下降,24小时划痕愈合率分别为69%和92%,差异具有显著性(P＜0.05)。
②Transwell侵袭实验:HMEC-1~(Egfl7RNAi+)较对照组HMEC-1~(Egfl7RNAi-)细胞侵袭能力明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为49±6和70±3,差异具有显著性意义(P＜0.05)。
3.体外实验中抑制Egfl7的表达不影响人微血管内皮细胞(HMEC-1)的增殖和凋亡。
①MTT法比较HMEC-1~(Egfl7RNAi+)和HMEC-1~(Egfl7RNAi-)的增殖能力,两组细胞增殖能力差异不具有显著性意义(P＞0.05)。
②AnnexinⅤ和PI双染法结合流式细胞仪技术检测HMEC-1~(Egfl7RNAi+)较对照组HMEC-1~(Egfl7RNAi-)的凋亡情况,两组细胞的凋亡差异不具有显著性意义(P＞0.05)。
4.体外实验中抑制Egfl7的表达可以抑制人微血管内皮细胞(HMEC-1)的血管生成。
①二维血管新生模型结果:实验组HMEC-1~(Egfl7RNAi+)内皮细胞不能在Matrigel上形成网状结构;对照组成管指数为2397±726,实验组成管指数为489±215,两组差异具有显著性(P＜0.05)。
②三维血管新生模型结果:对照组HMEC-1~(Egfl7RNAi-)内皮细胞在三维环境中增殖并且形成三维管状结构,而实验组HMEC-1~(Egfl7RNAi+)在三维环境中仅仅发生增殖而很少形成三维管状结构,对照组和实验组中每10个微载体成管数分别为45±9和9±7,差异具有显著性意义(P＜0.05)。
5.体内实验中抑制Egfl7的表达可以抑制VEGF诱导的血管生成。
①比较Mice~(Egfl7RNAi+)和Mice~(Egfl7RNAi-)两组裸鼠模型Hep-2原发瘤中的微血管密度(MVD),Mice~(Egfl7RNAi+)和Mice~(Egfl7RNAi-)两组裸鼠模型Hep-2原发瘤中每高倍镜视野平均MVD值分别为29±2和37±3,差异具有显著性(P＜0.05)。
②实验组和对照组的包装细胞种植于裸鼠皮下并成瘤,同时将混合了VEGF的Matrigel种植于裸鼠皮下做为血管新生的诱生物,实验组Matrigel中的新生血管少于对照组,实验组和对照组Matrigel中新生血管总长度分别为23634±6421μm和32145±4314μm,差异具有显著性意义(P＜0.05)。
结论:
1.Egfl7可能通过调控血管成管分子机制参与了喉癌的侵袭转移;
2.Egfl7可能成为抗喉癌血管生成的基因治疗新靶点。
PartyⅠThe study on clinical significance of the expression of Egfl7 in laryngeal squamous cell carcinoma
Objective:To investigate the expression of Egfl7 in laryngeal squamous cell carcinoma(LSCC)and its significance.
Methods:The expression of Egfl7 mRNA and protein were detected by RT-PCR and Western-blot in 33 fresh cases laryngeal cancer tissue; The expression of Egfl7 protein in 116 cases laryngeal carcinoma were detected by immunohistochemical methods.To research the expression of Egfl7 relation to clinicopathologic characteristics and prognosis,the multi-factor COX and the combination of clinical pathology and clinical follow-up data were analysis.
Results:
1.The expression of Egfl7 mRNA positive rate is significantly difference between 33 cases fresh laryngeal squamous cell carcinoma(LSCC) tissues 87.9%(29/33) to non-carcinoma laryngeal tissues(NCLT) 33.3%(11/33)(P＜0.01).The expression average level of Egfl7 mRNA in LSCC was significantly higher than NCLT(1.42±0.21/ 0.86±0.11,P=0.008).The expression of Egfl7 protein positive rate is significantly difference between 33 cases fresh laryngeal squamous cell carcinoma(LSCC) tissues 90.9%(30/33) to non-carcinoma laryngeal tissues(NCLT) 27.3%(9/33)(P＜0.01).The expression average level of Egfl7 protein in LSCC was significantly higher than NCLT(0.97±0.21/ 0.41±0.13,P=0.001).Furthermore,the expression of Egfl7 mRNA between protein is significant positive correlation(R=0.786,P＜0.01). Egfl7 mRNA and protein expression were significantly correlated with LSCC clinical stage,tumor size and the presence of lymph node metastasis(P＜0.05).The expression of Egfl7 mRNA and protein is significant positive correlation with MVD in 33 cases LSCC(R=0.842, P=0.231).
2.The positive immue staining of Egfl7 protein expression is majority located in the cytoplasm.Among 116 cases LSCC tissues,the expression of Egfl7 protein positive rate is significantly difference between the positive expression 81.03%(94/116) and negative expression rate 18.97%(22/116).
3.The expression Egfl7 protein in 116 cases LSCC is significantly related the clinical stage(P=0.003),tumor size in diameter(P=0.001) and lymph node metastasis(P=0.002).However,the expression Egfl7 protein in LSCC is unrelated sex,age and to the primary site(P＞0.05). Among 116 patients with LSCC,67 in Egfl7~(++/+++) group,49 in Egfl7~(-/+) group,the average survival time was 36.9 months,median survival time was 43.2 months and 55cases of them died in five years.Of which 55 died cases,40 cases was in Egfl7~(++/+++) group,15 in Egfl7~(-/+) group.Egfl7 high-expression group post-operative survival rate is significant difference lower than Egfl7 low expression group(P=0.007).Cox analysis result is show that Egfl7 expression(RR,1.74;P=0.012),lymph node metastases(RR,1.52;P=0.015) is an independent predictor of the prognosis laryngeal squamous cell carcinoma.
Conclusion:
1.Egfl7 may have been involved in the occurrence and development of LSCC.
2.Egfl7 might be a tumor marker as a predict prognosis of laryngeal cancer.
