散发性胆囊癌nm23-H1基因遗传不稳定性与错配修复基因hMLH1和hMSH2蛋白表达的研究

Study on genetic instability of nm23-H1 gene and expression of hMLH1, hMSH2 in sporadic gallbladder carcinoma

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。

AIM： To examine the MSI and LOH of locus D17S396 and their influence on the expression of nra23 -H1 in gallbladder tumors, and to examine the protein expression of hMLH1/hMSH2, which may provide experi- mental evidence for the tumor occurrence and metastasis. METHODS ： Techniques such as DNA extraction, CR - SSCP, ordinary silver stain were used to study MSI and LOH of locus D17S396. Envision IHC was used to assess the expression of nra23- H1 and hMLH1/hMSH2. RESULTS： ①The frequency of heredity instability of gallbladder carcinoma was 42. 55%. The frequency of LOH in liver and lymph node metastasis cases and in stage Nevin IV and V was significantly higher than that without metastasis and stage I, Ⅱ and Ⅲ. However, the frequency of MSI showed contrary correlation with some clinicopathologic characteristics.② The expression of nm23 - H1 was 46. 81%. The case with lymph node metastasis and Nevin stage Ⅳ and V showed significantly lower expression than that without lymph node metastasis and stage I, Ⅱ and Ⅲ. ③The expressions of hMLH1 and hMSH2 were 51.06% and 42. 55% respectively, hMLH1 in lymph node and liver metastasis cases and in stage Nevin IV and V were significantly lower than that without metastasis and in stage I, Ⅱ and Ⅲ④Positive frequency of hMLH1 in MSI positive group was higher than that in MSI negative group. The positive frequency of nm23 - H1 and hMSH2 protein in LOH positive group was lower than that in negative group. CONCLUSION： The heredity instability of nm23 -Hl gene may be implicated pathogenesis and progression of gallbladder carcinoma. Both MSI and LOH of nm23 - H1 control the development of gallbladder carcinoma independently in different paths. Abnormal expression of hMLH1/hMSH2 may be a molecule marker in early stage of gallbladder carcinoma.