摘要
研究背景和目的
胃癌是世界范围内第四位的恶性肿瘤,继肺癌之后占据全世界恶性肿瘤死亡率的第二位。淋巴结转移是导致胃癌治疗失败和患者死亡的主要原因,是胃癌最强烈的预后因素。大约60%的可切除胃癌在诊断之时已经发生了淋巴结转移,因此进展期胃癌总的五年生存率不足20%。因此,预测肿瘤转移倾向、判断肿瘤预后的标志物,寻找预防和治疗的有效途径,提高病人的5年生存率,降低死亡率是胃癌研究的重点内容,也是肿瘤防治研究中的一个重点和难点。
肿瘤的生长和转移有赖于血管新生,促进血管新生的关键因子是血管内皮生长因子家族成员(vascular endothelial growth factor family VEGFs)。VEGFs包括VEGF-A、B、C、D、E和PIGF (placenta growth factor,胎盘生长因子)。近年来,许多文献报道了血管内皮生长因子(VEGF)在多种恶性肿瘤中异常表达,并与肿瘤的生长、转移及预后相关。其中VEGF-B及其受体Flt-1的表达已见于人神经系统肿瘤、乳腺癌、肾癌、大肠癌、肺癌等,与大肠癌和肺癌的淋巴浸润和淋巴转移密切相关；可以调节肾癌中肿瘤血管的形成；在恶性的神经胶质瘤血管生成过程中,可能作为辅助因子而发挥作用；在乳腺癌中其主要作用不是促进血管形成,而是与淋巴转移密切相关,目前对于抗VEGF受体的治疗,为乳腺癌的治疗提供新的途径。但VEGF-B及其受体在胃癌中的表达及意义尚未见于报道。
血管内皮生长因子C(vascular endothelial growth factor C, VEGF-C)及其受体血管内皮生长因子受体-3(vascular endothelial growth factor receptor-3, VEGFR-3),亦称Flt-4,是近年来发现的淋巴管生长因子,研究认为VEGF-C是相对特异的淋巴管内皮生长因子,与VEGFR-3结合后可发生淋巴管内皮扩张,促使淋巴管形成,从而为研究淋巴结转移机制开辟了新的途径。血管内皮生长因子C与其受体VEGFR-3结合后,具有强大特异的淋巴管生成作用,并认为与肿瘤的淋巴结转移有密切关系。
T淋巴瘤侵袭转移诱导因子1(T lymphoma invasion and metastasis1,Tiam-1)基因足Dbl家族的鸟嘌呤核酸转换因子(guanine nucleotide exchange factor, GEFs)之,GEFs主要功能是调节Rho三膦酸鸟嘌呤核苷酸水解酶(Rho GTPases)家族活性,是普遍存在的二磷酸鸟嘌呤核仆酸(GDP)(?)三膦酸鸟嘌呤核苷酸(GTP)(?)专换因子,通过参与Tiam1-Rac(?)号传递通路调细胞骨架重组、细胞周期进程、基因转录、细胞迁移和粘附等细胞生命活动,在肿瘤增殖、侵袭和转移过程中发挥重要作用。早期研究表明Tiam-1是诱导人类T细胞淋巴瘤侵袭和转移的基因。Tiam1(?)基因最初在小鼠T淋巴瘤细胞高侵袭变异株中分离鉴定。随后,在乳腺襄胃癌及Ras诱导的皮肤癌等肿瘤证实Tiam1具有明显的促进肿瘤进展和转移的作用。在Tiam-1(?)敲除的动物模型中,小鼠皮肤癌发生率明显减低并且肿瘤进展缓慢,Malliri等认为Tiam-1在Ras诱导皮肤癌发生的的启动和进展阶段发挥关键作用,这一效应与Tiam-1表达量有明显的关系。上述的研究表明,Tiam-1是多种肿瘤的促癌基因和促转移基因。
淋巴结转移是胃癌患者预后的十分重要的不良因素,其诊断尤为重要。而且前要的临床诊断手.段却对胃癌淋巴结转移存在与否的预测缺乏特异性和敏感性。尽管常规病理理HE染色对于淋巴结转移是金标准,但是,对于早期的淋巴结微转移却缺乏敏感性,对胃癌淋巴结转移的基础实验研究则可能有助于我们找到解决问题的金钥匙。VEGF-C作为调控淋巴管发育及淋巴管生成的特异因子及其与淋巴结转移的相关性在动物实验及临床病理实验中已经得到证实。并且Tiam-1被认为是多种肿瘤促癌基因和促转移基因,这可能对于胃癌患者早期诊断、准确分期、改善手术方式和术后治方案,甚至研制相相关新药,促进肿瘤治疗进展将具有重大意义但两者的相关性及在外周血中的表达与转移的相关性尚没有明确研究。本研究通过实时荧光定量RT-PCR、免疫组化和酶联免疫法检测外周血及病理组织中VEGF-C和Tiam-1(?)的表达和基因水平变化,结合临床病理资料进行统计分析,旨在探讨VEGF-C和Tiam-1的表达和基因水平变化及其相关性与胃癌淋巴结转移和淋巴结微转移的相相关性，以及在胃癌患者肿瘤组织外周血中的表达有无相关性,明确能否用外周血中的表达水平来预测肿瘤组织的表达,寻找一种更简便的检测方法,并研究VEGF-C和Tiam-1的表达水平和肿瘤治疗及预后之间的关系,分析其作为肿瘤标记物的意义。
研究方法
1.临床病理参数采集：采集研究对象年龄、性别、肿瘤直径、TNM分期、血管浸润、浆膜浸润、淋巴管浸润资料信息。
2.血清中VEGF-B、VEGF-C水平的检测：ELISA检测试剂盒检测正常人群、胃癌患者术前术后及随访患者血清蛋白水平
3.病理组织中VEGF-B、VEGF-C和Tiam-1蛋白/基因水平的检测：应用免疫组化技术、Western blotting和荧光定量PCR检测胃癌标本、淋巴结及距病灶约3.5cm的癌旁正常胃粘膜组织中VEGF-B、VEGF-C和Tiam-1蛋白表达和基因水平变化。