PartyⅡStudy on the effect of biological behavior of laryngeal cancer to interfere with RNAi target to Egfl7 in vivo and in vitro
Objective:To investiga the mechanisms of the expression of Egfl7 in LSCC invasion and metastasis by interfence targeting Egfl7 silence.
Methods:PT67 packaging cells were transferred construction of retroviral pSUPER.Retro.neo small RNA interference plasmid,the virus particles and instantaneous access to double infection Hep-2 laryngeal cancer cell lines,the intervention of silence Egfl7 in Hep-2 expression in vitro Use the scratch test,Transwell invasion experiments,MTT and flow cytometry and other methods to study the laryngeal cancer cell line Hep-2 secretion Egfl7 control the proliferation of laryngeal cancer cells, laryngeal attack the molecular mechanism of movement.To examine the role of the expression of Egfl7 in laryngeal cancer cells is effected the ability of development of LSCC,Hep-2 cell lines were by RNAi interfered and were carry out a subcutaneous injection into node mice body in vivo.
Results:
1.In our present study,targeting of Egfl7siRNA plasmid and the control group expression vector were successfully constructed.
2.We obtained the stability of the expressed sequence 1 and C sequences of Hep-2 cell line,and named Hep-2~(Egfl7RNAi+)and Hep.2~(Egfl7RNAi-).
3.The expression of Egfl7 protein in Hep-2~(Egfl7RNAi+) cell lines was significantly inhibited by Western blot detection.
4.The expression of Egfl7 is inhibited in laryngeal cancer cells can lead to inhibition the invasion of migration is decreased in vitro.
①There is significant difference between Hep-2~(Egfl7RNAi+) (62%)and Hep-2~(Egfl7RNAi-)group(77%)(P＜0.05) by wound healing assay.
②There is significant difference Hep-2~(Egfl7RNAi+)(63±11) than Hep-2~(Egfl7RNAi-)cell invasion(129±13)(P＜0.05) by Transwell invasion experiment:
5.The expression of Egfl7 is inhibited in laryngeal cancer cells without affected cell proliferation and apoptosis.
①There is no significant difference between Hep-2~(Egfl7RNAi+) and Hep-2~(Egfl7RNAi-) proliferation by MTT detection method(P＞0.05).
②AnnexinⅤand PI double staining combined with flow cytometry detection technology show that in Hep-2~(Egfl7RNAi+) and Hep-2~(Egfl7RNAi-) groups apoptosis difference is not significant(P＞0.05).
6.To inhibit the expression of Egfl7 in laryngeal cancer cells can inhibit the ability of tumor development.The size of Mice~(Egfl7RNAi+)group is smaller(3.01±0.22) compare to Mice~(Egfl7RNAi-) group(3.45±0.23) after 45 days by subcutaneous injection,(P＜0.05)
Conclusion:
1.Egfl7 may be play a key role in LSCC invasion and metastasis.
2.Egfl7 would to be a new target for gene therapy in LSCC invasion and metastasis.
PartyⅢStudy on the effect of angiogenesis of laryngeal cancer to interfere with RNAi target to Egfl7 in vivo and in vitro
Objective:To investiga the mechanism of the expression of Egfl7 in LSCC angiogenesis by interfering targeted Egfl7 silence.
Methods:Silent targert to Egfl7 expression in human microvascular endothelial cells(HMEC-1) was interfered by silencing specific Retrovirus-mediated small interfering RNA;The molecular mechanism that secreted Egfl7 in Hep-2 regulate the cell differentiation,proliferation, invasion and angiogenesis of LSCC was studied by using scratch test, Transwell invasion test,two and three-dimensional vasicular tubulogenesis test,MTT and flow cytometry assay and other detection methods in vitro;The mechanisms which Egfl7 regulate vasicular tubulogenesis leading to tumor biological behavior change in LSCC was confirmed by using three-dimensional co-culture of tumor cells into nude mice experiments in vivo.
Results:
1.The HMEC-1 infection efficiency is about to 80%to 90%.The expression of Egfl7 in HMEC-1 lines were silencing specific Retrovirus-mediated small interfering RNA.The expression of Egfl7 protein is significantly decreased in HMEC-1~(Egfl7RNAi+)(0.36±0.07) group than that in the control HMEC-1~(Egfl7RNAi-Egfl7)(0.86±0.02) group by Western blot decation(P＜0.05).
2.The expression of Egfl7 is inhibited in HMEC-1 can lead to inhibition the invasion of migration is decreased in vitro.
①There is significant difference between HMEC-1~(Egfl7RNAi+) (69%)and HMEC-1~(Egfl7RNAi-)group(92%)(P＜0.05) by wound healing assay.
②There is significant difference in HMEC-1~(Egfl7RNAi+) invasion(49±6) compared with HMEC-1~(Egfl7RNAi-) invasion(70±3)(P＜0.05) by Transwell invasion experiment.
3.The expression of Egfl7 is inhibited in laryngeal cancer cells cannot be effected cell proliferation and apoptosis.
①There is no significant difference between HMEC-1~(Egfl7RNAi+) and HMEC-1~(Egfl7RNAi-) proliferation by MTT detection method(P＞0.05).
②AnnexinⅤand PI double staining combined with flow cytometry detection technology show that apoptosis in HMEC-1~(Egfl7RNAi+) and HMEC-1~(Egfl7RNAi-) groups is not significant(P＞0.05).
4.Egfl7 inhibition in vitro can inhibit the expression of HMEC-1 angiogenesis.
①The HMEC-1~(Egfl7RNAi+) endothelial cells can not form a mesh structure on Matrigel.There is a significant difference between the two groups(2397±726 vs 489±215)(P＜0.05).
②The control group HMEC-1~(Egfl7RNAi-)endothelial cell proliferation in the three-dimensional environment and the formation of threedimensional structure of the tube,while the HMEC-1~(Egfl7RNAi+)group in the three-dimensional environment and the proliferation took place only a few three-dimensional structure of the tube,In the control group and experimental group of 10 micro-carrier into a pipe a few were 45±9 and 9±7,with the difference was significant(P＜0.05).
