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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 ,该缺陷不足以单独引起可观察的某些生物学效应
Objective To establish and identify DNA double-strand break (DSB) repair protein hKu70-deficient cell lines and to observe some biological effects of the defective cells on hKu70 gene function and occupational harmful factors on DNA double-strand break repair. Methods Human embryonic lung fibroblasts (HLFs) were transfected with the constructed hKu70 antisense RNA green fluorescent protein eukaryotic expression vector (pEGFP C1 K). The expression of hKu70 gene in transfected cells was identified by Western blotting. At the same time, the growth morphology of transfected cells was observed and the growth curve was drawn. The soft agar culture method was used to identify the malignant degree. Results The expression of pEGFP C1 K in transfected cells was stable, the protein expression level of hKu70 in hKu70 protein-deficient cell line was reduced by 42%, and the morphology and growth rate of hKu70-deficient cells did not change significantly after transfection. No cell colonies. Conclusion The hKu70 protein deficient cell line was successfully established and identified and was not sufficient to cause some observable biological effects alone