CAJ | 학술논문

目的探讨REIC/Dkk-3基因是否具有杀伤肾癌细胞786-0的作用及其机制.方法采用REIC/Dkk-3基因过表达载体(pEZ-M29-REIC/Dkk-3)转染786-0细胞,分别于转染24、48、72、96 h后行细胞计数试剂盒(CCK-8)检测其对786-0细胞的增殖抑制作用;流式细胞仪膜联蛋白V(Annexin V)/碘化丙锭(PI)双染法检测其对786-0细胞诱导细胞凋亡的影响,PI单染法检测周期阻滞效应,Western blot法检测凋亡和周期阻滞相关蛋白的表达.结果 Western blot和实时定量聚合酶链反应(Real-time PCR)结果显示转染REIC/Dkk-3基因后其表达量较对照组升高5倍多.CCK-8结果显示,随着作用时间的增加,786-0细胞的增殖抑制率逐渐增加,处理24、48、72、96 h后对照组细胞存活率为92.0％、90.0％、84.0％、80.5％,REIC/Dkk-3过表达组分别为89.50％、73.75％、63.00％、58.50％,与空质粒组比较,差异有统计学意义(P＜0.05).流式Annexin V/PI双染法显示REIC/Dkk-3可诱导肾癌细胞凋亡,过表达组、空载体组、空白对照组凋亡率分别为28.5％、7.2％、0.6％,Western blot结果显示REIC/Dkk-3可上调活化酪半胱氨酰天冬氨酸特异性蛋白酶(cleavedCaspase-3)和下调B细胞淋巴瘤/白血病-2(bcl-2)的表达.细胞周期结果显示REIC/Dkk-3可诱导肾癌细胞发生G1期阻滞,过表达组、空载体组、空白对照组G1期比例分别为(69.90±3.40)％、(59.57±1.20)％和(52.57±1.20)％;Western blot结果显示REIC/Dkk-3过表达可上调p21和下调细胞周期素D1(Cyclin Dl).结论 REIC/Dkk-3可抑制肾癌细胞786-0的增殖,可能是通过诱导发生细胞凋亡和G1期阻滞实现的.
목적 탐토REIC/Dkk-3기인시부구유살상신암세포786-0적작용급기궤제.방법 채용REIC/Dkk-3기인과표체재체(pEZ-M29-REIC/Dkk-3)전염786-0세포,분별우전염24、48、72、96 h후행세포계수시제합(CCK-8)검측기대786-0세포적증식억제작용;류식세포의막련단백V(Annexin V)/전화병정(PI)쌍염법검측기대786-0세포유도세포조망적영향,PI단염법검측주기조체효응,Western blot법검측조망화주기조체상관단백적표체.결과 Western blot화실시정량취합매련반응(Real-time PCR)결과현시전염REIC/Dkk-3기인후기표체량교대조조승고5배다.CCK-8결과현시,수착작용시간적증가,786-0세포적증식억제솔축점증가,처리24、48、72、96 h후대조조세포존활솔위92.0％、90.0％、84.0％、80.5％,REIC/Dkk-3과표체조분별위89.50％、73.75％、63.00％、58.50％,여공질립조비교,차이유통계학의의(P＜0.05).류식Annexin V/PI쌍염법현시REIC/Dkk-3가유도신암세포조망,과표체조、공재체조、공백대조조조망솔분별위28.5％、7.2％、0.6％,Western blot결과현시REIC/Dkk-3가상조활화락반광안선천동안산특이성단백매(cleavedCaspase-3)화하조B세포림파류/백혈병-2(bcl-2)적표체.세포주기결과현시REIC/Dkk-3가유도신암세포발생G1기조체,과표체조、공재체조、공백대조조G1기비례분별위(69.90±3.40)％、(59.57±1.20)％화(52.57±1.20)％;Western blot결과현시REIC/Dkk-3과표체가상조p21화하조세포주기소D1(Cyclin Dl).결론 REIC/Dkk-3가억제신암세포786-0적증식,가능시통과유도발생세포조망화G1기조체실현적.
Objective To investigate the killing effects of DEIC/Dkk-3 on renal cancer cell line 786-0 and its potential mechanism.Methods We transfected the pEZ-M29-REIC/Dkk-3 plasmid into 786-0 cells to overexpress REIC/Dkk-3.The inhibitory effects of REIC/Dkk-3 on cell proliferation was assessed using cell countiong kit-8 (CCK-8) at 24,48,72 and 96 h after transfection.Apoptosis of 786-0 cells was detected by AnnexinV/PI double staining,and cell cycle was detected by PI staining using flow cytometry.Changes of protein expression related to apoptosis and cell cycle arrest were measured by Westem blotting.Results The results of immunoblotting (Western blotting) and real-time polymerase chain reaction (Real-time PCR) showed that REIC/Dkk-3 gene expression was increased five folds after transfection as compared with control group.The results of CCK-8 method indicated that with the prolongation of transfection time,inhibitory effect of 786-0 cell proliferation was increased.At 24,48,72 and 96 h,cell survival rate in gene overexpression group and control vector group was 92.0％,90.0％,84.0％ and 80.5％,and 89.50％,73.75％,63.00％ and 58.50％,respectively (P ＜0.05).The apoptosis rate in gene overexpression group,control vector group and blank control group was 28.5％,7.2％ and 0.6％ respectively.Overexpression of REIC/Dkk-3 could up-regulate the expression of Cleaved-Caspase-3,and down-regulate the expression of B cell lymphoma/leukemia-2 (bcl-2).In addition,overexpression of REIC/Dkk-3 caused G1 phase arrest of 786-0 cells.The percentage of G1 phase in pEZ-M29-REIC/Dkk-3 group,pEZ-M29-Flag group and control group was (69.90 ± 3.40) ％,(59.57 ± 1.20) ％ and (52.57 ± 1.20) ％ respectively.Results of Western blotting showed that overexpression of REIC/Dkk-3 could increase the expression of p21 and decrease the expression of Cyclin D1.Conclusion REIC/Dkk-3 can inhibit the proliferation of renal cancer cell 786-0 probably by inducing cell apoptosis and G1 phase arrest.