5.In vivo inhibition Egfl7 can inhibit the expression of VEGF-induced angiogenesis.
①To compare Mice~(Egfl7RNAi+) with Mice~(Egfl7RNAi-) two nude models the primary tumor microvessel density(MVD),There is a significant difference MVD values between the two groups(29±2 vs 37±3)(P＜0.05).
②Matrigel cultivation of VEGF in nude mice as a bio-induced angiogenesis,the experimental group in the Matrigel neovascularization was less than the control group,experimental and control groups of and, with the difference was significant(P＜0.05).There is a significant difference in Matrigel neovascularization of the total length between the two groups(23634±6421μm vs 32145±4314μm)(P＜0.05).
Conclusion:
1.Egfl7 may be involved in angiogenesis of LSCC invasion and metastasis;
2.Egfl7 could be as a key anti-angiogenesis target for LSCC gene therapy.
引文
[1] Chatni SS, Thankappan K. Lymph node status cannot be ignored in survival analysis of laryngeal cancer.Arch Otolaryngol Head Neck Surg. 2008;134(7):786; author reply 786-787.
[2] Idasiak A, Maslyk B, Blamek S, Suwinski R. Risk of distant metastases after postoperative radiation therapy for locally advanced laryngeal cancer Otolaryngol Pol. 2008;62(2): 149-57.
[3] Alhakeem M, Arabi A, Arab L, Guerra RA. Unusual sites of metastatic involvement: intracardiac metastasis from laryngeal carcinoma. Eur J Echocardiogr. 2008; 9(2): 323-325.
[4] Carinci F, Arcelli D, Lo Muzio L, Molecular classification of nodal metastasis in primary larynx squamous cell carcinoma.Transl Res. 2007;150(4):233-245.
[5] Kang SY. Regulation of tumor dormancy as a function of tumor-mediated paracrine regulation of stromal Tsp-1 and VEGF expression.APMIS. 2008; 116(7-8): 638-647.
[6] Udagawa T. Tumor dormancy of primary and secondary cancers.APMIS. 2008; 116(7-8): 615-628.
[7] Parker LH, Schmidt M, Jin SW, The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation.Nature. 2004,15;428(6984):754-758.Fitch
[8] MJ, Campagnolo L, Kuhnert F, Stuhlmann H.Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells.Dev Dyn. 2004; 230(2): 316-324.
[9] Musiyenko A, Bitko V, Barik S.Ectopic expression of miR-126~*, an intronic product of the vascular endothelial EGF-like 7 gene, regulates prostein translation and invasiveness of prostate cancer LNCaP cells.J Mol Med. 2008;86(3):313-322.
[10] Soncin F, Mattot V,Lionneton F,et al.VE-statin,an endothelial repressor of smooth muscle cell migration. EMBO J.2003, 3:22(21):5700-5711
[11] Guo Y, Liu J, Xu Z, Sun K, Fu W. HLA-B gene participates in the NF-kappaB signal pathway partly by regulating S100A8 in the laryngeal carcinoma cell line Hep2. Oncol Rep. 2008;19(6):1453-9.
[12] Sun Y, Liu M, Yang B, Li B, Lu J. Role of siRNA silencing of MMP-2 gene on invasion and growth of laryngeal squamous cell carcinoma.Eur Arch Otorhinola ryngol. 2008 ; 265(11):1385-91.
[13] Chen XM, Luan XY, Lei DP, Suppression of survivin expression by short hairpin RNA induces apoptosis in human laryngeal carcinoma cells. ORL J Otorhinolaryngol Relat Spec. 2008; 70(3): 168-75.
[14] Wang Y, Tao ZZ, Chen SM, Xiao BK, Application of combination of short hairpin RNA segments for silencing VEGF, TERT and Bcl-xl expression in laryngeal squamous carcinoma. Cancer Biol Ther. 2008;7(6):896-901.
[15] Shimizu M, Saitoh Y, Itoh H. Immunohistochemical staining of Ha-ras oncogene product in normal, benign, and malignant human pancreatic tissues. Hum Pathol. 1990; 21:607-612.
[16] Lee WT, Tubbs RR, Teker AM, Use of in situ hybridization to detect human papillomavirus in head and neck squamous cell carcinoma patients without a history of alcohol or tobacco use.Arch Pathol Lab Med. 2008;132(10),1653-1656
[17] Cheng L, Zhou L, Tao L, Zhang M, Preliminary study of proteomic shift from normal to premalignant laryngeal lesions and to laryngeal squamous cell carcinoma. Acta Otolaryngol. 2008 27:1-5
[18] Idasiak A, Maslyk B, Blamek S, Risk of distant metastases after postoperative radiation therapy for locally advanced laryngeal cancer. Otolaryngol Pol. 2008; 62(2): 149-157.
[19] Tu CF, Yan YT, Wu SY, Djoko B, Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1.J Biol Chem. 2008, 2;283(18):12478-12488.
[20] Tao L, Zhou L, Zheng L, Yao M. Elemene displays anti-cancer ability on laryngeal cancer cells in vitro and in vivo.Cancer Chemother Pharmacol. 2006; 58(1): 24-34.
[21] Kumar RV, Shenoy AM, Daniel R, Cyclin Dl, p53, MIB1, intratumoral microvessel density, and human papillomavirus in advanced laryngeal carcinoma: association with nodal metastasis.Otolaryngol Head Neck Surg. 2004;131(4):509-13.Wang Z, Ji W, Tang Q, Pan Z. Relationship among expression of the VEGF gene and
[22] MVD with cervical lymph nodes metastasis in laryngeal squamous cell carcinoma Lin Chuang Er Bi Yan Hou Ke Za Zhi. 2004;18(2):100-2.
