CAJ | 학술논문

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1～6 d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Western bloting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48 h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Western bloting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.
목적 통과구건만병독개도적파향수소기인적소간우RNA(siRNA),탐토수소대A549세포증식화조망적영향.방법 건립siRNA-a조、siRNA-b조급기각자음성대조조화공백대조조(A549)5조세포,분별우형광현미경하관찰세포5d화11d적생장정황.새서람(MTT)비색법측정공백대조조、siRNA-a조급기음성대조조세포1～6 d적생장정황,회제생장곡선,비교각조세포증식차이.류식세포의분석각조세포조망솔,관찰수소적siRNA대A549세포조망적영향.RT-PCR화Western bloting방법검측각조세포수소mRNA화단백표체수평.결과 siRNA-a세포경하형태기본정상,5d후형광효솔잉교고,감염은정,단생장교음성대조조급공백대조조세포완만.siRNA-b세포감염48 h후,경하견세포종창、변형,공포출현,5d후견다량세포쇄편,조망소체형성,정현조망상태,11d후가견세포쇄편,이근완전조망.MTT실험siRNA-a조세포종제3천기생장비기타각조균완만(P＜0.01).류식세포의검측세포조망솔결과:siRNA-a조、siRNA-b조세포조망솔명현고우음성대조조화공백대조조.RT-PCR화Western bloting결과 현시간우조세포수소mRNA화단백표체수평교음성대조조화공백대조조명현하조.결론 만병독개도적siRNA능하조폐선암A549세포수소기인적표체,종이억제A549세포증식,촉진기조망.유차가견,수소적siRNA유망성위폐선암기인치료적신수단.
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 three groups 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 骟efficiency was still high,which showed it was a stable infection.But it grew slower than the blank and negative control groups.After 48 hours of siRNA-b cell's constructing,the cell swelling,deformation and cavitation could be seen under microscope and after five days,the cell debris,apoptotic bodies could be seen,then eleven days later,siRNA-b cells were almost dead completely.In MTT test,the siRNA-a group cells 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.Conclusions Lentiviral leptin siRNA can decrease leptin expression in human lung adenocarcinoma 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.