中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
2期
248-251
,共4页
施华%王爱军%郑宝军%冯俊伟%王红钰%吴肖
施華%王愛軍%鄭寶軍%馮俊偉%王紅鈺%吳肖
시화%왕애군%정보군%풍준위%왕홍옥%오초
缺氧诱导因子-2α%胃癌%RNA干扰%凋亡
缺氧誘導因子-2α%胃癌%RNA榦擾%凋亡
결양유도인자-2α%위암%RNA간우%조망
Hypoixa inducible factor-2α%Gastric carcinoma%RNA interference%Apoptosis
目的 探讨缺氧诱导因子-2α(HIF-2α)沉默对胃癌细胞株SGC7901凋亡的影响及其机制.方法 常规培养SGC7901细胞,实时荧光定量聚合酶链反应(FQ-PCR)和Western blot法检测低氧组(90%氮气+5%氢气+5%二氧化碳)和正常氧条件下HIF-2α、B淋巴细胞/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3的表达;合成针对HIF-2α的小干扰RNA(HIF-2α-siRNA);Western blot检测HIF-2α-siRNA的转染效果;细胞计数试剂盒(CCK)法检测HIF-2α-siRNA转染后的细胞增殖,流式细胞仪(FCM)检测细胞凋亡率;FQ-PCR和Western blot检测转染前后bcl-2、bax、Caspase-3的表达.结果 低氧条件下,HIF-2α(2.94±0.31、0.61±0.08)、bcl-2 (2.0l±0.29、1.05±0.09) mRNA和蛋白表达与正常氧条件下HIF-2α(1.06±0.15、0.23±0.04)、bcl-2(1.08±0.16、0.34±0.05)比较明显升高,bax (0.54±0.06、0.48 ±0.05)、Caspase-3(0.45±0.05、0.58 ±0.07)mRNA和蛋白表达与正常氧条件bax (0.97 ±0.13、0.74±0.12)、Caspase-3 (0.96±0.12、0.89 ±0.14)比较则明显降低(P<0.05);HIF-2α-siRNA转染后与转染前比较能有效沉默HIF-2α;HIF-2α-siRNA转染后[(0.47±0.05)%]与转染前[(0.89±0.12)%]比较能抑制增殖水平,与转染前[(8.15±0.97)%]比较,转染后[(20.16±3.09)%]凋亡率升高(P<0.05);与转染前(0.96 ±0.09、1.37 ±0.21、1.13±0.19、0.82±0.16、0.97 ±0.13、0.69±0.11)比较,转染后可降低bcl-2 mRNA和蛋白表达(0.43±0.06、0.78±0.12),升高bax(2.91 ±0.28、1.51 ±0.28)、Caspase-3 mRNA和蛋白表达(2.42 ±0.34、1.34 ±0.21) (P<0.05).结论 低氧下,HIF-2α-siRNA可通过降低bcl-2表达、升高bax、CasDase-3表达而诱导SGC7901细胞凋亡.
目的 探討缺氧誘導因子-2α(HIF-2α)沉默對胃癌細胞株SGC7901凋亡的影響及其機製.方法 常規培養SGC7901細胞,實時熒光定量聚閤酶鏈反應(FQ-PCR)和Western blot法檢測低氧組(90%氮氣+5%氫氣+5%二氧化碳)和正常氧條件下HIF-2α、B淋巴細胞/白血病-2(bcl-2)、bcl-2相關X蛋白(bax)、半胱氨酰天鼕氨痠特異性蛋白酶(Caspase)-3的錶達;閤成針對HIF-2α的小榦擾RNA(HIF-2α-siRNA);Western blot檢測HIF-2α-siRNA的轉染效果;細胞計數試劑盒(CCK)法檢測HIF-2α-siRNA轉染後的細胞增殖,流式細胞儀(FCM)檢測細胞凋亡率;FQ-PCR和Western blot檢測轉染前後bcl-2、bax、Caspase-3的錶達.結果 低氧條件下,HIF-2α(2.94±0.31、0.61±0.08)、bcl-2 (2.0l±0.29、1.05±0.09) mRNA和蛋白錶達與正常氧條件下HIF-2α(1.06±0.15、0.23±0.04)、bcl-2(1.08±0.16、0.34±0.05)比較明顯升高,bax (0.54±0.06、0.48 ±0.05)、Caspase-3(0.45±0.05、0.58 ±0.07)mRNA和蛋白錶達與正常氧條件bax (0.97 ±0.13、0.74±0.12)、Caspase-3 (0.96±0.12、0.89 ±0.14)比較則明顯降低(P<0.05);HIF-2α-siRNA轉染後與轉染前比較能有效沉默HIF-2α;HIF-2α-siRNA轉染後[(0.47±0.05)%]與轉染前[(0.89±0.12)%]比較能抑製增殖水平,與轉染前[(8.15±0.97)%]比較,轉染後[(20.16±3.09)%]凋亡率升高(P<0.05);與轉染前(0.96 ±0.09、1.37 ±0.21、1.13±0.19、0.82±0.16、0.97 ±0.13、0.69±0.11)比較,轉染後可降低bcl-2 mRNA和蛋白錶達(0.43±0.06、0.78±0.12),升高bax(2.91 ±0.28、1.51 ±0.28)、Caspase-3 mRNA和蛋白錶達(2.42 ±0.34、1.34 ±0.21) (P<0.05).結論 低氧下,HIF-2α-siRNA可通過降低bcl-2錶達、升高bax、CasDase-3錶達而誘導SGC7901細胞凋亡.