[23] Kiss J, Tfmar J, Somlai B, Gilde K, Association of microvessel density with infiltrating cells in human cutaneous malignant melanoma.Pathol Oncol Res. 2007;13(1):21-31.
[24] Wang YD, Wu P, Mao JD, Huang H, Zhang F. Relationship between vascular invasion and microvessel density and micrometastasis.World J Gastroenterol. 2007,14;13(46):6269-73.
[25] Nie F, Xu HX, Lu MD, Wang Y, Tang Q. Anti-angiogenic gene therapy for hepatocellular carcinoma mediated by microbubble-enhanced ultrasound exposure: an in vivo experimental study.J Drug Target. 2008;16(5):389-95.
[26] Kamijo T, Yokose T, Hasebe T, Potential role of microvessel density in predicting radiosensitivity of Tl and T2 stage laryngeal squamous cell carcinoma treated with radiotherapy.Clin Cancer Res. 2000;6(8):3159-65.
[27] Sullivan CA, Ghosh S, Ocal IT, Microvessel area using automated image analysis is reproducible and is associated with prognosis in breast cancer.Hum Pathol. 2008 15.346-359
[28] Raghuwanshi SK, Nasser MW, Chen X, Strieter RM, Depletion of beta-arrestin-2 promotes tumor growth and angiogenesis in a murine model of lung cancer.J Immunol. 2008 15;180(8):5699-706.
[29] Oliveira-Neto HH, Silva ET, Leles CR, Involvement of CXCL12 and CXCR4 in lymph node metastases and development of oral squamous cell carcinomas.Tumour Biol. 2008;29(4):262-71.
[30] Hotokezaka M, Jimi S, Hidaka H, Ikeda T, Factors influencing outcome after surgery for stage IV colorectal cancer.Surg Today. 2008;38(9):784-9.
[31] Kischel P, Guillonneau F, Dumont B, Cell membrane proteomic analysis identifies proteins differentially expressed in osteotropic human breast cancer cells. Neoplasia. 2008; 10(9): 1014-20.
[32]高进.痛细胞转移模型的建立及应用.见:高进.主编 癌的侵袭和转移—基础研究与临床 第1版.北京.北京医科大学中国协和医科大学联合出版社.1996.53-63.
[33]Albini A,Iwamoto Y,Kleinman HK,et al.A rapid in vitro assay for quantitating the invasive potential of tumor cells.Cancer Res.1987;47:3239-3245.
[34]Vernon RB,Sage EH.Between molecules and morphology.Extracellular matrix and creation of vascular form.Am J Pathol.1995;147:873-883.
[35]Li WW,Grayson G,Folkman J,et al.Sustained-release endotoxin.A model for inducing corneal neovascularization.Invest Ophthalmol Vis Sci.1991;32:2906-2911.
[36]Kuhn R,Streif S,Wurst W.RNA interference in mice.Handb Exp Pharmacol.2007;178:149-176
[37]Liu Y,Wang B,Wang J,Wan W,own-regulation of PKCzeta expression inhibits chemotaxis signal transduction in human lung cancer cells.Lung Cancer.200811.1244-1250
[38]Hu L,Roth JM,Brooks P,Luty J,Thrombin up-regulates cathepsin D which enhances angiogenesis,growth,and metastasis.Cancer Res.2008 15;68(12):4666-73.
[39]Li X,Lim B.RhoGTPases and their role in cancer.Oncol Res 2003;13:323-331
[40]Bishop AL,Hall A.Rho GTPases and their effector proteins.Biochem J 2000;348:241-255
[41]Rana TM.Illuminating the silence:understanding the structure and function of small RNAs Nat Rev Mol Cell Biol.2007;8:23-36.
[42]Aharinejad S,Sioud M,Lucas T,et al.Target validation using RNA interference in solid tumors.Methods Mol Biol.2007;361:227-238.
[43]Barik S,Bitko V.Prospects of RNA interference therapy in respiratory viral diseases:update 2006.Expert Opin Biol Ther.2006;6:1151-1160.
[44]Putral LN,Gu W,McMillan NA.RNA interference for the treatment of cancer.Drug News Perspect.2006;19:317-324.
[45]Putnam D,Doody A.RNA-interference effectors and their delivery.Crit Rev Ther Drug Carrier Syst.2006;23:137-164.
[46]Wiznerowicz M,Szulc J,Trono D.Tuning silence:conditional systems for RNA interference. Nat Methods. 2006;3:682-688.
[47] Miyano-Kurosaki N, Takaku H. Gene silencing of virus replication by RNA interference. Handb Exp Pharmacol. 2006;173:151-171.
[48] Barquinero J, Eixarch H, Perez-Melgosa M. Retroviral vectors: new applications for an old tool. Gene Ther. 2004; 11 Suppl l:S3-9.
[49] Kissler S, Van Parijs L. Exploring the genetic basis of disease using RNA interference. Expert Rev Mol Diagn. 2004;4:645-651.
[50] Devroe E, Silver PA. Therapeutic potential of retroviral RNAi vectors. Expert Opin Biol Ther. 2004;4:319-327.
[51]Kuhn R, Streif S, Wurst W. RNA interference in mice. Handb Exp Pharmacol. 2007; 178:149-176
[52]Rana TM. Illuminating the silence: understanding the structure and function of small RNAs Nat Rev Mol Cell Biol. 2007;8:23-36.
[53] Nakatsu MN, Sainson RC, Aoto JN, et al. Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and Angiopoietin-1. Microvasc Res. 2003;66:102-112.
[54] Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer. 2003; 3:401-410.
[55] Chigurupati S, Kulkarni T, Thomas S, et al. Calcitonin stimulates multiple stages of angiogenesis by directly acting on endothelial cells. Cancer Res. 2005; 65: 8519- 829.
[56] Ji W, Guan C, Pan Z. Analysis of curative effects on laryngeal carcinoma patients in the northeast region of China. Acta Otolaryngol. 2008 ;128(5):574-7.
