siRNA靶向抑制PKR基因及其对宫颈癌Hela细胞凋亡的影响  

siRNA can inhibit PKR gene and facilitate apoptosis of Hela cells of uterine cervix cancer:a randomized and controlled trial

作　　者：徐扬[1] 王建[1] 辛晓燕[1] 张潍[1] 

机构地区：[1]第四军医大学西京医院妇产科,西安710033

出　　处：《西南国防医药》2010年第5期465-467,共3页Medical Journal of National Defending Forces in Southwest China

基　　金：国家自然科学基金(30672235);全军"十一五"医药卫生科研基金(06M260)

摘　　要：目的研究siRNA(small interfering RNA,siRNA)对宫颈癌Hela细胞中双链RNA(dsRNA)激活的蛋白激酶(protein kinase regulated by dsRNA,PKR)基因的抑制作用,观察其对dsRNA诱导的细胞凋亡的影响。方法采用RNAi技术,对宫颈癌Hela细胞PKR基因进行特异性抑制;用阳离子脂质体与化学合成的Pre-designed anti-PKR siRNA构建转染复合体,反转染Hela细胞72h后提取总蛋白,用Western blot检测PKR蛋白表达水平的变化,用流式细胞仪检测细胞的凋亡率,激酶活性检测法测定半胱氨蛋白水解酶-3(caspase-3)活性变化。结果 siRNA能降低PKR基因的蛋白表达;未转染组的Hela细胞在加入dsRNA后,细胞凋亡明显增加,caspase-3激酶活性增强(P<0.05)。与对照组相比,转染组的Hela细胞在加入dsRNA后,细胞凋亡显著减少,caspase-3激酶活性降低(P<0.05)。结论 siRNA可特异有效地干扰宫颈癌Hela细胞内PKR基因的表达,从而影响肿瘤细胞凋亡。dsRNA可通过激活PKR,增强caspase-3激酶活性,而促进Hela细胞的凋亡。Objective To study the inhibitory effects of small interfering RNA（siRNA）of protein kinase regulated by dsRNA（PKR）gene on apoptosis of PKR-related Hela cell line of uterine cervix cancer,and to observe its effect on dsRNA-induced apoptosis.Methods RNA interference（RNAi）was employed to inhibit PKR gene specifically.The transfection compound of cationic lipsome and chemically synthesized Pre-designed anti-PKR siRNA were constructed,and then the total protein was extracted at 72 h after Hela cells were reversely transfected.The expression level of PKR protein was detected by Western blot,caspase-3 activity was assessed by kinase activity test and apoptotic rate by flow cytometry.Results siRNA could decrease the expression of PKR protein.In response to dsRNA addition,the apoptotic rate and the caspase-3 activity of the untransfected cells were enhanced significantly（P0.05）.However,the above two indexes of transfected cells decreased significantly compared with the control group following dsRNA addition（P0.05）.Conclusion siRNA can effectively and specifically interfere the expression of PKR gene,which may play a significant role in the apoptosis of Hela cells.dsRNA can enhance the caspase-3 activity and facilitate the apoptosis of Hela cells by the targeted activation of PKR.

关 键 词：PKR HELA细胞 DSRNA 凋亡 RNA干扰 

分 类 号：R737.33[医药卫生—肿瘤]