中华血液学杂志
中華血液學雜誌
중화혈액학잡지
Chinese Journal of Hematology
2009年
12期
825-828
,共4页
李方方%郑改焕%徐酉华%罗庆
李方方%鄭改煥%徐酉華%囉慶
리방방%정개환%서유화%라경
RNA干扰%血管内皮生长因子%细胞凋亡%K562细胞
RNA榦擾%血管內皮生長因子%細胞凋亡%K562細胞
RNA간우%혈관내피생장인자%세포조망%K562세포
Adenovirus%recombinant%Vascular endothelial growth factor%Inhibitor apoptosis proteins%Gene%survivin
目的 应用腺病毒介导的血管内皮生长因子(VEGF) siRNA沉默K562细胞中VEGF的表达,观察其对K562细胞凋亡及凋亡抑制基因survivin表达的影响.方法 应用成功构建的携带特异性VEGF siRNA的重组腺病毒(Ad5-VEGF siRNA)感染K562细胞,实验分为3组:实验组(K562/Ad5-VEGF siRNA组)、空载体组(K562/Ad5组)、对照组(K562组).通过RT-PCR法检测细胞内VEGF及survivin mRNA的表达,ELISA法检测细胞培养上清中VEGF蛋白表达,Western blot法检测细胞survivin蛋白表达,流式细胞术检测细胞凋亡.结果 实验组细胞VEGF及survivin mRNA表达水平较对照组均有明显降低(P＜0.01),且细胞培养上清中VEGF蛋白表达水平[(1121±15)pg/ml]低于空载体组[(1290±28)pg/ml]和对照组[(1303±28)pg/ml](P＜0.01).Western blot法检测结果显示,实验组survivin蛋白表达水平(0.26 ± 0.11)较对照组(0.74±0.10)也显著降低(P＜0.01).实验组细胞凋亡率与对照组比较明显增加(P＜0.01).结论 应用RNA干扰沉默K562细胞中VEGF基因表达后,survivin的表达也随之降低,同时细胞凋亡率相应增加.VEGF可诱导K562细胞中survivin基因表达,可能是survivin基因表达的上游调控因子.
目的 應用腺病毒介導的血管內皮生長因子(VEGF) siRNA沉默K562細胞中VEGF的錶達,觀察其對K562細胞凋亡及凋亡抑製基因survivin錶達的影響.方法 應用成功構建的攜帶特異性VEGF siRNA的重組腺病毒(Ad5-VEGF siRNA)感染K562細胞,實驗分為3組:實驗組(K562/Ad5-VEGF siRNA組)、空載體組(K562/Ad5組)、對照組(K562組).通過RT-PCR法檢測細胞內VEGF及survivin mRNA的錶達,ELISA法檢測細胞培養上清中VEGF蛋白錶達,Western blot法檢測細胞survivin蛋白錶達,流式細胞術檢測細胞凋亡.結果 實驗組細胞VEGF及survivin mRNA錶達水平較對照組均有明顯降低(P＜0.01),且細胞培養上清中VEGF蛋白錶達水平[(1121±15)pg/ml]低于空載體組[(1290±28)pg/ml]和對照組[(1303±28)pg/ml](P＜0.01).Western blot法檢測結果顯示,實驗組survivin蛋白錶達水平(0.26 ± 0.11)較對照組(0.74±0.10)也顯著降低(P＜0.01).實驗組細胞凋亡率與對照組比較明顯增加(P＜0.01).結論 應用RNA榦擾沉默K562細胞中VEGF基因錶達後,survivin的錶達也隨之降低,同時細胞凋亡率相應增加.VEGF可誘導K562細胞中survivin基因錶達,可能是survivin基因錶達的上遊調控因子.
목적 응용선병독개도적혈관내피생장인자(VEGF) siRNA침묵K562세포중VEGF적표체,관찰기대K562세포조망급조망억제기인survivin표체적영향.방법 응용성공구건적휴대특이성VEGF siRNA적중조선병독(Ad5-VEGF siRNA)감염K562세포,실험분위3조:실험조(K562/Ad5-VEGF siRNA조)、공재체조(K562/Ad5조)、대조조(K562조).통과RT-PCR법검측세포내VEGF급survivin mRNA적표체,ELISA법검측세포배양상청중VEGF단백표체,Western blot법검측세포survivin단백표체,류식세포술검측세포조망.결과 실험조세포VEGF급survivin mRNA표체수평교대조조균유명현강저(P＜0.01),차세포배양상청중VEGF단백표체수평[(1121±15)pg/ml]저우공재체조[(1290±28)pg/ml]화대조조[(1303±28)pg/ml](P＜0.01).Western blot법검측결과현시,실험조survivin단백표체수평(0.26 ± 0.11)교대조조(0.74±0.10)야현저강저(P＜0.01).실험조세포조망솔여대조조비교명현증가(P＜0.01).결론 응용RNA간우침묵K562세포중VEGF기인표체후,survivin적표체야수지강저,동시세포조망솔상응증가.VEGF가유도K562세포중survivin기인표체,가능시survivin기인표체적상유조공인자.
Objective To study the effect of adenovirus vector-mediated siRNA targeting vascular endothelial growth factor (VEGF) on apoptosis and the expression of survivin iu K562 cells.Methods K562 cells were infected with recombinant adenovirus Ad5-VEGFsi for 72 hours as experimental group (K562/Ad5-VEGFsi),and empty vector group (K562/Ads) and blank control group (K562) as controls.VEGF mRNA and survivin mRNA expression were determined by RT-PCR.The protein levels of VEGF and survivin were measured by ELISA and Western blot,respectively.The apoptosis of K562 cells wss detected by flow cytometry.Results The levels of VEGF and survivin mRNA expression in experimental group cells were signitlcanfly decreased (P＜0.01).The protein concentration of VEGF in experimental group supernatant was (1121±15)pg/ml,being lower than that in empty vector group[(1290±28)pg/ml] and black control group [(1303±28)pg/ml] (P＜0.01),and the level of survivin protein in experimental group (0.26±0.11) was significantly reduced compared with that in blank control group(0.74±0.10)(P＜0.01).The apoptosis rate of K562/Ad5-VEGFsi cells(16.45±0.14)% was higher than those of K562/Ad5 cells (3.54±0.17)% and K562 cells(2.56±0.20)% (P＜0.01).Conclusions VEGF can up-regulate the expression of survivin.After inhibition of VEGF by RNAi,the expression of survivin is decreased subsequently and the rates of cell apoptosis are increased.