[57] Lichtenberg J, Hjarnaa PJ, Kristjansen PE, et al. The rat Subcutaneous Air Sac model: a quantitative assay of antiangiogenesis in induced vessels. Pharmacol Toxicol.1999;84:34-40.
[58] Xu Y, Swerlick RA, Sepp N, et al. Characterization of expression and modulation of cell adhesion molecules on an immortalized human dermal microvascular endothelial cell line (HMEC-1). J Invest Dermatol. 1994; 102:833-837.
[59] Nguyen M, Shing Y, Folkman J. Quantitation of angiogenesis and antiangioge nesis in the chick embryo chorioallantoic membrane. Microvasc Res. 1994; 47: 31-40.
[60] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000; 6: 389-395.
[61] Ruoslahti E. Specialization of tumour vasculature. Nat Rev Cancer. 2002; 2: 83-90.
[62] Lu X, Le Noble F, Yuan L, et al. The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature. 2004; 432: 179-186.
[63] Ades EW, Candal FJ, Swerlick RA, et al. establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 1992; 99:683 -690.
[64] Matsushita S, Nitanda T, Furukawa T, The effect of a thymidine phosphorylase inhibitor on angiogenesis and apoptosis in tumors. Cancer Res. 1999;59:1911-1916.
[65] Nakatsu MN, Sainson RC, Aoto JN, et al. Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and Angiopoietin-1. Microvasc Res. 2003; 66:102-112.
[66] Nguyen M, Shing Y, Folkman J. Quantitation of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane. Microvasc Res. 1994;47: 31-40.
[67] Lichtenberg J, Hansen CA, Skak-Nielsen T, et al. The rat subcutaneous air sac model: a new and simple method for in vivo screening of antiangiogenesis. Pharmacol Toxicol. 1997;81:280-284.
[68] Lichtenberg J, Hjarnaa PJ, Kristjansen PE, et al. The rat Subcutaneous Air Sac model: a quantitative assay of antiangiogenesis in induced vessels. Pharmacol Toxicol.1999; 84:34-40.
[69]Wiktorowska-Owczarek A, Namiecinska M, Balcerczyk A, Human micro- and macrovessel-derived endothelial cells: a comparative study on the effects of adrenaline and a selective adenosine A2-type receptor agonist under normoxic and hypoxic conditions.Pharmacol Rep. 2007;59(6):800-6.
[70]Hoang MV, Whelan MC, Senger DR. Rho activity critically and selectively regulates endothelial cell organization during angiogenesis.Proc Natl Acad Sci U S A.2004;101:1874-1879.
[1] Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2002 ;2(8):563-72
[2] Bernards R. Cancer: cues for migration. Nature. 2003 ;425(6955): 247-8
[3] Moore MA. The role of chemoattraction in cancer metastases. Bioessays. 2001; 23(8): 674-6
[4] Campagnolo L, Leahy A, Chitnis S, et al. EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury.Am J Pathol. 2005; 167(1): 275-84
[5] Parker LH, Schmidt M, Jin SW, et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature 2004 ;428:754-758
[6] Soncin F, Mattot V, Lionneton F, et al. VE-statin, an endothelial repressor of smooth muscle cell migration. EMBO J 2003;22:5700- 5711
[7] Doliana R, Bot S, Bonaldo P, et al. EMI, a novel cysteine-rich domain of EMILINs and other extracellular proteins, interacts with the gC1q domains and participates in multimerization.FEBS Lett. 2000;484,164-168
[8] Callebaut I, Mignotte V. Souchet M, et al. EMI domains are widespread and reveal the probable orthologs of the Caenorhabditis elegans CED-1 protein. Biochem.Biophys. Res. Commun. 2003;300,619-623
[9] Steno J, Stenberg Y, Muranyi A. Calcium-binding EGFlike modules in coagulation proteinases: function of the calcium ion in module interactions. Biochim. Biop- hys Acta 2000;1477:51-63
[10] Fitch MJ, Campagnolo L, Kuhnert F, et al.. Egfl7, a Novel Epidermal Growth Factor-Domain Gene Expressed in Endothelial Cells. DEVELOPMENTAL DYNAMICS 2004; 230:316-324
[11] Drake CJ, Fleming PA. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 2000 ;95:1671-1679
[12] Uyttendaele H, Marazzi G, Wu G, et al. Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development 1996; 122:2251-2259
[13] Shutter JR, Scully S, Fan W, et al. D114, a novel Notch ligand expressed in arterial endothelium.Genes Dev 2000;14:1313-1318
[14]Lengauer C,Kinzler KW,Vogelstein B.Genetic instabilities in human cancers.Nature.1998;396(6712):643-9
[15]Oppenheimer SB.Cellular basis of cancer metastasis:A review of fundamentals and new advances.Acta Histochem.2006;108(5):327-34
[16]Woodhouse EC,Chuaqui RE Liotta LA.General mechanisms of metastasis.Cancer.1997;80(8 Suppl):1529-37
[17]Mundy GR,DeMartino S,Rowe DW.Collagen and collagen-derived fragments are chemotactic for tumor cells.J Clin Invest 1981;68:1102-5
[18]Waas JA,Varani J,Piontek GE,et al.Responses of normal and malignant cells to collagen,collagen-derived peptides and the C5-related tumor cell chemotactic peptide.Cell Differ 1981;10:329-32
[19]Vicente-Manzanares M,Sanchez-Madrid F.Role of the cytoskeleton during leukocy-te responses.Nat Rev Immunol.2004;4:110-122
[20]Rossi D,Zlotnik A.The biology of chemokines and their receptors.Annu Rev Imm-unol.2000;18:217-242
[21]Robert C,Johnson,Hellmut G,et al.Endothelial Cell Membrane Vesicles in the Study of Organ Preference of Metas-tasis.CANCER RESEARCH 1991;51:394-399
[22]Pauli BU,Augustin-Voss HG,el-Sabban ME,et al.Organ-preference of metastasis.The role of endothelial cell adhesion molecules.Cancer Metastasis Rev.1990;9(3):175-89
[23]Muller A,Homey B,Soto H,et al.Involvement of chemokine receptors in breast cancer metastasis.Nature.2001;410(6824):50-6
[24]Folkman,J.How is blood vessel growth regulated in normal and neoplastic tissue?G.H.A.Clowes memorial award lecture.Cancer Res.1986;46:467-743
[25]Hashizume H,Baluk P,Morikawa S,et al.Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol.2000;156(4):1363-80
[26]Lee YT,Geer DA.Primary liver cancer:pattern of metastasis.J Surg Oncol.1987;36(1):26-31
[2] Idasiak A, Maslyk B, Blamek S, Suwinski R. Risk of distant metastases after postoperative radiation therapy for locally advanced laryngeal cancer Otolaryngol Pol. 2008;62(2): 149-57.
