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目的:研究人类17号染色体D17S396位点微卫星不稳定性和杂合性缺失,对nm23-H1蛋白表达的影响,同时检测错配修复基因hMLH1和hMSH2蛋白的表达,为揭示nm23-H1基因、hMLH1和hMSH2基因与肿瘤发生和转移机制提供实验依据。方法:采用石蜡包埋组织抽提DNA、PCR-SSCP、常规银染、Envision免疫组织化学等方法,对50例胆囊癌及其相应的正常组织,进行D17S396位点MSI、LOH的检测和nm23-H1、hMLH1和hMSH2蛋白表达研究。结果:①原发性胆囊癌D17S396位点遗传不稳定发生率为42.55%,LOH的发生率与肿瘤组织分化程度差异显著(P<0.05);在肝脏侵润和淋巴转移组高于无肝脏侵润和无淋巴转移组(P<0.01),在Ne-vinⅣ+Ⅴ期高于Ⅰ+Ⅱ+Ⅲ期(P<0.01);而MSI发生率则相反;②nm23-H1蛋白阳性率为46.81%,在淋巴转移组低于无淋巴转移组(P<0.01);NevinⅣ+Ⅴ期低于Ⅰ+Ⅱ+Ⅲ期(P<0.05);③hMLH1和hMSH2蛋白阳性率分别为51.06%和42.55%,hMLH1蛋白表达在有无淋巴转移组和Nevin分期有显著差异(P<0.01),肝脏侵润组低于无肝脏侵润组(P<0.05);④MSI阳性组中hMLH1蛋白阳性率显著高于MSI阴性组(P<0.05)。LOH阳性组中nm23-H1和hMSH2蛋白阳性率显著低于LOH阴性组(P<0.05);⑤hMSH2蛋白阳性组中nm23-H1蛋白表达明显高于hMSH2蛋白阴性组(P<0.05)。结论:nm23-H1基因的遗传不稳定性可能是胆囊癌发生、发展的一个重要分子机制。nm23-H1基因的MSI和LOH,通过相互独立的途径调控胆囊癌的发生和转移。hMLH1/hMSH2表达异常可能是胆囊癌的早期分子事件。提高胆囊癌局部nm23-H1、hMLH1和hMSH2蛋白的表达,可减缓肿瘤的侵润转移并提高预后率。
Objective: To investigate the effect of microsatellite instability and heterozygosity deletion on chromosome 17173939 on the expression of nm23-H1 protein, and to detect the expression of mismatch repair genes hMLH1 and hMSH2. To reveal the relationship between nm23-H1 gene, hMLH1 and hMSH2 genes and tumorigenesis and metastasis mechanisms provide experimental evidence. Methods: Fifty cases of gallbladder carcinoma and its corresponding normal tissues were stained with paraffin-embedded tissue DNA, PCR-SSCP, conventional silver staining and Envision immunohistochemistry. MSI and LOH at D17S396 and nm23- H1, hMLH1 and hMSH2 Protein Expression Studies. Results: ① The incidence of genetic instability in D17S396 locus was 42.55% in primary gallbladder cancer, the incidence of LOH was significantly different from that of tumor tissue (P <0.05), but higher in liver invasion and lymph node metastasis than in non-hepatic invasion The positive rate of nm23-H1 protein was 46.81% in the group of Ne-vin Ⅳ + Ⅴ (P <0.01) (P <0.01); NevinⅣ + Ⅴ phase was lower than that of Ⅰ + Ⅱ + Ⅲ phase (P <0.05); ③ The positive rates of hMLH1 and hMSH2 protein were 51.06% and 42.55%, respectively, hMLH1 protein The positive rate of hMLH1 protein in MSI positive group was significantly higher than that in MSI negative group (P <0.01), and the positive rate of hMLH1 protein in MSI positive group was significantly lower than that in non-liver infiltration group (P <0.05). The positive rate of nm23-H1 and hMSH2 protein in LOH-positive group was significantly lower than that in LOH-negative group (P <0.05). ⑤The expression of nm23-H1 protein in hMSH2-positive group was significantly higher than that in hMSH2-negative group (P <0.05). Conclusion: The genetic instability of nm23-H1 gene may be an important molecular mechanism for the occurrence and development of gallbladder carcinomas. The MSI and LOH of nm23-H1 gene regulate the occurrence and metastasis of gallbladder carcinoma through independent pathways. Abnormal expression of hMLH1 / hMSH2 may be an early molecular event in gallbladder cancer. To improve the expression of nm23-H1, hMLH1 and hMSH2 protein in gallbladder carcinoma can reduce the invasion and metastasis of tumor and improve the prognosis.