论文标题:Kai1/CD82在大肠癌细胞系的表达及其表达下调机制的初步研究
Study on Expression of Kai1/CD82 and Possible Mechanisms of Its Down-regulation in Colorectal Carcinoma Cell Lines
论文作者 杨光之
论文导师 丁彦青,论文学位 硕士,论文专业 病理学
论文单位 第一军医大学,点击次数 88,论文页数 59页File Size3950k
2002-05-01论文网 http://www.lw23.com/lunwen_288688157/ 大肠癌;转移;Kail／CD82;点突变;甲基化;p53
Human colorectal carcinoma,metastasis,Kail/CD82,Mutation,p53,methylation
大肠癌转移是一个极其复杂的过程，是肿瘤细胞和宿主相互作用的共同结果。Kai1基因在这一过程中起抑制作用。该基因于1995年发现，其表达产物为CD82，是TM4SF(transmembrane 4 superfamily)或称TST(tetra span transmembrane)家族成员。该家族结构的最大特点是由4个疏水保守区构成跨膜区(transmembrane 1～transmembrane 4，TM1～TM4)。在TM1与TM2之间、TM3与TM4之间分别有一个较小与较大的膜外半环样结构(extracellular loop 1、extracellular loop 2，EC1、EC2)，EC2区还有3个潜在的糖基结合位点，膜内则有较短的氨基和羧基末端。结构分析提示它在细胞与细胞间粘附、细胞与基质的连接中可能起有一定作用，研究也证实它可以增强肿瘤细胞之间的聚集能力，降低其吞噬能力及侵袭能力，而不影响细胞的增殖力。 目前研究证实Kai1／CD82对多种肿瘤转移具有抑制作用，但其与大肠癌转移相关性的研究尚不多，特别是其表达下调的机制未有报道。为探讨Kai1基因与大肠癌转移之间的相关性，本研究利用四种不同转移潜能的大肠癌细胞系HCT116、HT29、LoVo与SW480，从mRNA与蛋白质水平，用原位核酸杂交、免疫组织化学与Western Blot三种方法检测Kai1／CD82的表达，得到了一致的结果：Kai1／CD82在HT29与SW480细胞系的表达较LoVo与HCT116为高。 为进一步探讨Kai1基因表达下调的机制，本研究用PCR-SSCP技术分析了Kai1／CD82表达较低的LoVo与HCT116细胞系的Kai1基因第3～10外显子是否存在点突变，检测Kai1基因启动子区域CpG岛的甲基化状况，并用免疫组化与Western Blot等方法研究Kai1基因表达与p53异常之间的相关性。实验结果表明：基因点突变、启动子CpG岛甲基化、p53蛋白异常与Kai1基因表达下调不存在相关性。 本研究结果表明：Kai1基因与大肠癌细胞转移潜能有关；基因点突变、启动子CpG岛甲基化与p53异常在Kai1基因表达下调中不起重要作用。
The metastasis of human colorectal carcinoma (HCC) is a complicated process involving the interaction between carcinoma cells and the host. Kail gene,cloned in 1995,has been approved to related to the progress of many kinds of neoplasms. Its protein product,CD82,belongs to TM4SF (transmembrane four superfamily) or TST (terra span transmembrane) superfamily. The protein has four highly conserved hydrophobic domains spanning the liquid bilayer. Then two extracellular domains are formed,a smaller one named EC1 between TM1 and TM2,the other called EC2 between TM3 and TM4 with three potential N-glycosylation sites. The short N- and C- terminus lies in cytoplasm. According to the specialty of the structure,it may be predicted that the function of Kail gene come down to cell-cell adherence and cell-matrix connection. Previous studies also showed that the Kail contributed to increasing the carcinoma cells aggregation,decreasing the phagokinetic activity and invasive ability,but no influence of the cell proliferation.Although many efforts have been made to elucidate the role of Kail gene in many kinds of carcinoma,there are little reports concerning the role of Kail in human colorectal carcinoma,especially regarding the mechanism of its down-regulation in metastasis carcinoma. In present study,the expression of Kail was detected by in situ hybridization,immumohistochemistry and Western Blot in four cell lines of HCC with different metastatic potential. The results indicated that Kail/CD82 is higher expressed in HT29 and SW480 than in LoVo and HCT116 whatever in mRNA or protein level.The possible mechanism of down regulation of Kail was also analyzed. By PCR-SSCP,8 exons of Kail,exon 3 to exon 10,were screened in LoVo and HCT116 cell lines. The methylation of CpG islands in promoter region was analyzed,and the expression level of p53 was detected by IH and Western Blot. The results showed that mutation of the Kail gene,methylation of CpG islands and the abnormity of p53 are not related to low expression of Kail.In conclusion,this research has gotten some significant results:firstly Kail gene is highly related with the metastasis potential of colorectal carcinoma cells,secondly mutation of Kail gene,methylation of CpG islands in the promoter and abnormal expression of p53 have no important effects in the down-regulation of Kail gene.
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