4.统计分析：应用SPSS13.0软件进行统计学处理,采用x2检验分析VEGF-B、 VEGF-C和Tiam-1蛋白/基因水平与各临床病理参数之间的关系；采用Spearman相关分析VEGF-C和Tiam-1蛋白/基因水平相关性；采用Kaplan-Meier法回顾性分析VEGF-C和Tiam-1蛋白表达及基因水平与胃癌患者生存期的关系；以COX回归对各项指标单因素或多变量联合与生存期的关系进行统计学分析。
研究结果
1.胃癌患者术前血清VEGF-C水平较对照组血清VEGF-C升高,差异具有显著意义(P<0.01)。胃癌术后患者血清VEGF-C水平与术前相应指标比较,差异具有显著性(P<0.05)。但胃癌手术后血清VEGF-C水平与健康对照组相比,无明显差异(P>0.05)。VEGF-B在术前术后胃癌患者血清中无明显变化。
2.复发转移组的血清VEGF-C水平较未复发转移的血清VEGF-C水平升高,两组相比,差异具有显著性(P<0.05)。复发转移组与对照组间的比较,差异具有显著性(P<0.05)。对照组和无复发转移组间的比较,无显著差异。VEGF-B在复发转移组与对照组间的比较无明显差异。
3.胃癌患者血清VEGF-C水平与淋巴结转移、浸润浆膜及临床分期有关,无淋巴结转移组、未侵润浆膜组、TNM Ⅰ期组及高中分化组患者血清VEGF-C水平均明显低于淋巴结转移组,浸润浆膜组、TNM Ⅱ Ⅲ Ⅳ期及低分化未分化组(均P<0.05), VEGF-B关系不明显。
4. VEGF-C和Tiam-1蛋白主要表达于胃癌组织原发灶及淋巴结转移灶的癌细胞的胞浆或/和胞膜上,伴淋巴结转移的胃癌的VEGF-C和Tiam-1表达的阳性率明显高于无淋巴结转移的胃癌的阳性率,差异具有显著性(P<0.05)。胃癌患者癌组织中VEGF-C和Tiam-1蛋白与淋巴结转移、浸润浆膜及临床分期有关,无淋巴结转移组、未侵润浆膜组、TNM ⅠⅡ期组患者癌组织中VEGF-C和Tiam-1蛋白均明显低于淋巴结转移组,浸润浆膜组、TNMⅢⅣ期组(均P<0.05),
5.实时荧光定量RT-PCR结果显示,正常胃粘膜组织与各实验组胃癌组织及淋巴结组织的VEGF-C和Tiam-1基因水平差别有统计学意义(P<0.05); VEGF-C、Tiam-1mRNA相对含量与淋巴结转移、临床分期密切相关，在有淋巴结转移和TNMⅢⅣ期的癌组织中,VEGF-C、Tiam-1mRNA相对含量较没有淋巴结转移、TNM Ⅰ Ⅱ期的癌组织中显著增高(P<0.01),并且淋巴结转移越远,VEGF-C、 Tiam-1mRNA含量增高越明显(p<0.01)。
6. Spearman相关分析显示VEGF-C和Tiam-1在胃癌中的表达成正关(r=0.567,P<0.01),且两者的表达水平均随胃癌组织分化程度降低、浸润深度增加、TNM分期升高及淋巴结转移的发生而逐渐增强(P<0.05)。
7. Kaplan-Meier生存曲线显示,VEGF-C和Tiam-1表达阳性患者与未表达患者患者生存时间有显著性差异(P<0.05)。将VEGF-C和Tiam-1表达水平和临床病理参数综合与癌患者的的生存时间进行单因素分析,结果显示VEGF-C在癌组织中,高表达和Tiam-1在淋巴结中高表达、肿瘤分期是影响患者生存时间的重要因素(P值分别为：0.003、0.007、0.028)。
结论
1.血清VEGF-C水平检测可作为胃癌的血清学标记物,对筛选胃癌及观察胃癌复发转移具有一定的临床意义。
2. VEGF-B在胃癌组织有一定的表达,但与胃癌的各病理参数无相相关性
3. VEGF-C和Tiam-1在胃癌组织中及转移淋巴结中高表达,在正常组织中表达,提示VEGF-B、VEGF-C和TIAM-1可作为临床监测胃癌转移和复发的重要标志物；
4. VEGF-C和Tiam-1的表达强度与胃癌病理分期关系密切,其表达水平可作为判断胃癌侵袭性的参考指标之一。
5. VEGF-C和Tiam-1高表达与患者的生存期密切相关,提示VEGF-C和Tiam-1可作为胃癌预后判断的重要指标,对判断胃癌预后有重要意义。
6. VEGF-C和Tiam-1有一定的相关性,说明两者在促进肿瘤发生、发展及转移中具有一定的协同作用,对胃癌治疗靶点的选择具有重要参考价值。
Background and objective
Gastric cancer is the forth most common malignancy in the world, and is the second leading cause of cancer death after lung cancer. Lymph node metastasis is the strongest prognostic factor, and the major cause of treatment failure and death of patients with gastric cancer. About60%of resecable gastric cancers have lymph node metastasis at the time of diagnosis, and