摘要
肿瘤是危害人类健康的重大疾病之一,特别是恶性肿瘤,最易发生转移,远处器官转移是肿瘤病人死亡的首要原因。鼻咽癌(nasopharyngeal carcinoma,NPC)是我国发病率最高的十大恶性肿瘤之一,严重危害着我国人民的健康。NPC发病隐匿,发现之前常常已有颈部淋巴结转移和远处转移,临床确诊的患者据报道约64.1%发生颈部淋巴结转移。在某些癌症如头颈部肿瘤发生在口腔和口咽时的治疗过程中,早期探测原发瘤的区域淋巴结转移是选择合适治疗方案的核心问题。因此,探索癌症淋巴结转移候选基因,对深入了解肿瘤淋巴结转移和远处转移机制有着重要的意义。
肿瘤并不是均一的整体,是由各种不同转移潜能的细胞亚群构成的。肿瘤的转移是一个复杂的过程,转移的发生发展是宿主因子与恶性肿瘤细胞内在的特性相互作用的结果,是一个高选择性的过程,只有具有转移潜能的细胞亚群才能发生转移。肿瘤的定向转移是指特定的肿瘤能够向特定的器官转移,是转移的肿瘤细胞与特定的器官微环境相互作用的结果。根据肿瘤转移的这一特性,有不少研究人员实验性的施加选择压力,分离出适宜特定器官微环境的肿瘤转移亚群,为研究肿瘤的定向转移提供更为纯净的研究对象。
肿瘤细胞原位移植技术的出现,极大的推动了肿瘤动物模型的发展。将人类肿瘤组织或肿瘤细胞原位种植到免疫缺陷的动物体内,能够很好的模拟肿瘤在体内的生长、侵袭、转移过程,复制出与患者十分接近的临床特征,为研究肿瘤的发生发展、转移以及开展实验性的治疗干预,提供了良好的研究对象。近年来新兴的肿瘤分子成像技术,使人们能够在活体动物体内观察肿瘤的侵袭转移过程,提高人们对微小肿瘤和肿瘤微转移的认识。绿色荧光蛋白(greenfluorescent protein,GFP)广泛应用于可视化动物模型的研究,使得人们可以在活体状态下观察肿瘤的演进规律,极大地推动了肿瘤的早期生长、发展规律的研究,有效的弥补了人体肿瘤仅能通过活检、手术标本和尸检等进行分析的不足。大多数其它的恶性肿瘤可以从手术切除标本中获得较大量组织以供研究,但NPC研究仅能依赖微小的活检组织。本实验通过对已标记GFP的人鼻咽癌高转移细胞株5-8F-EGFP进行裸鼠体内连续传代,建立鼻咽癌淋巴结转移动物模型并且筛选获得高淋巴结转移潜能的鼻咽癌5-8F-LN(EGFP)NPC细胞,对进一步探索淋巴结转移有重要的作用。人为地施加选择压力,成功的建立了人鼻咽癌细胞淋巴结转移动物模型,并筛选到高淋巴结转移潜能的鼻咽癌5-8F-LN细胞。
基因芯片(Gene Chip)技术是基于核酸探针互补杂交的原理而开发的一种检测基因表达水平的新技术。它具有灵敏度高、检测量大等优点,可同时检测成千上万个基因的表达情况或平行对比这些基因在不同样本中的表达,被认为是一种高通量检测基因表达水平的方法。因此,基因芯片技术可以成为探讨肿瘤转移的分子机制的主要手段。
鼻咽癌5-8F细胞株成瘤且具有高转移潜能,6-10B细胞株成瘤不发生转移,将两株细胞作芯片分析获得307个差异性表达基因,它们的表达差异均在两倍以上;我们通过文献挖掘近5年的资料获得大量癌症如:乳腺癌,头颈部鳞癌等肿瘤的基因芯片分析得到的转移标签基因1638个和42个其它文献公布淋巴结转移相关基因共计1680个。将这些文献筛选得到的肿瘤淋巴结转移签名基因和我们5-8F/6-10B差异性表达基因做比较得到21个重叠的基因(来源于10篇文献)。接着利用MILANO软件进行文献调研发现TPM2、ADM、IRF1、KRT19、EGR1、CAV1和IGFBP6在肿瘤的研究方面的出现的文献较多(表1)。当我们结合GenCLIP3.3软件分析这21个基因后,得到IGFBP6,ELF3,DUSP1,CXCL2,CAV1,EGR1,IRF1,GAS1,ADM,KRT19和KRT13文献联系比较密切(图1.)。最后,我们结合基因功能分析筛选获得与淋巴结转移有关的3个高表达的签名基因ADM,IRF1和CAV1可能和鼻咽癌5-8F细胞株高转移特性有关。
采用SYBR Green I qRT-PCR(quantitatie realtime PCR)技术对靶基因进行实时荧光定量分析是近年来发展起来的一项新技术。SYBR Green I是一种能与双链DNA结合发光的荧光染料。因此,它的荧光信号强度与双链DNA的数量相关,可以根据荧光信号检测出PCR体系中存在的双链DNA数量,并且通过熔解曲线的分析,排除非特异性扩增的干扰,对靶基因进行定量分析,准确性和灵敏性都比普通RT-PCR高。通过△△Ct方法利用SYBR Green I qRT-PCR技术能够很好的分析基因表达相对差异。用△△CT统计分析基因表达相对定量的方法在很多文献中有报道,由于这种方法无需做标准曲线,因此比绝对定量方法更加简便可行。
使用qRT-PCR的方法验证ADM,IRF1和CAV1在5-8F-EGFP,6-10B-EGFP,5-8F-LN和NP69四种细胞中的表达情况,我们发现CAV1在5-8F细胞株的表达比其在6-10B细胞株中的表达高2.153±0.120倍,且两者之间表达有统计学差别(p=0.014);而IRF1在3株细胞(5.8F-EGFP,6-10B-EGFP和5-8F-LN)中表达均明显高出永生化细胞株NP69的4.823±1.131、3.390±1.004和4.263±0.965倍;ADM在四种细胞中的表达情况无明显差别。
结合qRT-PCR对鼻咽癌细胞株的分析结果,在5-8F和6-10B鼻咽癌组织中用免疫组织化学的方法检测IRF1和CAV1的表达情况,发现IRF1在5-8F肿瘤组织块中的表达强于在6-10B中的表达;而CAV1在5-8F肿瘤组织的边缘表达明显强于6-10B,CAV1在5-8F肿瘤组织边缘相对高表达可能与其高转移和侵袭能力有关。
Neoplasm is one of the most hazard diseases which harm human's health, especially for the malignant tumor.It is the principal cause of death in individuals suffering from cancer.The incidence of nasopharyngeal carcinoma(NPC) is one of the ten highest in our country;and it seriously jeopardizes people's health.NPC is a mysterious tumor and often has lymph node or distant metastasis before diagnosis. It is reported that there are 64.1%outpatients who had lymph node metastasis. Detection of local lymph node metastasis is pivotal for choosing appropriate treatment,especially for individuals diagnosed with head and neck squamous cell carcinomas(HNSCC) in the oral cavity or oropharyngeal carcinoma.So it is meaningful to explore the metastasis-related signature genes for understanding the metastatic mechanism.
Tumor is composed of many subpopulation cells with different metastatic potential that is well known as tumor heterogeneity.The development of tumor metastasis is a complicated process.Metastasis is a highly selective process resulted from tumor cells interacting with a specific microenvironment.According to the characteristic of tumor cells,many researchers have isolated some organ tropic subpopulations.
