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目的 探索TNF α基因能否成功导入耐药细胞并获得表达 ,发挥生物学效应。方法 以重组逆转录病毒为载体将TNF α基因导入具有多药耐药 (MDR )表型的人乳腺癌细胞系MCF7/ADR ,经G 418抗性筛选获 4株阳性克隆。PCR、Elisa法鉴定并检测TNF α基因的整合和表达。细胞计数法观察细胞生长速度的改变 ,流式细胞仪检测细胞周期变化。结果 4株阳性克隆整合了TNF α基因并获得表达 ,分泌TNF α量分别为 341pg/ml(10 6cell/48h)、5 40 pg/ml、1737pg/ml、2 875 pg/ml。与对照细胞相比 ,TNF α分泌量较低的 2株细胞克隆生长速度无明显减慢 ,而 2株高表达TNF α的细胞生长速度明显减慢 ,生长抑制率分别为 32 .4%、5 4.8% ,且细胞周期受阻于S +G2 /M期。结论 TNF α基因能成功导入耐药细胞并获得表达。高表达TNF α的细胞克隆生长明显受抑。
Objective To explore whether the TNFα gene can be successfully introduced into drug-resistant cells and obtain the expression and exert the biological effect. Methods TNFα gene was introduced into human breast cancer cell line MCF7 / ADR with multidrug resistance (MDR) phenotype by recombinant retrovirus. Four positive clones were screened by G 418 resistance. PCR, Elisa method to identify and detect TNFα gene integration and expression. Cell counting method was used to observe the change of cell growth rate, and cell cycle was detected by flow cytometry. Results The four positive clones integrated the TNFα gene and expressed it. The amounts of TNFα secreted by the four positive clones were 341pg / ml (106cell / 48h), 5 40 pg / ml, 1737pg / ml and 2875 pg / ml, respectively. Compared with the control cells, the growth of 2 cells with lower TNFα production did not slow down significantly, while the growth of 2 cells with high expression of TNFα slowed down significantly with growth inhibition rates of 32.4% and 5% respectively 4.8%, and the cell cycle was blocked in S + G2 / M phase. Conclusion TNF α gene can be successfully introduced into drug-resistant cells and expressed. Cell clones highly expressing TNFα were significantly inhibited.