节点文献
microRNA-21在大鼠心肌缺血再灌注早期的抗凋亡作用及机制
The Effect and Mechanism of Anti-apoptosis of MicroRNA-21in Rat Myocardium during Early Phase of Ischemia-repersusion
【作者】 杨琼;
【导师】 杨侃;
【作者基本信息】 中南大学 , 临床医学, 2013, 博士
【摘要】 目的:细胞凋亡是缺血再灌注(ischemia-reperfusion, I/R)损伤的重要环节,抑制细胞凋亡可以防止或减轻I/R损伤。文献报道microRNA-21(miR-21)在I/R动物模型表达异常,本研究探讨miR-21在I/R早期表达异常是否与细胞凋亡相关及其可能机制。曲美他嗪(trimetazidine, TMZ)是经典抗缺血药物,本研究还将探讨TMZ在I/R早期抗细胞凋亡作用是否由miR-21及其靶基因介导。方法:1.体内实验:48只SD大鼠随机分为假手术(Sham)、I/R2h、 I/R4h、I/R6h组,每组12只大鼠。Sham组大鼠仅开胸,其余组分别开胸结扎前降支45分钟后再灌注2h、4h、6h。比较各组的细胞凋亡水平及miR-21水平,以Bcl-2/Bax、Caspase-3为心肌细胞凋亡指标。HE染色观察心肌组织形态结构,Westernblot方法检测Bcl-2/Bax、 Caspase-3的蛋白水平,Realtime PCR检测miR-21变化。2.体内miR-21过表达实验:成功构建重组腺相关病毒rAAV9-ZsGreen-pre-miR-21及空白对照病毒,滴度为5.0×1012vg/ml。30只SD大鼠,随机分为①对照组:大鼠转染空白病毒;②miR-21组:大鼠转染rAAV9-ZsGreen-pre-miR-21;③Sham组:大鼠转染空白病毒后开胸;④I/R组:大鼠转染空白病毒后I/R2h;⑤I/R+miR-21组:大鼠转染rAAV9-ZsGreen-premiR-21后I/R2h。以PTEN/AKT通路作为拟验证的miR-21的下游通路。比较过表达miR-21后细胞凋亡水平及miR-21下游通路水平变化。Realtime PCR方法检测miR-21、PTENmRNA水平,免疫组化观察缺血区Bcl-2、Bax、Caspase-3的表达,Westernblot方法检测缺血区凋亡指标水平及下游通路PTEN、 p-AKT的蛋白水平。3.体外实验:用LipofectamineTM2000将30nM,50nM,100nM的miR-21抑制剂(inhibitors)及30nM,50nM,100nM的miR-21模拟物(mimics)转染H9C2细胞,摸索最佳转染浓度。将细胞随机分为①Vehicle Control(VC).②抑制物阴性对照(inhibitors negative control,INC)、③模拟物阴性对照(mimics negative control, MNC)、④缺氧-复氧(hypoxia-reoxygenation,H/R)+VC、⑤H/R+INC、⑥H/R+MNC、⑦H/R+inhibitors、⑧H/R+mimics。H/R处理为低氧培养4小时后常氧培养3小时。比较正、负调控miR-21表达时miR-21、下游通路PTEN/AKT水平及细胞凋亡水平的变化。Realtime PCR检测miR-21, PTENmRNA水平,Westernblot方法检测Bcl-2/Bax, Caspase-3, PTEN, p-AKT的蛋白水平,流式细胞仪检测细胞凋亡。4.体外TMZ抗凋亡实验:H9C2细胞随机分为Control、H/R+Control、 H/R+TMZ组;同时另设H/R+TMZ+INC、H/R+TMZ+inhibitors组。观察TMZ对H/R后细胞miR-21、下游通路PTEN/AKT水平及细胞凋亡水平的影响,进一步观察miR-21表达抑制后上述指标的变化。结果:1.与Sham组比较,I/R2h,4h,6h组大鼠心肌缺血区miR-21进行性降低(P<0.05),抑制凋亡指标Bcl-2/Bax水平降低(P<0.05),促凋亡因子Caspase-3水平逐渐升高(P<0.05)。非缺血区miR-21表达增高(P<0.05)。2.SD大鼠过表达miR-21时PTEN mRNA表达水平未明显变化(P>0.05),但PTEN蛋白水平下降(P<0.01)。与I/R组比较,过表达miR-21使I/R组大鼠miR-21表达升高、PTEN下降、p-AKT水平升高‘(P<0.05),抑制凋亡的Bcl-2/Bax升高、促凋亡因子Caspase-3表达降低(P<0.05)。3.与VC组比较,H/R可使Bcl-2/Bax降低,Caspase-3升高,细胞凋亡率升高(P<0.05);与H/R+INC组比较,miR-21表达抑制进一步降低Bcl-2/Bax水平,促进Caspase-3表达,细胞凋亡率进一步升高(P<0.05);与H/R+MNC组比较,miR-21过表达可以升高Bcl-2/Bax水平、抑制Caspase-3水平,降低细胞凋亡率(P<0.05)。4.与H/R+Control组比较, H/R+TMZ组可以升高Bcl-2/Bax.抑制Caspase-3水平,改善细胞凋亡率(P<0.05);但与H/R+TMZ组比较,抑制miR-21表达则使Bcl-2/Bax降低,Caspase-3升高,细胞凋亡率升高(P<0.05)。结论:1.在I/R早期大鼠心肌缺血区miR-21表达随再灌注时间延长进行性下降,并伴随细胞凋亡进展。2.rAAV9在大鼠心脏可以持久、高效表达,作用安全。3.过表达miR-21可改善心肌细胞凋亡,抑制miR-21表达可促进细胞凋亡。4.miR-21通过PTEN/AKT信号通路调控I/R早期细胞凋亡。5. miR-21/PTEN/AKT部分介导了曲美他嗪的抗凋亡作用。图32幅,表7个,参考文献112篇
