中华实用儿科临床杂志
中華實用兒科臨床雜誌
중화실용인과림상잡지
Journal of Applied Clinical Pediatrics
2015年
9期
707-710
,共4页
张朝霞%刘子勤%陈燕飞%玄立田%王天有
張朝霞%劉子勤%陳燕飛%玄立田%王天有
장조하%류자근%진연비%현립전%왕천유
肝癌%Survivin%小分子干扰RNA
肝癌%Survivin%小分子榦擾RNA
간암%Survivin%소분자간우RNA
Hepatocellular carcinoma%Survivin%Small interfering RNA
目的 探讨小分子干扰RNA (siRNA)对人肝癌细胞株MHCC-97H Survivin基因表达及其增殖和凋亡的影响.方法 设计、合成针对Survivin基因序列特异性siRNA,用脂质体包裹转染人肝癌细胞株MHCC-97H,实验分为3组:Survivin siRNA转染组(Si-survivin组),无关siRNA转染组(NC组)和无转染组(正常对照组).采用反转录-聚合酶链反应、Western blot技术检测Survivin mRNA及蛋白表达;四甲基偶氮唑蓝比色法检测细胞增殖情况;分别以终浓度为12.5、25.0、50.0 nmol/L的siRNA作用于细胞后24h,采用流式细胞仪AnnexinV/PI双染法检测细胞凋亡.结果 转染后48 h,Si-survivin组、NC组、正常对照组Survivin mRNA表达水平分别为0.55±0.16、0.85±0.28、0.93±0.40,组间比较差异有统计学意义(F=414,P＜0.01);Si-survivin组最低,与NC组、正常对照组比较差异均有统计学意义(t=-20.56、-28.37,P均＜0.001).siRNA转染48 h后,Si-survivin组、NC组、正常对照组Survivin蛋白相对表达量分别为0.602±0.005、0.835±0.007、0.993±0.003,组间比较差异有统计学意义(F =238,P＜0.01);Si-survivin组最低,与NC组、正常对照组比较差异均有统计学意义(t=-40.17、-66.03,P均＜0.001).转染后72 h和96 h,Si-survivin组细胞抑制率[(19.5±3.6)％、(12.0±0.9)％]显著高于NC组[(3.6±0.9)％、(-1.3±6.1)％],差异均有统计学意义(t=36.18、42.53,P均＜0.05).不同浓度siRNA作用于细胞后24h,12.5、25.0、50.0 nmol/L siRNA组、NC组和正常对照组细胞凋亡率[分别为(22.64±2.54)％、(35.37±3.28)％、(53.28±4.35)％、(8.77±i.25)％、(9.72±1.37)％]比较,组间差异有统计学意义(F=35.93,P＜0.01),不同浓度siRNA间细胞凋亡率两两比较,差异有统计学意义(t=-29.73、-38.57,P均＜0.001).结论 Survivin特异性siRNA能抑制人肝癌细胞株MHCC-97H Survivin基因表达,抑制细胞增殖,促进细胞凋亡.
目的 探討小分子榦擾RNA (siRNA)對人肝癌細胞株MHCC-97H Survivin基因錶達及其增殖和凋亡的影響.方法 設計、閤成針對Survivin基因序列特異性siRNA,用脂質體包裹轉染人肝癌細胞株MHCC-97H,實驗分為3組:Survivin siRNA轉染組(Si-survivin組),無關siRNA轉染組(NC組)和無轉染組(正常對照組).採用反轉錄-聚閤酶鏈反應、Western blot技術檢測Survivin mRNA及蛋白錶達;四甲基偶氮唑藍比色法檢測細胞增殖情況;分彆以終濃度為12.5、25.0、50.0 nmol/L的siRNA作用于細胞後24h,採用流式細胞儀AnnexinV/PI雙染法檢測細胞凋亡.結果 轉染後48 h,Si-survivin組、NC組、正常對照組Survivin mRNA錶達水平分彆為0.55±0.16、0.85±0.28、0.93±0.40,組間比較差異有統計學意義(F=414,P＜0.01);Si-survivin組最低,與NC組、正常對照組比較差異均有統計學意義(t=-20.56、-28.37,P均＜0.001).siRNA轉染48 h後,Si-survivin組、NC組、正常對照組Survivin蛋白相對錶達量分彆為0.602±0.005、0.835±0.007、0.993±0.003,組間比較差異有統計學意義(F =238,P＜0.01);Si-survivin組最低,與NC組、正常對照組比較差異均有統計學意義(t=-40.17、-66.03,P均＜0.001).轉染後72 h和96 h,Si-survivin組細胞抑製率[(19.5±3.6)％、(12.0±0.9)％]顯著高于NC組[(3.6±0.9)％、(-1.3±6.1)％],差異均有統計學意義(t=36.18、42.53,P均＜0.05).不同濃度siRNA作用于細胞後24h,12.5、25.0、50.0 nmol/L siRNA組、NC組和正常對照組細胞凋亡率[分彆為(22.64±2.54)％、(35.37±3.28)％、(53.28±4.35)％、(8.77±i.25)％、(9.72±1.37)％]比較,組間差異有統計學意義(F=35.93,P＜0.01),不同濃度siRNA間細胞凋亡率兩兩比較,差異有統計學意義(t=-29.73、-38.57,P均＜0.001).結論 Survivin特異性siRNA能抑製人肝癌細胞株MHCC-97H Survivin基因錶達,抑製細胞增殖,促進細胞凋亡.
