双链断裂修复蛋白hKu70缺陷细胞株的建立及其生物学特性  

Construction of double-strand break repair protein hKu70 deficient cell strain and its biologic characters

作　　者：刘起展[1] 江高峰[1] 何云[1] 杜柳涛[1] 庄志雄[2] 

机构地区：[1]中山大学公共卫生学院卫生毒理学教研室,广州510089 [2]深圳市疾病预防控制中心

出　　处：《中华劳动卫生职业病杂志》2003年第2期105-107,共3页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家"973"计划资助项目(2002CB512 90 4 );国家自然科学基金资助项目(39970 6 36 )

摘　　要：目的　建立并鉴定DNA双链断裂 (DSB)修复蛋白hKu70缺陷细胞株 ,并观察该缺陷细胞的某些生物学效应 ,用于hKu70基因功能及职业有害因素对DNA双链断裂修复影响的研究。方法　用构建的hKu70基因反义RNA绿色荧光蛋白真核表达载体 (pEGFP C1 K)转染人胚肺成纤维细胞 (HLF) ,用蛋白免疫印迹法鉴定转染细胞中hKu70基因的表达水平。同时观察转染细胞生长形态 ,绘制生长曲线 ,软琼脂培养法鉴定恶性程度。结果　pEGFP C1 K载体在转染细胞内可较稳定表达 ,hKu70蛋白缺陷细胞株hKu70基因的蛋白表达水平下降了 42 % ,转染后hKu70蛋白缺陷细胞生长形态、生长速度无明显变化 ,软琼脂培养未见细胞集落。结论　成功建立和鉴定了hKu70蛋白缺陷细胞株 。Objective To construct DNA double strand break(DSB) repair protein hKu70 deficient cell strain and to observe its biological characters for studying the functions of hKu 70 gene and the effects of occupational harmfulness factors on DSB repair. Methods Human lung fibroblasts(HLF) were transfected with the eukaryotic expression plasmids of hKu 70 gene antisense RNA(pEGFP C1 K) to construct hKu70 protein deficient cells(named as “HLFK”).The protein expression levels of hKu 70 gene in HLFC and HLFK were detected by the Western blotting to estimate the effects of antisense inhibition.Morphology,growth character and growth status in soft agar of transfected HLFK were observed. Results pEGFP C1 K vector was successfully expressed in HLF.The protein expression level of hKu 70 gene in HLFK was decreased by 42% as compared with that in HLFC.No obvious changes of the biologic characters were observed in HLFK. Conclusion The hKu70 protein deficient cell strain was successfully constructed.The hKu70 protein deficiency alone didn't induce obvious changes of the biological characters in HLFK.

关 键 词：DNA双链断裂 hKu70基因 蛋白兔疫印迹法 基因表达 

分 类 号：R346[医药卫生—基础医学]