hPARP-1蛋白缺陷细胞株建立及生长特性

Construction of the hPARP-1 protein deficient cell strain and its growth characters

目的建立hPARP-1蛋白缺陷细胞株,观察该缺陷所引起的生物学效应。方法用构建的hPARP-1基因反义RNA绿色荧光蛋白真核表达载体(pEGFP-C1-P)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hPARP-1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,用流式细胞术观察细胞周期,软琼脂培养法鉴定转染细胞恶性程度。结果pEGFP-C1-P载体在转染细胞内可较稳定表达,hPARP-1蛋白缺陷细胞株hPARP-1基因的蛋白表达水平比HLF下降了47%,比绿色荧光蛋白载体(HLFC)低45%,HLFC比HLF仅少3%。转染后hPARP-1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。hPARP-1缺陷细胞生长形态无明显变化,细胞倍增时间为0.89 d,与HLF细胞0.93 d接近,软琼脂培养未见细胞集落。结论成功建立和鉴定了hPARP-1蛋白缺陷细胞株,在正常生长环境中,该缺陷不足以单独引起可观察的生长特性改变。

Objective To observe biological characters of hPARP-1 protein deficient cell strain. Methods Human lung fibroblasts（HLF） were transfected with the eukaryotic expression plasmids of hPARP-1 gene antisense RNA （pEGFP-C1-P）. The inhibition effect of the antisense RNA on hPARP-1 gene in HLFP and HLFC was verified by the Western blot. Morphology, growth character, cell cycle and growth status in soft agar of transfected HLF were observed. Results pEGFP-C1-P vector was successfully expressed in HLF. The protein expression level of hPARP-1 gene in HLFP and HLFC were decreased by 47 % and 3 % respectively as compared with that in HLF, No obvious changes of the biologic character were observed in HLFP, Conclusion The hPARP-1 protein deficient cell strain is successfully constructed and that the level of hPARP-1 in HLFP cells does not change their growth characters in normal condition,