[3] Alhakeem M, Arabi A, Arab L, Guerra RA. Unusual sites of metastatic involvement: intracardiac metastasis from laryngeal carcinoma. Eur J Echocardiogr. 2008; 9(2): 323-325.
[4] Carinci F, Arcelli D, Lo Muzio L, Molecular classification of nodal metastasis in primary larynx squamous cell carcinoma.Transl Res. 2007;150(4):233-245.
[5] Kang SY. Regulation of tumor dormancy as a function of tumor-mediated paracrine regulation of stromal Tsp-1 and VEGF expression.APMIS. 2008; 116(7-8): 638-647.
[6] Udagawa T. Tumor dormancy of primary and secondary cancers.APMIS. 2008; 116(7-8): 615-628.
[7] Parker LH, Schmidt M, Jin SW, The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation.Nature. 2004,15;428(6984):754-758.Fitch
[8] MJ, Campagnolo L, Kuhnert F, Stuhlmann H.Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells.Dev Dyn. 2004; 230(2): 316-324.
[9] Musiyenko A, Bitko V, Barik S.Ectopic expression of miR-126~*, an intronic product of the vascular endothelial EGF-like 7 gene, regulates prostein translation and invasiveness of prostate cancer LNCaP cells.J Mol Med. 2008;86(3):313-322.
[10] Soncin F, Mattot V,Lionneton F,et al.VE-statin,an endothelial repressor of smooth muscle cell migration. EMBO J.2003, 3:22(21):5700-5711
[11] Guo Y, Liu J, Xu Z, Sun K, Fu W. HLA-B gene participates in the NF-kappaB signal pathway partly by regulating S100A8 in the laryngeal carcinoma cell line Hep2. Oncol Rep. 2008;19(6):1453-9.
[12] Sun Y, Liu M, Yang B, Li B, Lu J. Role of siRNA silencing of MMP-2 gene on invasion and growth of laryngeal squamous cell carcinoma.Eur Arch Otorhinola ryngol. 2008 ; 265(11):1385-91.
[13] Chen XM, Luan XY, Lei DP, Suppression of survivin expression by short hairpin RNA induces apoptosis in human laryngeal carcinoma cells. ORL J Otorhinolaryngol Relat Spec. 2008; 70(3): 168-75.
[14] Wang Y, Tao ZZ, Chen SM, Xiao BK, Application of combination of short hairpin RNA segments for silencing VEGF, TERT and Bcl-xl expression in laryngeal squamous carcinoma. Cancer Biol Ther. 2008;7(6):896-901.
[15] Shimizu M, Saitoh Y, Itoh H. Immunohistochemical staining of Ha-ras oncogene product in normal, benign, and malignant human pancreatic tissues. Hum Pathol. 1990; 21:607-612.
[16] Lee WT, Tubbs RR, Teker AM, Use of in situ hybridization to detect human papillomavirus in head and neck squamous cell carcinoma patients without a history of alcohol or tobacco use.Arch Pathol Lab Med. 2008;132(10),1653-1656
[17] Cheng L, Zhou L, Tao L, Zhang M, Preliminary study of proteomic shift from normal to premalignant laryngeal lesions and to laryngeal squamous cell carcinoma. Acta Otolaryngol. 2008 27:1-5
[18] Idasiak A, Maslyk B, Blamek S, Risk of distant metastases after postoperative radiation therapy for locally advanced laryngeal cancer. Otolaryngol Pol. 2008; 62(2): 149-157.
[19] Tu CF, Yan YT, Wu SY, Djoko B, Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1.J Biol Chem. 2008, 2;283(18):12478-12488.
[20] Tao L, Zhou L, Zheng L, Yao M. Elemene displays anti-cancer ability on laryngeal cancer cells in vitro and in vivo.Cancer Chemother Pharmacol. 2006; 58(1): 24-34.
[21] Kumar RV, Shenoy AM, Daniel R, Cyclin Dl, p53, MIB1, intratumoral microvessel density, and human papillomavirus in advanced laryngeal carcinoma: association with nodal metastasis.Otolaryngol Head Neck Surg. 2004;131(4):509-13.Wang Z, Ji W, Tang Q, Pan Z. Relationship among expression of the VEGF gene and
[22] MVD with cervical lymph nodes metastasis in laryngeal squamous cell carcinoma Lin Chuang Er Bi Yan Hou Ke Za Zhi. 2004;18(2):100-2.
[23] Kiss J, Tfmar J, Somlai B, Gilde K, Association of microvessel density with infiltrating cells in human cutaneous malignant melanoma.Pathol Oncol Res. 2007;13(1):21-31.
[24] Wang YD, Wu P, Mao JD, Huang H, Zhang F. Relationship between vascular invasion and microvessel density and micrometastasis.World J Gastroenterol. 2007,14;13(46):6269-73.
[25] Nie F, Xu HX, Lu MD, Wang Y, Tang Q. Anti-angiogenic gene therapy for hepatocellular carcinoma mediated by microbubble-enhanced ultrasound exposure: an in vivo experimental study.J Drug Target. 2008;16(5):389-95.
