西安交通大学学报（医学版）
西安交通大學學報（醫學版）
서안교통대학학보（의학판）
JOURNAL OF XI'AN JIAOTONG UNIVERSITY(MEDICAL SCIENCES)
2015年
4期
439-443
,共5页
张杰波%刘映峰%缪绯%刘芃
張傑波%劉映峰%繆緋%劉芃
장걸파%류영봉%무비%류봉
缺氧复氧%促红细胞生成素(EPO)%细胞凋亡%Omi/HtrA2%RNA 干扰%caspase-3%缺血/再灌注损伤
缺氧複氧%促紅細胞生成素(EPO)%細胞凋亡%Omi/HtrA2%RNA 榦擾%caspase-3%缺血/再灌註損傷
결양복양%촉홍세포생성소(EPO)%세포조망%Omi/HtrA2%RNA 간우%caspase-3%결혈/재관주손상
hypoxia-reoxygenation%EPO%apoptosis%Omi/HtrA2%RNA interference%caspase-3%ischemia reperfusion injury
目的：观察促红细胞生成素(EPO)及/或进一步沉默 Omi/HtrA2表达时对缺氧复氧(H/R)心肌细胞的影响,探索相关抗细胞凋亡机制。方法培养乳鼠心肌细胞株(H9C2细胞),分组(对照组、H/R 组及各浓度的 EPO 干预组)处理后,酶标仪检测各组细胞上清液中乳酸脱氢酶(LDH)释放率,Western blot 检测细胞内 cleaved caspase-3、Omi/HtrA2蛋白表达变化；并观察 Omi/HtrA2在胞质和线粒体的表达变化(转位情况)。再经脂质体法将 Omi/HtrA2特异性 siRNA 干扰片段转染至 H9C2细胞中,RT-PCR 和 Western blot 验证 siRNA 对 Omi/HtrA2表达的沉默效应后,分组同前干预,分别检测各组细胞 LDH 释放率及 cleaved caspase-3表达变化。结果与 H/R 组相比, EPO 干预组细胞上清液 LDH 释放减少,cleaved caspase-3表达减弱；H/R 组 Omi/HtrA2蛋白表达在胞质中表达较对照组增多(向胞质发生转位),而 EPO(20 IU/mL)组其胞质转位减少。经 siRNA 干扰后,与 H/R 组相比,LDH 释放降低,cleaved caspase-3表达减弱,差异均有统计学意义(P <0.05)。结论EPO 可能通过减少 Omi/HtrA2蛋白的转位,抑制 caspases-3通路激活,而发挥细胞保护作用。
目的：觀察促紅細胞生成素(EPO)及/或進一步沉默 Omi/HtrA2錶達時對缺氧複氧(H/R)心肌細胞的影響,探索相關抗細胞凋亡機製。方法培養乳鼠心肌細胞株(H9C2細胞),分組(對照組、H/R 組及各濃度的 EPO 榦預組)處理後,酶標儀檢測各組細胞上清液中乳痠脫氫酶(LDH)釋放率,Western blot 檢測細胞內 cleaved caspase-3、Omi/HtrA2蛋白錶達變化；併觀察 Omi/HtrA2在胞質和線粒體的錶達變化(轉位情況)。再經脂質體法將 Omi/HtrA2特異性 siRNA 榦擾片段轉染至 H9C2細胞中,RT-PCR 和 Western blot 驗證 siRNA 對 Omi/HtrA2錶達的沉默效應後,分組同前榦預,分彆檢測各組細胞 LDH 釋放率及 cleaved caspase-3錶達變化。結果與 H/R 組相比, EPO 榦預組細胞上清液 LDH 釋放減少,cleaved caspase-3錶達減弱；H/R 組 Omi/HtrA2蛋白錶達在胞質中錶達較對照組增多(嚮胞質髮生轉位),而 EPO(20 IU/mL)組其胞質轉位減少。經 siRNA 榦擾後,與 H/R 組相比,LDH 釋放降低,cleaved caspase-3錶達減弱,差異均有統計學意義(P <0.05)。結論EPO 可能通過減少 Omi/HtrA2蛋白的轉位,抑製 caspases-3通路激活,而髮揮細胞保護作用。
목적：관찰촉홍세포생성소(EPO)급/혹진일보침묵 Omi/HtrA2표체시대결양복양(H/R)심기세포적영향,탐색상관항세포조망궤제。방법배양유서심기세포주(H9C2세포),분조(대조조、H/R 조급각농도적 EPO 간예조)처리후,매표의검측각조세포상청액중유산탈경매(LDH)석방솔,Western blot 검측세포내 cleaved caspase-3、Omi/HtrA2단백표체변화；병관찰 Omi/HtrA2재포질화선립체적표체변화(전위정황)。재경지질체법장 Omi/HtrA2특이성 siRNA 간우편단전염지 H9C2세포중,RT-PCR 화 Western blot 험증 siRNA 대 Omi/HtrA2표체적침묵효응후,분조동전간예,분별검측각조세포 LDH 석방솔급 cleaved caspase-3표체변화。결과여 H/R 조상비, EPO 간예조세포상청액 LDH 석방감소,cleaved caspase-3표체감약；H/R 조 Omi/HtrA2단백표체재포질중표체교대조조증다(향포질발생전위),이 EPO(20 IU/mL)조기포질전위감소。경 siRNA 간우후,여 H/R 조상비,LDH 석방강저,cleaved caspase-3표체감약,차이균유통계학의의(P <0.05)。결론EPO 가능통과감소 Omi/HtrA2단백적전위,억제 caspases-3통로격활,이발휘세포보호작용。
Objective To observe the expression of caspase-3 and transposition of Omi/HtrA2 in H9C2 by erythropoietin and/or Omi/HtrA2 silencing to explore the related anti-apoptotic mechanisms.Methods The cultured H9C2 cardiomyocytes were divided into control group,H/R group (anoxia 2 h and re-oxygenation 24 h), and different concentrations of EPO treatment groups.The release rate of lactate dehydrogenase (LDH)in cell supernatant was measured in each group.Expressions of cleaved caspase-3 and Omi/HtrA2 were measured by Western blot;then the transposition of Omi/HtrA2 between cytoplasm and mitochondria was observed.Specific siRNA interfering fragment was transfected into H9C2 cardiomyocytes by liposome method.Its silencing effect on Omi/HtrA2 was measured by RT-PCR and Western blot.The survival rate,release rate and expression of cleaved caspase-3 were measured.And the expression of Omi/HtrA2 was measured in cytoplasm and mitochondria in H9C2 (transposition of Omi/HtrA2 ).Results Compared with H/R group,the release of LDH and expression of cleaved caspase-3 were decreased; the transposition of Omi/HtrA2 from mitochondria to cytoplasm in H/R treatment groups was increased compared with control group,while that in EPO (20 IU/mL)group decreased.si-HtrA2 group transfected with siRNA showed a decreased release of LDH and expression of cleaved caspase-3 with all significant variances (P <0.05).Conclusion EPO exerts a cytoprotective effect by inhibiting the transposition of Omi/HtrA2 and hence the activation of caspases-3 pathway.