Orthotopic implantation of tumor cells technique has greatly facilitated the development of animal model of tumors.When human tumor cells or tissues were inoculated orthotopically into immunodeficiency animals,they would simulate the serial progression process of tumors in vivo.These animals behaved just like small patients,and we could use these animal models for clinical research.In recently years,the molecular imagining technique has been developed and applied to study the process of tumor invasion and micro metastasis.Green fluorescent protein (GFP) has been widely used in following tumor growth and metastasis,it made us can visualize the tumor progression in live animal.It obviously affords us a method to continuously monitor the early growth and spread of tumors without taking biopsies or killing some animals at fixed intervals.Moreover,it is not allowed to take biopsy specimens from NPC patient's metastatic lymph nodes,so it is important to establish a stable NPC animal model for nodal metastasis and selected a NPC cell line with preferential potentiality of lymph node metastasis for research.
Gene chip is a new technique to detect gene expression level based on hybridization of nucleic acid.It has high sensitivity to test the gene expression level in both qualitatively and quantitatively,especially in simultaneously monitoring thousands of gene expression in the same tissue or parallelly analyzing the same gene among different tissues.It is one of the most important techniques used in studying the mechanism of tumor metastasis.
5-8F-EGFP cell line with high metastatic potentiality of human NPC was inoculated into nude mice and selected in vivo.We selected successfully the subpopulation of NPC cell line,which preferentially metastasized to the lymph node, and was named 5-8F-LN-EGFP.
NPC 5-8F-EGFP cell line has high tumorigenic and strong metastatic potentiality.Oppositely,NPC 6-10B cell line has lower tumorigenic and almost has no metastatic potentiality.There are 307 differential expression genes between 5-8F and 6-10B by microarray analysis,and all of them changed twofold or higher.And we also have got lots of lymphatic metastasis signature genes of breast cancer as well as HNSCCs using data mining method.Based on the above-mentioned results we have got 21 genes overlapped.Then we found out there were a lot of literatures about genes of TPM2,ADM,IRF1,KRT19,EGR1,CAV1 and IGFBP6(table.1).3 genes with high expression level related to metastasis probably.They are ADM,IRF1,and CAV1 genes.These genes might be concerned with the high metastatic potentiality of 5-8F NPC cell line.
Then we used qRT-PCR to validate the data-mining results in the 5-8F-EGFP, 6-10B-EGFP and NP69 as well as 5-8F-LN NPC cell lines.We have found that the expression level of CAV1 in 5-8F cell lines was 2.153±0.120 times higher than that in 6-10B(p=0.014).The expression level of IRF1 in the NPC cell lines of 5-8F-EGFP, 6-10B-EGFP and 5-8F-LN are 4.823±1.131、3.390±1.004 and 4.263±0.965 times respectively higher than that in NP69 cell line(p=0.008,0.022,0.006).No significant differences can be demonstrated in the expressions of ADM gene among these cell lines.
Based on the analytical results using qRT-PCR method,we validated these differential expression genes in 5-8F and 6-10B NPC cell line xenografs by the immunohistochemistry methods.The expressions of IRF1 in 5-8F NPC tissue are stronger than that in 6-10B tissue.The expression of CAV1 at borders of 5-8F tumor is significantly stronger than that of 6-10B.The relatively higher expression of CAV1 in 5-8 F tumor's edge may be correlated with the invasion and metastasis potentiality.
引文
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