[26] Kamijo T, Yokose T, Hasebe T, Potential role of microvessel density in predicting radiosensitivity of Tl and T2 stage laryngeal squamous cell carcinoma treated with radiotherapy.Clin Cancer Res. 2000;6(8):3159-65.
[27] Sullivan CA, Ghosh S, Ocal IT, Microvessel area using automated image analysis is reproducible and is associated with prognosis in breast cancer.Hum Pathol. 2008 15.346-359
[28] Raghuwanshi SK, Nasser MW, Chen X, Strieter RM, Depletion of beta-arrestin-2 promotes tumor growth and angiogenesis in a murine model of lung cancer.J Immunol. 2008 15;180(8):5699-706.
[29] Oliveira-Neto HH, Silva ET, Leles CR, Involvement of CXCL12 and CXCR4 in lymph node metastases and development of oral squamous cell carcinomas.Tumour Biol. 2008;29(4):262-71.
[30] Hotokezaka M, Jimi S, Hidaka H, Ikeda T, Factors influencing outcome after surgery for stage IV colorectal cancer.Surg Today. 2008;38(9):784-9.
[31] Kischel P, Guillonneau F, Dumont B, Cell membrane proteomic analysis identifies proteins differentially expressed in osteotropic human breast cancer cells. Neoplasia. 2008; 10(9): 1014-20.
[32]高进.痛细胞转移模型的建立及应用.见:高进.主编 癌的侵袭和转移—基础研究与临床 第1版.北京.北京医科大学中国协和医科大学联合出版社.1996.53-63.
[33]Albini A,Iwamoto Y,Kleinman HK,et al.A rapid in vitro assay for quantitating the invasive potential of tumor cells.Cancer Res.1987;47:3239-3245.
[34]Vernon RB,Sage EH.Between molecules and morphology.Extracellular matrix and creation of vascular form.Am J Pathol.1995;147:873-883.
[35]Li WW,Grayson G,Folkman J,et al.Sustained-release endotoxin.A model for inducing corneal neovascularization.Invest Ophthalmol Vis Sci.1991;32:2906-2911.
[36]Kuhn R,Streif S,Wurst W.RNA interference in mice.Handb Exp Pharmacol.2007;178:149-176
[37]Liu Y,Wang B,Wang J,Wan W,own-regulation of PKCzeta expression inhibits chemotaxis signal transduction in human lung cancer cells.Lung Cancer.200811.1244-1250
[38]Hu L,Roth JM,Brooks P,Luty J,Thrombin up-regulates cathepsin D which enhances angiogenesis,growth,and metastasis.Cancer Res.2008 15;68(12):4666-73.
[39]Li X,Lim B.RhoGTPases and their role in cancer.Oncol Res 2003;13:323-331
[40]Bishop AL,Hall A.Rho GTPases and their effector proteins.Biochem J 2000;348:241-255
[41]Rana TM.Illuminating the silence:understanding the structure and function of small RNAs Nat Rev Mol Cell Biol.2007;8:23-36.
[42]Aharinejad S,Sioud M,Lucas T,et al.Target validation using RNA interference in solid tumors.Methods Mol Biol.2007;361:227-238.
[43]Barik S,Bitko V.Prospects of RNA interference therapy in respiratory viral diseases:update 2006.Expert Opin Biol Ther.2006;6:1151-1160.
[44]Putral LN,Gu W,McMillan NA.RNA interference for the treatment of cancer.Drug News Perspect.2006;19:317-324.
[45]Putnam D,Doody A.RNA-interference effectors and their delivery.Crit Rev Ther Drug Carrier Syst.2006;23:137-164.
[46]Wiznerowicz M,Szulc J,Trono D.Tuning silence:conditional systems for RNA interference. Nat Methods. 2006;3:682-688.
[47] Miyano-Kurosaki N, Takaku H. Gene silencing of virus replication by RNA interference. Handb Exp Pharmacol. 2006;173:151-171.
[48] Barquinero J, Eixarch H, Perez-Melgosa M. Retroviral vectors: new applications for an old tool. Gene Ther. 2004; 11 Suppl l:S3-9.
[49] Kissler S, Van Parijs L. Exploring the genetic basis of disease using RNA interference. Expert Rev Mol Diagn. 2004;4:645-651.
[50] Devroe E, Silver PA. Therapeutic potential of retroviral RNAi vectors. Expert Opin Biol Ther. 2004;4:319-327.
[51]Kuhn R, Streif S, Wurst W. RNA interference in mice. Handb Exp Pharmacol. 2007; 178:149-176
[52]Rana TM. Illuminating the silence: understanding the structure and function of small RNAs Nat Rev Mol Cell Biol. 2007;8:23-36.
[53] Nakatsu MN, Sainson RC, Aoto JN, et al. Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and Angiopoietin-1. Microvasc Res. 2003;66:102-112.
[54] Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer. 2003; 3:401-410.
[55] Chigurupati S, Kulkarni T, Thomas S, et al. Calcitonin stimulates multiple stages of angiogenesis by directly acting on endothelial cells. Cancer Res. 2005; 65: 8519- 829.
[56] Ji W, Guan C, Pan Z. Analysis of curative effects on laryngeal carcinoma patients in the northeast region of China. Acta Otolaryngol. 2008 ;128(5):574-7.
[57] Lichtenberg J, Hjarnaa PJ, Kristjansen PE, et al. The rat Subcutaneous Air Sac model: a quantitative assay of antiangiogenesis in induced vessels. Pharmacol Toxicol.1999;84:34-40.
[58] Xu Y, Swerlick RA, Sepp N, et al. Characterization of expression and modulation of cell adhesion molecules on an immortalized human dermal microvascular endothelial cell line (HMEC-1). J Invest Dermatol. 1994; 102:833-837.
[59] Nguyen M, Shing Y, Folkman J. Quantitation of angiogenesis and antiangioge nesis in the chick embryo chorioallantoic membrane. Microvasc Res. 1994; 47: 31-40.
