靶向瘦素基因的siRNA对人肺腺癌A549细胞增殖和凋亡的影响

Effects of siRNA targeting ieptin gene on proliferation and apoptosis in human lung adenocarcinoma A549 cell line

目的通过构建慢病毒介导的靶向瘦素基因的小干扰RNA（siRNA），探讨瘦素对A549细胞增殖和凋亡的影响。方法建立siRNA-a组、siRNA-b组及其各自阴性对照组和空白对照组（A549）5组细胞，分别于荧光显微镜下观察细胞5d和11d的生长情况。噻唑蓝（MTT）比色法测定空白对照组、siRNA-a组及其阴性对照组细胞1-6d的生长情况，绘制生长曲线，比较各组细胞增殖差异。流式细胞仪分析各组细胞凋亡率，观察瘦素的siRNA对A549细胞凋亡的影响。RT-PCR和Westernbloting方法检测各组细胞瘦素mRNA和蛋白表达水平。结果siRNA-a细胞镜下形态基本正常，5d后荧光效率仍较高，感染稳定，但生长较阴性对照组及空白对照组细胞缓慢。siRNA-b细胞感染48h后，镜下见细胞肿胀、变形，空泡出现，5d后见多量细胞碎片，凋亡小体形成，呈现凋亡状态，11d后可见细胞碎片，已近完全凋亡。MTT实验siRNA-a组细胞从第3天起生长比其他各组均缓慢（P〈0．01）。流式细胞仪检测细胞凋亡率结果：siRNA-a组、siRNA-b组细胞凋亡率明显高于阴性对照组和空白对照组。RT-PCR和Westernbloting结果显示干扰组细胞瘦素mRNA和蛋白表达水平较阴性对照组和空白对照组明显下调。结论慢病毒介导的siRNA能下调肺腺癌A549细胞瘦素基因的表达，从而抑制A549细胞增殖，促进其凋亡。由此可见，瘦素的siRNA有望成为肺腺癌基因治疗的新手段。

Objective To construct a lentiviral leptin small interfering RNA （siRNA） vector to silence leptin gene expression, and to study its effect on proliferation and apoptosis in lung carcinoma cells. Methods Five groups of cells were established,including siRNA-a group,siRNA-b group,respective negative control groups and blank control group （A549）. Cell growth was observed under fluorescence microscope after five days and eleven days. Through MTT colorimetric, we assayed the growth of threegroups of cells,including blank control group, siRNA-a group and negative control group from the first day to the sixth day, and drew a growth curve to compare the proliferations of cells. The rates of apoptosis of the cells were assayed by flow cytometry to observe the impact of siRNA on apoptosis in A549 cells. Expression of leptin mRNA and protein in three groups of cells was examined by RT-PCR and Western bloting analysis. Results siRNA-a cells＇ appearance was almost normal. After five days,the fluorescence efficiency was still high, which showed it was a stable infection. But it grew slower than the blank andnegative control groups. After 48 hours of siRNA-b cellrs constructing,the cell swelling, deformation and cavitation could be seen under microscope and after five days, the cell debris, apoptotie bodies could be seen,then eleven days later,siRNA-b cells were almost dead completely. In MTT test,the siRNA-a groupcells grew slower than the other groups from the third day （ P〈 0.01 ）, but there were no significant differences among other groups. With flow cytometry, the apoptosis rates of siRNA-a （38.7%） and siRNA-b （48.9%） groups was higher than blank （21.1%） and negative control groups （22.1%）. Expression of leptin mRNA and protein in interfence group was signigicantly lower than other groups as showed by RT-PCR and Western bloting analysis. Cenelusiens Lentiviral leptin siRNA can decrease leptin expression in human lung adenoearcinoma A549 cells, and inhibit proliferation and promote apoptosis in A549 cells. It is expected that siRNA targeting leptin will be a new gene therapy for lung adenocarcinoma.