摘要
目的 观察肿瘤坏死因子 α(TNF α)基因对耐药细胞生长的抑制作用及机制。方法以重组逆转录病毒为载体将TNF α基因导入耐药性人乳腺癌细胞系MCF7/ADR ,获阳性克隆MCF7/ADR TNF1与MCF7/ADR TNF2。体外观察并比较野生型和转基因癌细胞的生长速度 ,流式细胞仪分析细胞周期的变化 ,端粒酶重复扩增实验 (TRAP)综合非变性聚丙烯酰胺凝胶电泳银染法测定端粒酶活性的变化。结果 MCF7/ADR TNF1与MCF7/ADR TNF2细胞中有TNF α基因的整合和表达 ,病毒上清中TNF α含量分别为 173 7μg/L(10 6个细胞 /4 8h)、2 875 μg/L。与阴性对照细胞相比 ,MCF7/ADR TNF1与MCF7/ADR TNF2细胞的生长速度明显减慢 ,生长抑制率分别为 3 2 .4%、5 4.8% ,细胞周期阻滞于S +G2 /M期 ,端粒酶活性降低。结论 外源性TNF α基因的导入能有效抑制耐药细胞 ,阻滞细胞周期、抑制端粒酶活性可能为其主要机制。
Objective To observe the effects and mechanism of tumor necrosis factof α (TNF α) gene therapy for drug resistant cancer cells.Methods Using recombinant retrovirus vector,TNF α gene was transfected into multidrug resistant human breast cancer cell line MCF7/ADR to obtain the TNF α secreting cell clones MCF7/adr TNF1 and MCF7/ADR TNF2.The growth of wild type and gene transfected cancer cells was observed and compared in vitro.The cell cycle distrioution was assayed by FCM.The change of telomerase activity was detected by using telomerase repeat amplification protocol (TRAP) combined with PAGE silver staining.Results The level of TNF α secreted by MCF7/ADR TNF1 and MCF7/ADR TNF2 was 1?737?μg/L (10 6 cells/48?h) and 2?875?μg/L respectively.Cancer cells showed lower growth rate after transfection,and the inhibition of growth rate were 32.4% and 54.8% in MCF7/ADR TNF1 and MCF7/ADR TNF2 in comparison with the control,respectively.At the same time,the cell cycle was arrested at S and G 2/M phase,and the telomerase activity inhibited.Conclusion Exogenous TNF α gene can effectively inhibit the proliferation of durg resistant caneer cells.Arresting cell cycle and inhibiting telomerase activity may be the major mechanism.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2002年第1期31-33,共3页
Chinese Journal of Experimental Surgery
