【摘要】 背景与目的:有资料提示K-ras基因突变可能与子宫内膜癌的发生有关。本研究在发现子宫内膜腺癌HEC-1A细胞有K-ras基因第12密码子突变的基础上,探讨犤12Asp犦K-ras4B突变基因的致瘤性及其发生机制。方法:(1)用RT-PCR方法扩增犤12Asp犦K-ras4B基因全长cDNA,将此cDNA插入真核表达载体pcDI,构建pcDI-犤12Asp犦K-ras4B真核表达质粒;(2)观察真核表达质粒pcDI-犤12Asp犦K-ras4B转染前后细胞形态学改变,用MTT方法、3H掺入实验、集落形成实验检测转染前后细胞体外生长的变化,以及用转基因肿瘤细胞裸鼠异种接种成瘤实验,了解犤12Asp犦K-ras4B的致瘤作用。结果:(1)成功构建了真核表达质粒pcDI-犤12Asp犦K-ras4B。(2)通过转染前后细胞形态学变化、体外增殖变化、软琼脂集落形成实验以及裸鼠皮下转染细胞接种实验,证明犤12Asp犦K-ras4B基因具有致瘤作用。(3)将pCMV-RasN17质粒转染到pcDI-犤12Asp犦K-ras4B细胞,与未转染细胞比较,发现pcDI-犤12Asp犦K-ras4B细胞增生受到明显抑制。(4)分别将pCMV-RafS621A和pCMV-RafCAAX质粒转染到pcDI-犤12Asp犦K-ras4BNIH3T3细胞,与未转染的细胞比较,转染pCMV-RafS621A质粒的pcDI-犤12Asp犦K-ras4B细胞增殖受到抑制,与对照组比较差异显著(P<0.05);转染pCMV-RafCAAX质粒的pcDI-犤12Asp?更多还原

【Abstract】 Background & Objectives: Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type K-ras4B gene. Methods: (1) Full-length K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3)To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and thymidine incorporation method. Results: (1)The authors have successfully constructed eukaryotic expression plasmid pcDI-K-ras4B; (2)To confirm that K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3)After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-K-ras4B cell (P< 0.05). However, RafCAAX mutant can enhance pcDI-K-ras4B cell growth (P< 0.05). Conclusions: (1) K-ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.更多还原