在石蜡切片中进行微切割-PCR-银染的方法  

Methods for microdissection PCR silver stain technique in paraffin embedded tissue sections

作　　者：黄智达[1] 黄琼[1] 来茂德[1] 

机构地区：[1]浙江大学医学院病理教研室,杭州310031

出　　处：《中华医学遗传学杂志》2000年第6期429-431,共3页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金!(39770 2 97)

摘　　要：目的　探索在石蜡切片中应用微切割 - PCR-银染技术 ,检测大肠癌的微卫星不稳定性和杂合性缺失。方法　用微切割技术在 2 8例石蜡切片中提取淋巴细胞和间质细胞、癌旁粘膜腺管、不典型增生腺管、腺瘤腺管和腺癌细胞 ,每例至少包含 3种类型细胞 ,经蛋白酶 K消化后 ,直接用于 PCR扩增 ,产物进行8%变性聚丙烯酰胺凝胶电泳 -银染。共进行了 TGF-βR (A) 1 0 、h MSH2 (A) 2 6 - Bat2 6 ,h MSH3(A) 8,h MSH6(C) 84个微卫星位点的检测。结果　 h MSH3(A) 8和 h MSH6 (C) 8位点在 2 8例标本全部扩增出目的片段 ,而TGF-βR (A) 1 0 和 h MSH2 (A) 2 6 位点则分别有 2 3例、2 2例扩增出目的片段 ,且 h MSH2 (A) 2 6 位点有 5例癌细胞呈阳性 ,其中 1例同时有腺瘤细胞阳性。结论　微切割 - PCR-银染技术应用于石蜡切片 ,可获得较满意的结果 。Objective To use the microdissection PCR silver stain technique in the paraffin embedded tissue sections and analyze the microsatellite instability in colorectal cancer. Methods By microdissection technique, the lymphocytes and mesenchymal cells, normal mucous epithelial cells, displasia, adenoma, and carcinoma cells were recovered from paraffin embedded tissue sections. The cells were digested by protinase K;the cell lysates were used as PCR template and the PCR products were analysed by denatured polyacrylamide gel electrophoresis and silver stain. Four microsatellite loci, TGF βRⅡ(A) 10 , hMSH 2(A) 26 Bat 26, hMSH 3(A) 8, hMSH 6(C) 8,were analyzed. Results In all 28 specimens, hMSH 3(A) 8 and hMSH 6(C) 8 loci were amplified successfully.TGF βRⅡ(A) 10 , Bat 26 had consistent amplification in 23/28, 22/28 specimens, respectively. And Bat 26 microsatellite instability was found in carcinoma cells in 5 specimens, and in adenoma cells in one of the 5 specimens. Conclusion Microdissection PCR silver stain technique can be used in paraffin embedded tissue sections with satisfactory results. This method can be employed in analyzing microsatellite instability in colorectal carcinoma.

关 键 词：微切割 石蜡切片 大肠癌 微卫星不稳定性 PCR 

分 类 号：Q78[生物学—分子生物学] R735.34[医药卫生—肿瘤]