用显微切割技术定点检测诱发大鼠肺癌发生发展各阶段p53、K-ras基因突变与表达

Microdissection and PCR-SSCP detected mutation and expression of p53 and K-ras gene in carcinogenesis and development of induced rat lung carcinoma

目的 探讨p5 3、K ras基因突变、蛋白表达在 3 甲基胆蒽 (3 methylcholanthrene ,MCA)和二乙基亚硝胺 (diethylinitrosamine ,DEN)诱发大鼠肺鳞癌发生演进中的作用 ,及其突变与蛋白表达的关系。方法 将大鼠诱发肺癌石蜡标本连续切片 ,切片用于HE染色确定肺癌发生发展的病变阶段 ,及免疫组织化学 (SP法 )检测各阶段p5 3、K ras蛋白表达 ,并用于显微切割 ,定点对位分别切割由正常支气管黏膜上皮细胞演变成癌细胞 ,癌浸润、转移各阶段病灶的主质 ,提取DNA ,用聚合酶链反应 单链构象多态性 (PCR SSCP)检测各阶段p5 3、K ras基因的突变。结果 30例正常支气管黏膜上皮未检测到p5 3、K ras基因突变及其蛋白表达。在 32例支气管黏膜增生和鳞状化生、2 1例不典型增生、12例原位癌、4 3例浸润癌及 17例转移癌组织中 ,p5 3基因突变率分别为 3 1% ,2 8 6 % ,30 0 % ,5 1 2 % ,5 2 9% ;p5 3蛋白阳性表达率分别为 0 ,4 2 9% ,5 0 0 % ,6 0 5 % ,6 4 7% ;不典型增生阶段与增生、鳞状化生阶段相比 ,p5 3基因突变率及蛋白表达率增高 ,差异均有显著性意义 (P <0 0 2 5 ,P <0 0 0 5 ) ,p5 3基因突变及蛋白阳性表达高度相关 (P <0 0 0 5 ,Pearson′sR =0 5 996 )。K ras基因突变率分别为 0 ,4 8% ,8 3% ,9

Objective To investigate the roles of p53 and K ras gene in carcinogenesis and development of the lung carcinoma induced by 3 methylcholanthrene (MCA) and diethylinitrosamine (DEN) in Wistar rats, and to elucidate the relationships between the protein expression and gene mutation of p53 and K ras Methods Microdissection was used to obtain pure cell populations of each phase in the carcinogenesis and development of lung carcinoma induced by MCA and DEN DNA of the microdissected cell populations was extracted and used to analyze the mutations of p53 exons 5～8 and K ras exons 1～2 by PCR SSCP The expressions of p53 and K ras protein in each phase were detected by immunohistochemistry Results No mutation and protein expression of p53 and K ras was found in the 30 cases with normal bronchial epithelium Mutation of p53 was detected in 3 1% of 18 hyperplasia and 14 squamous metaplasia cases, 28 6% of 21 dysplasia, 30 0% of 12 carcinomas in situ, 51 2% of 43 infiltration carcinomas, 52 9% of 17 metastases The positive immunostaining rate of p53 protein was 0, 42 9%, 50 0%, 60 5% and 64 7% respectively K ras mutation rate was 0, 4 8%, 8 3%, 9 3%, 11 8% respectively, while the overexpression rate of K ras protein was 15 6%, 19 0%, 25 0%, 41 9%, 52 9% respectively p53 protein expression was closely related with p53 mutation ( P <0 005, Pearson′s R =0 599 6) There was no relationship between the protein expression and gene mutation of K ras ( P >0 500) Conclusions p53 gene mutation and K ras overexpression were early events in the carcinogenesis anddevelopment of rat lung carcinoma induced by MCA and DEN, while K ras mutation does not play any important role