Bcl-2 siRNA增强HepG2细胞对5-氟尿嘧啶敏感性

Bcl-2 siRNA Increased Sensitivity to 5-Fluorouracil in HepG2 Cells

目的观察Bcl-2 siRNA对人类肝癌细胞HepG2 5-氟尿嘧啶(5-FU)敏感性的影响。方法Bcl-2 siRNA表达载体构建并稳定转染至肝癌细胞HepG2中,用G418筛选稳定的阳性细胞克隆。用RT-PCR检测Bcl-2 mRNA水平,免疫荧光检测目的基因Bcl-2蛋白水平的表达。用131、30、1300和13 000 mg/L 5-FU处理稳定转染细胞,用MTT观察细胞对药物敏感性的影响,用流式细胞术观察细胞凋亡情况。Western blot检测Bcl-2和Bax蛋白水平。结果5-FU处理细胞24 h,Bcl-2 siRNA转染组的细胞生长抑制率明显高于阴性载体转染组和正常细胞;Bcl-2 siRNA转染组细胞凋亡率高于正常细胞和阴性载体转染组;在Bcl-2表达下调时,Bax蛋白表达水平不变。结论siRNA下调目的基因Bcl-2能增强人类肝癌细胞HepG2对5-FU的敏感性;HepG2细胞对5-FU的敏感性增强与Bax/Bcl-2的比例增高有关。

Objective To investigate the changes of sensitivity to 5 - fluorouracil （5 - FU）in Bcl- 2 siRNA transfected Hepg2 cells. Method Bcl - 2 siRNA expression vector were constructed and stably transfected into HepG2 cells. RT- PCR and Immunofluorescence were used to detect the target gene expression. Westem Blotting was used to detect Bcl - 2, Bax expressiom. Drug sensitivity of the cells to 5 - fluorouracil were analyzed with MTT and flow cytometry. Results MTT results showed that Bcl - 2 transfectants had a higher cell inhibition rate than negative vector or untreated cells after treated withl3, 130, 1 300, 13 000 mg/L 5 - FU; Moreover, Flow cytometry results demonstrated that Bcl - 2 siRNA cells had increased apoptosis rate compared with negative siRNA or untreated cell; The protein expression of Bax had no change when Bcl - 2 protein was reduced. Conclusion siRNA targeting Bcl - 2 gene can specifically down - regulate Bcl - 2 expression, increased Bax/Bcl - 2 ratio expression which lead to increase cell spontaneous apoptosis and sensitize cells to 5 - FU.