목적 탐토소분자간우RNA (siRNA)대인간암세포주MHCC-97H Survivin기인표체급기증식화조망적영향.방법 설계、합성침대Survivin기인서렬특이성siRNA,용지질체포과전염인간암세포주MHCC-97H,실험분위3조:Survivin siRNA전염조(Si-survivin조),무관siRNA전염조(NC조)화무전염조(정상대조조).채용반전록-취합매련반응、Western blot기술검측Survivin mRNA급단백표체;사갑기우담서람비색법검측세포증식정황;분별이종농도위12.5、25.0、50.0 nmol/L적siRNA작용우세포후24h,채용류식세포의AnnexinV/PI쌍염법검측세포조망.결과 전염후48 h,Si-survivin조、NC조、정상대조조Survivin mRNA표체수평분별위0.55±0.16、0.85±0.28、0.93±0.40,조간비교차이유통계학의의(F=414,P＜0.01);Si-survivin조최저,여NC조、정상대조조비교차이균유통계학의의(t=-20.56、-28.37,P균＜0.001).siRNA전염48 h후,Si-survivin조、NC조、정상대조조Survivin단백상대표체량분별위0.602±0.005、0.835±0.007、0.993±0.003,조간비교차이유통계학의의(F =238,P＜0.01);Si-survivin조최저,여NC조、정상대조조비교차이균유통계학의의(t=-40.17、-66.03,P균＜0.001).전염후72 h화96 h,Si-survivin조세포억제솔[(19.5±3.6)％、(12.0±0.9)％]현저고우NC조[(3.6±0.9)％、(-1.3±6.1)％],차이균유통계학의의(t=36.18、42.53,P균＜0.05).불동농도siRNA작용우세포후24h,12.5、25.0、50.0 nmol/L siRNA조、NC조화정상대조조세포조망솔[분별위(22.64±2.54)％、(35.37±3.28)％、(53.28±4.35)％、(8.77±i.25)％、(9.72±1.37)％]비교,조간차이유통계학의의(F=35.93,P＜0.01),불동농도siRNA간세포조망솔량량비교,차이유통계학의의(t=-29.73、-38.57,P균＜0.001).결론 Survivin특이성siRNA능억제인간암세포주MHCC-97H Survivin기인표체,억제세포증식,촉진세포조망.
Objective To observe the effect of small interfering RNA(siRNA) targeting Survivin gene on survivin expression,proliferation and apoptosis of hepatocellular carcinoma cell line MHCC-97H.Methods Survivin sequence specific siRNA was designed and synthesized.siRNA/liposome complex was transfected into hepatocellular carcinoma cell line MHCC-97H.The MHCC-97H cells were divided into Survivin siRNA group(Si-survivin),negative control siRNA group(NC group)and blank group (normal control group).Survivin mRNA and protein expressions were detected by reverse transcription-PCR and Western blot,respectively.The proliferation of MHCC-97H was measured by methythiazolydiphenyl-tetrazolium bromide assay.The Annexin V/PI double labeled flow cytometry was employed to measure the apoptosis at 24 h after transfection in different concentrations of Survivin siRNA(12.5,25.0 and 50.0 nmoL/L,respectively).Results After 48 h of transfection,the Survivin mRNA levels were 0.55 ± 0.16 (Si-survivin group),0.85 ± 0.28 (NC group) and 0.93 ± 0.40 (normal control group),respectively,which were significantly different among 3 groups (F =414,P ＜ 0.01).The level of Survivin mRNA was the lowest in Si-survivin group,which was statistically different with NC group and normal control group (t =-20.56,-28.37,all P ＜ 0.001).The levels of Survivin protein expression in 3 groups were 0.602 ± 0.005 (Si-survivin group),0.835 ± 0.007 (NC group) and 0.993 ± 0.003 (normal control group) at 48 h after transfection,which were statistically different among 3 groups (F =238,P ＜0.01).The lowest level of protein expression was in Si-survivin group,which was statistically different with NC group and normal control group (t =-40.17,-66.03,all P ＜ 0.001).After 72 h and 96 h of transfection,the inhibitory rate of cell growth was significantly higher in Si-survivin group [(19.5 ± 3.6)％,(12.0 ± 0.9)％] compared with that in NC group [(3.6 ± 0.9) ％,(-1.3 ± 6.1) ％] (t--36.18,42.53,all P ＜ 0.05).The apoptosis rates in 12.5,25.0,50.0 nmol/L Survivin siRNA were (22.64 ± 2.54) ％,(35.37 ± 3.28) ％ and (53.28 ± 4.35) ％,respectively.However,in NC group and normal control group,the apoptosis rates were (8.77 ± 1.25) ％ and (9.72 ± 1.37) ％.The rates were statistically different among those 5 groups(F =35.93,P ＜0.01).And in the apoptosis rates of siRNA groups in different concentratiom were statistically different when compared between each two groups (t =-29.73,-38.57,all P ＜ 0.001).Conclusion Survivin specific siRNA can inhibit the proliferation and induce the apoptosis by blocking Survivin gene expression in hepatocellular carcinoma cell line MHCC-97H.