so, the5-year overall survival of advanced gastric cancer is lower than20%. Thus, to predict Lymph node metastasis tendency, identify the prognosis markers, find the effective way for prevention and therapy and raise five-year survival rate of Gastric cancer patients have been the hot spots for studies, and also been an emphasis and difficult point in treatment and prevention of tumor.
Growth and metastasis of tumor depend on angiogenesis; the most key factor is vascular endothelial growth factor family (VEGFs) in encouraging angiogenesis. VEGFs include VEGF-A、B、C、D、 and PIGF (placenta growth factor).Recently, many studies reveal that VEGF has abnormal expression in many malignant tumors. VEGF-B and its receptor Flt-1are found in human nervous system tumor, breast carcinoma, renal carcinoma, cancer of colon, lung cancer etc. They have relation to lymphatic vessel invasion and lymphatic metastasis of colon and lung cancer; Regulate formation of tumor vessel in renal carcinoma; they have correlation with lymphatic metastasis, not promote angiogenesis in breast carcinoma; in process of malignant neurospongi-oma vessel formation, they may play a role as a assistance factor. At present, the therapy with anti-VEGF receptor provides a new method for breast cancer. But, the expression and significance of VEGF-B and its receptor in gastric carcinoma haven't been found in reports.
Vascular endothelial growth factor C (VEGF-C) and its receptor VEGF receptor-3(VEGFR-3, also designated Flt-4) are recently identified lymphangiogenic stimulators that can selectively induce growth of the lymphatic vasculature. In recent years, the vascular endothelial growth factor C (VHGF-C) and its receptor VEGFR-3are found, and VEGF-C is known that it is a relatively specific lymphatic endothelial growth factor. The expansion of lymphatic endothelial to promote the formation of lymphatic vessels is occurred after VEGF-C and VEGFR-3combine together and thus to study the mechanism of lymph node metastasis has opened new avenues. They have strong special function of promoting the formation of lymphatic vessels after VEGF-C combined with VEGFR-3. And was known keep a close relationship with lymph node metastases.
