外源性TNF-α基因克服多药耐药性的初步研究

Preliminary Study on Conquering Multidrug Resistance with Exogenous TNF-α Gene Therapy

目的：探索外源性细胞因子 TNF-α基因克服多药耐药（ multidrug resistance,MDR）的新途径。方法：以重组逆转录病毒为载体，将 TNF-α基因导入具 MDR表型的人乳腺癌细胞系 MCF- 7/Adr，经 G418抗性筛选获阳性克隆 MCF- 7/Adr- TNF1与 MCF- 7/Adr- TNF2。以 PCR和 ELISA法检测目的基因的整合与表达。细胞计数法观察细胞生长速度的改变 , MTT法检测外源性 TNF-α基因的逆转 MDR作用，流式细胞仪分析细胞内 ADR积累的变化。结果： MCF- 7/Adr- TNF1与 MCF- 7/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 1 737 pg/ml（ 106 cells/48 h）、 2 875 pg/ml。与阴性对照细胞相比， MCF- 7/Adr- TNF1与 MCF- 7/Adr- TNF2细胞的生长速度明显减慢，生长抑制率分别为 32.4％、 54.8％，对 ADR的耐药性明显降低，耐药逆转倍数分别为 5.2倍、 19.3倍，细胞内 ADR的积累明显增加。结论：外源性 TNF-α基因的导入能有效克服耐药性，增加细胞内药物的积累为其逆转耐药性的主要机制。

Objective： This study was designed to evaluate TNF-α gene therapy for conquering multidrug resistance(MDR). Methods： By using recombinant retrovirus vector, TNF-α gene was transfected into multidrug- resistant human breast cancer cell line MCF- 7/Adr. The TNF-α secreting cell clone MCF- 7/Adr- TNF1 and MCF- 7/Adr- TNF2 were obtained by G418 selection. The integrating and secreting of TNF-α were analyzed by PCR and ELISA method. Cell growth inhibiting and reversal effect of MDR on MCF- 7/Adr cells by TNF-α gene were examined by cell account and MTT assay. The changing of intracellular ADR accumulation was analyzed by flow cytometric assay. Results： The level of TNF-α secreted by MCF- 7/Adr- TNF1 and MCF- 7/Adr- TNF2 were 1737 pg/ml (106 cells/48 h) and 2875 pg/ml respectively. Cancer cells showed lower growth rate after transfection, and the inhibition of growth rate were 32.4％ and 54.8％ in MCF- 7/Adr- TNF1 and MCF- 7/Adr- TNF2 in comparison with the control, respectively. At the same time, the resistance to ADR were reversed by 5.2 times and 19.3 times, and the intracellular ADR accumulation increased significantly. Conclusion： Drug resistance could be conquered by TNF-α gene therapy. Increasing intracellular drug accumulation may be the mechanism of reversing drug resistance.