靶向bcl-2基因Stealth^TM RNA干扰诱导肝癌细胞凋亡的作用

Targeted bcl-2 induces the apoptosis of human hepatoma carcinoma cells by Stealth^TM RNA interference

目的应用RNA干扰技术，构建靶向bcl-2基因Stealth^TMRNA，转染人人肝癌细胞株，探讨RNA干扰诱导人肝癌细胞凋亡的机制。方法确定并合成3对人肝癌bcl-2基因干扰序列；脂质体法转染入肝癌细胞株内；荧光显微镜下观察转染细胞并计算转染率；倒置显微镜下观察转染前后细胞形态学的改变；SP免疫细胞化学技术检测Stealth^TM RNA处理前后细胞bcl-2蛋白表达变化；流式细胞术检测Stealth^TM RNA干扰对癌细胞凋亡和周期的影响。结果各组转染成功，荧光镜下显清晰的绿色荧光；各转染组转染率与时间有关，各组48h的转染率最高（P〈0．01）；转染组间Stealth^TM RNAⅠ组的转染效率最高（P〈0．01）；倒置显微镜下观察见转染后见部分细胞边缘圆，折光度降低，细胞增殖减慢且易脱落；未转染组细胞呈上皮性贴壁生长，生长旺盛。免疫细胞化学显示，Stealth^TM RNA干扰后各转染组发现细胞的bcl-2表达减弱，与未转染组比较，差异有统计学意义（P〈0．01）。流式细胞术结果示，各转染组细胞凋亡率明显升高，并出现凋亡峰，Stealth^TM RNAⅠ、Ⅱ、Ⅲ组的凋亡率分别为（27．87±4．51）％、（24．50±0．89）％和（26．03±0．57）％，与未转染组细胞凋亡率（3．20±1．71）％，差异有统计学意义（P〈0．01）。各转染组细胞发生显著的细胞周期阻滞，表现为S期细胞明显减少，G0／G1细胞明显增多。结论靶向bcl-2基因Stealth^TM RNA成功的转染入肝癌细胞中，bcl-2蛋白表达受抑制，并明显的诱导癌细胞发生凋亡。

Objective To explore the influence on the apoptosis of human hepatoma carcinoma BEL-7402 cells by Stealth^TM RNA interference, after three pairs Stealth^TM RNA target to bcl-2 gene were designed and constructed, then transfected. Methods Interfering target of human hepatoma carcinoma bcl-2 gene was established and synthesized. Stealth^TM RNAs were transfected into ceils by using lipofectamine 2000. Cells were observed and transfection rate was evaluated under fluorescent microscopy. Cell morphological changes were investigated by reverse microscopy before and after transfection. Changes in bcl-2 protein were detected after Stealth^TM RNA interference by SP-immunocytochemical technique. Ap- optosis of cells was analyzed by flow cytometry. Results In experimental groups, transfection was carried out succeedfully and green fluorescence in the cells was seen clearly under the fluorescent microscope. Transfection rate of Stealth^TM RNA Ⅰ transfected group was the highest among the transfected groups （P 〈 0.01 ）. Transfection rate varied with time,and transfection rate reached the peak at 48 h in every transfected group （P 〈 0.01 ）. Under reverse microscopy, cells after transfection became round, diopter degraded, sheded easily, at the same time, cell proliferation was down-regnlated. In untransfected group, cells showed epithelial growth and proliferated productively. SP-immunocytochemical technique revealed that the expression of bcl-2 gene was decreased in the transfected groups as compared with untransfected group （P 〈 0.01 ）. The flow cytometry showed that in transfected groups apoptosis peak appeared and apoptosis rate was （27.87 ± 4.51 ） %, （24.50 ± 0.89） % and （26.03 ± 0.57） % respectively, significantly higher than in untransfected group （3.20 ± 1.71 ,P 〈 0.01 ）. Flow cytometry indicated in transfected groups the cell cyle arrest was found： percent of cells in G0/G1 was significantly increased and that in S decreased markedly. Conclusion Stealth^TM RNA interference could inhibit the expression of bcl-2 and induce apoptosis of cells.