Tiam-1(T lymphoma invasion and metastasis1) was originally identified as the invasion-and metastasis-inducing gene by proviral tagging in combination with in vitro selection for invasiveness in T lymphoma cells. Tiam1is one of guanine nucleotide exchange factors (GEFs), which activate GTPases by promoting the exchange of their inactive GDP-bound forms to their active GTP-bound forms. Whereas Tiam-1displays GEF activity towards all three Rho-like GTPases Racl, Cdc42and RhoA in vitro, Tiaml specifically activates Rac in vivo. Recent evidence suggests that Tiam-1could influence Rac GTPases signaling specificity in addition to promoting their activation.Tiam1has been implicated to directly bind to many different cytoplasmic and membrane-associated proteins, which couples Tiam-1-Rac activity to specific signaling pathways. The role of Tiam-1in cellular migration, invasion and metastasis may not be limited to T lymphoma. Related studies on breast cancer, lung cancer and skin cancer induced by Ras confirmed that Tiam1could accelerate tumor progression and metastasis significantly. In the Tiam-1-knockout animal model, the incidence rate of mouse skin cancer obviously reduced and tumors were grown slowly. The above studies showed that Tiam1might accelerate carcinogenesis and metastasis in many tumors.
Lymph node metastasis is one of the most important negative predictor to patients with gastric cancer. It's especially important to be diagnosis for metastases of lymph node. The most common clinical device used to diagnosis cannot give us a little useful advice about lymph node metastases. There is no specificity or sensitivity to lymph node metastasis. To study the basic theory about lymph node metastases in gastric cancer can be helpful to look for the gold key to see the essential qualities of lymph metastases. With the study progresses on VEGF-C and Lymph node metastasis, we can gradually master the matter used on early diagnosis, correct stage, improve operation style, select better treatment plan after surgery and investigate new drugs resist tumor. It will be improve the treatment about gastric cancer and have great significance. In this study, the VEGF-C and Tiam-1expression level was detected by RT-PCR, immunohistochemical streptavidin peroxidase method, and ELISA method; And all the data was analyzed statistically with clinical and pathological features. So we can investigate the relationship among the expression of VEGF-C and Tiam-land the relationship between them and the lymphatic metastasis or micro metastasis. To study the correlation ship of the expression level of VEGF-C and Tiam-1between cancer tissue and peripheral blood serum, so identify if it can be taken the place by VEGF-C and Tiam-1protein detected in serum, so we can find a easier method detection way. The relationship between the VEGF-C and Tiam-1expression's level and tumor treatment was selected. The clinical significance of VEGF-C and Tiam-1expression as a tumor marker was analyzed together.
Methods
1. Clinical-pathologic parameters:Study information of Age, gender, tumor size, Lauren classification, TNM stage, vascular invasion, serosal invasion, and lymphatic invasion is collected.
2. Measurement of serum VEGF-B、VEGF-C level in patients with gastric carcinoma: Serum VEGF-B、VEGF-C level of normal people, preoperative and postoperative patients and followed up patients was examined by using ELISA.
3. The expressions of VEGF-B、VEGF-C and Tiam-1gene/protein in tissue:Real time PCR, immunochemistry, Western blot were used to examine the expressions of VEGF-B、VEGF-C and Tiam-1in human gastric cancer tissue, surrounding lymph nodes and normal gastric tissue.
4. Statistical analysis:SPSS13.0software for statistical applications the relationship of VEGF-C and Tiam-1protein expressions with clinical-pathologic parameters was analyzed with x2test. Kaplan-Meier method was used to retrospectively analyze the relationship of VEGF-C and Tiam-1protein expressions with life span of gastric carcinoma patients. The relationship of single factor of each index or multi-variable combination with life span was analyzed by COX regression
Results
1. The serum VEGF-C level in patients with gastric cancer before surgery is higher than that in healthy controls. The concentrations were statistically significantly different in preoperation and postoperation, P<0.01; preoperation and health controls, P<0.01, respectively. But there is no statistically different in serum level between post-operation and healthy controls, P>0.05.There is no obvious difference of VEGF-B level in preoperation and postoperation
2.The level of serum VEGF-C in group with metastatic cancer is different from that in group without it, There is a significantly statistically different, P<0.01. There is a significantly statistically different in serum VEGF-C levels between group with metastatic cancer and control healthes, P<0.01.The level is no different between group without metastatic cancer and control healthes, P>0.05. Serum VEGF-B is not significantly different among control healthes, metastatic group and nonmetastatic cancer group.