[60] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000; 6: 389-395.
[61] Ruoslahti E. Specialization of tumour vasculature. Nat Rev Cancer. 2002; 2: 83-90.
[62] Lu X, Le Noble F, Yuan L, et al. The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature. 2004; 432: 179-186.
[63] Ades EW, Candal FJ, Swerlick RA, et al. establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 1992; 99:683 -690.
[64] Matsushita S, Nitanda T, Furukawa T, The effect of a thymidine phosphorylase inhibitor on angiogenesis and apoptosis in tumors. Cancer Res. 1999;59:1911-1916.
[65] Nakatsu MN, Sainson RC, Aoto JN, et al. Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and Angiopoietin-1. Microvasc Res. 2003; 66:102-112.
[66] Nguyen M, Shing Y, Folkman J. Quantitation of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane. Microvasc Res. 1994;47: 31-40.
[67] Lichtenberg J, Hansen CA, Skak-Nielsen T, et al. The rat subcutaneous air sac model: a new and simple method for in vivo screening of antiangiogenesis. Pharmacol Toxicol. 1997;81:280-284.
[68] Lichtenberg J, Hjarnaa PJ, Kristjansen PE, et al. The rat Subcutaneous Air Sac model: a quantitative assay of antiangiogenesis in induced vessels. Pharmacol Toxicol.1999; 84:34-40.
[69]Wiktorowska-Owczarek A, Namiecinska M, Balcerczyk A, Human micro- and macrovessel-derived endothelial cells: a comparative study on the effects of adrenaline and a selective adenosine A2-type receptor agonist under normoxic and hypoxic conditions.Pharmacol Rep. 2007;59(6):800-6.
[70]Hoang MV, Whelan MC, Senger DR. Rho activity critically and selectively regulates endothelial cell organization during angiogenesis.Proc Natl Acad Sci U S A.2004;101:1874-1879.
[1] Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2002 ;2(8):563-72
[2] Bernards R. Cancer: cues for migration. Nature. 2003 ;425(6955): 247-8
[3] Moore MA. The role of chemoattraction in cancer metastases. Bioessays. 2001; 23(8): 674-6
[4] Campagnolo L, Leahy A, Chitnis S, et al. EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury.Am J Pathol. 2005; 167(1): 275-84
[5] Parker LH, Schmidt M, Jin SW, et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature 2004 ;428:754-758
[6] Soncin F, Mattot V, Lionneton F, et al. VE-statin, an endothelial repressor of smooth muscle cell migration. EMBO J 2003;22:5700- 5711
[7] Doliana R, Bot S, Bonaldo P, et al. EMI, a novel cysteine-rich domain of EMILINs and other extracellular proteins, interacts with the gC1q domains and participates in multimerization.FEBS Lett. 2000;484,164-168
[8] Callebaut I, Mignotte V. Souchet M, et al. EMI domains are widespread and reveal the probable orthologs of the Caenorhabditis elegans CED-1 protein. Biochem.Biophys. Res. Commun. 2003;300,619-623
[9] Steno J, Stenberg Y, Muranyi A. Calcium-binding EGFlike modules in coagulation proteinases: function of the calcium ion in module interactions. Biochim. Biop- hys Acta 2000;1477:51-63
[10] Fitch MJ, Campagnolo L, Kuhnert F, et al.. Egfl7, a Novel Epidermal Growth Factor-Domain Gene Expressed in Endothelial Cells. DEVELOPMENTAL DYNAMICS 2004; 230:316-324
[11] Drake CJ, Fleming PA. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 2000 ;95:1671-1679
[12] Uyttendaele H, Marazzi G, Wu G, et al. Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development 1996; 122:2251-2259
[13] Shutter JR, Scully S, Fan W, et al. D114, a novel Notch ligand expressed in arterial endothelium.Genes Dev 2000;14:1313-1318
[14]Lengauer C,Kinzler KW,Vogelstein B.Genetic instabilities in human cancers.Nature.1998;396(6712):643-9
[15]Oppenheimer SB.Cellular basis of cancer metastasis:A review of fundamentals and new advances.Acta Histochem.2006;108(5):327-34
[16]Woodhouse EC,Chuaqui RE Liotta LA.General mechanisms of metastasis.Cancer.1997;80(8 Suppl):1529-37
[17]Mundy GR,DeMartino S,Rowe DW.Collagen and collagen-derived fragments are chemotactic for tumor cells.J Clin Invest 1981;68:1102-5
[18]Waas JA,Varani J,Piontek GE,et al.Responses of normal and malignant cells to collagen,collagen-derived peptides and the C5-related tumor cell chemotactic peptide.Cell Differ 1981;10:329-32
[19]Vicente-Manzanares M,Sanchez-Madrid F.Role of the cytoskeleton during leukocy-te responses.Nat Rev Immunol.2004;4:110-122
[20]Rossi D,Zlotnik A.The biology of chemokines and their receptors.Annu Rev Imm-unol.2000;18:217-242
[21]Robert C,Johnson,Hellmut G,et al.Endothelial Cell Membrane Vesicles in the Study of Organ Preference of Metas-tasis.CANCER RESEARCH 1991;51:394-399
[22]Pauli BU,Augustin-Voss HG,el-Sabban ME,et al.Organ-preference of metastasis.The role of endothelial cell adhesion molecules.Cancer Metastasis Rev.1990;9(3):175-89
[23]Muller A,Homey B,Soto H,et al.Involvement of chemokine receptors in breast cancer metastasis.Nature.2001;410(6824):50-6
[24]Folkman,J.How is blood vessel growth regulated in normal and neoplastic tissue?G.H.A.Clowes memorial award lecture.Cancer Res.1986;46:467-743
[25]Hashizume H,Baluk P,Morikawa S,et al.Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol.2000;156(4):1363-80
[26]Lee YT,Geer DA.Primary liver cancer:pattern of metastasis.J Surg Oncol.1987;36(1):26-31