목적 탐토결양유도인자-2α(HIF-2α)침묵대위암세포주SGC7901조망적영향급기궤제.방법 상규배양SGC7901세포,실시형광정량취합매련반응(FQ-PCR)화Western blot법검측저양조(90%담기+5%경기+5%이양화탄)화정상양조건하HIF-2α、B림파세포/백혈병-2(bcl-2)、bcl-2상관X단백(bax)、반광안선천동안산특이성단백매(Caspase)-3적표체;합성침대HIF-2α적소간우RNA(HIF-2α-siRNA);Western blot검측HIF-2α-siRNA적전염효과;세포계수시제합(CCK)법검측HIF-2α-siRNA전염후적세포증식,류식세포의(FCM)검측세포조망솔;FQ-PCR화Western blot검측전염전후bcl-2、bax、Caspase-3적표체.결과 저양조건하,HIF-2α(2.94±0.31、0.61±0.08)、bcl-2 (2.0l±0.29、1.05±0.09) mRNA화단백표체여정상양조건하HIF-2α(1.06±0.15、0.23±0.04)、bcl-2(1.08±0.16、0.34±0.05)비교명현승고,bax (0.54±0.06、0.48 ±0.05)、Caspase-3(0.45±0.05、0.58 ±0.07)mRNA화단백표체여정상양조건bax (0.97 ±0.13、0.74±0.12)、Caspase-3 (0.96±0.12、0.89 ±0.14)비교칙명현강저(P<0.05);HIF-2α-siRNA전염후여전염전비교능유효침묵HIF-2α;HIF-2α-siRNA전염후[(0.47±0.05)%]여전염전[(0.89±0.12)%]비교능억제증식수평,여전염전[(8.15±0.97)%]비교,전염후[(20.16±3.09)%]조망솔승고(P<0.05);여전염전(0.96 ±0.09、1.37 ±0.21、1.13±0.19、0.82±0.16、0.97 ±0.13、0.69±0.11)비교,전염후가강저bcl-2 mRNA화단백표체(0.43±0.06、0.78±0.12),승고bax(2.91 ±0.28、1.51 ±0.28)、Caspase-3 mRNA화단백표체(2.42 ±0.34、1.34 ±0.21) (P<0.05).결론 저양하,HIF-2α-siRNA가통과강저bcl-2표체、승고bax、CasDase-3표체이유도SGC7901세포조망.
Objective To observe the Mechanisms of silencing hypoixa inducible factor-2α (HIF-2α) gene on apoptosis of human gastric carcinoma cell lines SGC7901.Methods Human gastric carcinoma cell lines SGC7901 were cultured in vitro.The expression of HIF-2α,B lymphocytes/leukemia-2(bcl-2),bcl-2 associated X protein (bax),cysteinyl aspartate-specific protease (Caspase)-3 were detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting in normal or under hypoxia (90% N2 + 5% H2 + 5% CO2).Transfection effect of HIF-2α-small interfering RNA (siRNA)was detected by Western blotting.cell counting kit (CCK) was used to test the cell proliferation and flow cytometry (FCM) was used to test the cell apoptosis rates after transfection of HIF-2α-siRNA.And the expression of bcl-2,bax,Caspase-3 were detected by FQ-PCR and Western blotting.Results The expression of HIF-2α mRNA and protein (2.94 ± 0.31,0.61 ± 0.08),bcl-2 mRNA and protein (2.01 ± 0.29,1.05±0.09) increased and the expression of bax mRNA and protein (0.54 ±0.06,0.48 ±0.05),Caspase-3 mRNA and protein (0.45 ± 0.05,0.58 ± 0.07) decreased under hypoxia than under normal condition of HIF-2α (1.06 ±0.15,0.23 ±0.04),bcl-2 (1.08 ±0.16,0.34 ±0.05),bax (0.97 ±0.13,0.74±0.12) and Caspase-3 (0.96±0.12,0.89 ±0.14) mRNA and protein (P<0.05).HIF-2α expression was suppressed after HIF-2α-siRNA transfection.The proliferation [(0.47 ±0.05) %]decreased,the cell apoptosis rates [(20.16 ± 3.09)%] increased after HIF-2α-siRNA transfection [(0.89±0.12)%,(8.15 ±0.97)%,P<0.05].bcl-2 mRNA and protein (0.43±0.06,0.78 ±0.12) decreased,bax mRNA and protein (2.91 ± 0.28,1.51 ± 0.28),Caspase-3 mRNA and protein (2.42 ±0.34,1.34 ± 0.21) increased after HIF-2α-siRNA transfection (0.96 ± 0.09,1.37 ± 0.21,1.13±0.19,0.82±0.16,0.97±0.13,0.69±0.11) (P<0.05).Conclusion Under hypoxia,HIF-2α-siRNA could decrease the expression of bcl-2,increase the expression of bax,Caspase-3,and induce apoptosis.