3. The VEGF-C level of gastric cancer patients is related with the lymph node metastasis, serosal invasion and clinical stage, the VEGF-C level in patients without lymph node metastasis, no serosal invasion, TNM Ⅰ and well and moderate differentiated group phase was significantly lower than that suffering the lymph node metastasis, serosal invasion, TNM Ⅱ Ⅲ Ⅳ stage and poorly differentiated and undifferentiated group. The relationship between VEGF-B and them is not obvious
4. The expression of VEGF-C and Tiam-1mainly focus on primary foci in stomach tissue and lymph node metastases. The positive rate of the VEGF-C and Tiam-1expression with lymph node metastasis was significantly higher than those without lymph node metastasis of gastric cancer, the difference was significant. The VEGF-C and Tiam-1expression of gastric cancer tissue is related with the lymph node metastasis, serosal invasion and clinical stage, the VEGF-C level in patients without lymph node metastasis, no serosal invasion and TNM Ⅰ Ⅱ was significantly lower than that suffering the lymph node metastasis, serosal invasion, TNMⅢ Ⅳ stage and poorly differentiated and undifferentiated group.
5. Real-time quantitative PCR showed that the VEGF-C and Tiam-1gene level between normal gastric tissue and gastric cancer or lymph node tissue was significantly different (P<0.05); There was a close relationship between quantitaty of VEGF-C and Tiam-1mRNA and lymph node metastasis, quantitaty of VEGF-C and Tiam-1mRNA was higher in cases with lymph node metastasis than those without lymph node metastasis (P<0.01). With the degree of lymph node metastasis, the VEGF-C、TIAM-1mRNA got more obviously (p<0.01)
6. Spearman correlation analysis showed that there was a line relationship between VEGF-C and Tiam-1expression in gastric cancer; the expression level of VEGF-C and TIAM-1increased With the lower degree of differentiation, increased depth of invasion, worse TNM stage and Wider lymph node metastasis
7. Kaplan-Meier survival curves show the survival time had significant difference between patients with positive VEGF-C and Tiam-1expression and those without expression (P<0.05)..Multi-factor analysis showed that the high expression of VEGF-C in tumor tissue and Tiam-1in lymph node, TNM were independent factors affecting the survival prognosis of gastric carcinoma (P value was0.003、0.007、0.028respectively)
Conclusion
1. The serum VEGF-C rather than VEGF-B level is a reliable biologic marker in diagnosing gastric carcinoma, the VEGF-C levels up-regulation appears to be useful biologic marker for gastric cancer metastasis or recurrence.
2. VEGF-B were expressed in some cases but it had no relation with clinic parameter
2. VEGF-C and Tiam-1protein was up-regulated in gastric carcinoma tissues and metastatic lymph nodes, and had no expression in normal gastric tissue, indicating that VEGF-B、VEGF-C and Tiam-1may be regarded as the important marker for clinical detection of gastric carcinoma;
3. As tight correlations existed between the expressions of VEGF-C, Tiam-1and the pathobiological behavior of gastric cancer, both of them maybe acted as reference indexes for judging the invasive and metastatic state of gastric cancer.
4. Combined evaluation of VEGF-C and Tiam-1high expression and clinical follow-up data analysis showed that VEGF-C and Tiam-1had close relationship with prognosis of gastric carcinoma patients. VEGF-C and Tiam-1may act as the important marker for judging prognosis of gastric carcinoma;
5. The VEGF-C and Tiam-1play an important role in the course of development, progression and lymphatic metastasis of gastric cancer. The agreed expression of VEGF-C and Tiam-1was related to lymph node metastasis, it indicated the cooperation effection of VEGF-C and Tiam-1may promote lymphatic metastasis in gastric cancer, which provides a new target for the Treatment of lymph node metastasis of gastric cancer
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