【Abstract】 Objective:Cell apoptosis is the important mechanism in ischemia-reperfusion (I/R) injury. Protective effects on cell apoptosis can avoid from ischemia-reperfusion injury. Evidenced support that the abnormal expression of microRNA-21(miR-21) in I/R animal model, we demonstrated in the present study that the relationship between abnormal expression of miR-21and cell apoptosis in early I/R injury and the mechanism. Trimetazidine (TMZ) is a classic anti-ischemia drug, we plan to determin that miR-21mediated the anti-apoptosis effect of TMZ in early I/R injury.Methods:l.In vivo experiment:Forty-eight SD rats were randomly divided into Sham group, I/R2h group, I/R4h group, I/R6h group. There are12rats in each group. Rats in Sham group were deal with open-chest, rats in other groups suffered ligation of anterior descending coronary atrery for45min and reperfusion for2h,4h,6h respectively. To compare the expression level of miR-21and level of cell apoptosis between I/R groups and Sham group, and between in the diffrent I/R time point groups. Bcl-2/Bax and Caspase-3as classic factors to stand for the level of cell apoptosis. Realtime-PCR was used to assess the expression level of miR-21in ischemic and non-ischemic area; Immunohistochemistry and Western-blot were used to determine the expression of Bcl-2, Bax, Caspase-3and Bcl-2/Bax.2.Thirty rats were divided into five groups randomly:We constructed rAAV9-ZsGreen-pre-miR-21successfully with the titer5.0×1012vg/ml.①Control group(n=6):rats were transfected with rAAV9-ZsGreen by coronary injection;②miR-21group (n=6):rats were transfected rAAV9-ZsGreen-pre-miR-21by coronary injection;③Sham group:rats were dealt with open-chest after transfected with rAAV9-ZsGreen;④I/R group (n=6):rats were treated with I/R after transfected with rAAV9-ZsGreen;⑤I/R+miR-21(n=6):rats were dealt with I/R after transfected rAAV9-ZsGreen-pre-miR-21by coronary injection. Realtime-PCR was used to assess the expression level of miR-21; Immunohistochemistry and Western-blot were used to determine the expression of Bcl-2, Bax, Caspase-3, PTEN, p-AKT and Bcl-2/Bax.3.In vitro experiment:H9C2cell were transfected with different concentration of miR inhibitors and mimics(30nM,50nM and100nM respectively) by liposome LipofectamineTM2000, Realtime-PCR was used to assess the expression level of miR-21,we choose the best concentration. H9C2cell was randomly divided into Vehicle Control (NC),inhibitors negative control (INC), mimics NC (MNC), H/R+INC, H/R+MNC, H/R+inhibitors and H/R+mimics.Cell was treated with hypoxia for4h and reoxygenation for3h. Realtime-PCR was used to assess the expression level of miR-21; Immunohistochemistry and Western-blot were used to determine the expression of Bcl-2, Bax, Caspase-3, PTEN, p-AKT and Bcl-2/Bax. Flow cytometry evaluated apoptosis rate.4. H9C2cell was randomly divided into Control, H/R+Control and H/R+TMZ. There is another2groups, H/R+TMZ+INC and H/R+TMZ+inhibitors. Cell incubated with0μM or10μM TMZ for48hours. Realtime-PCR was used to assess the expression level of miR-21; Immunohistochemistry and Western-blot were used to determine the expression of Bcl-2, Bax, Caspase-3, PTEN, p-AKT and Bcl-2/Bax. Flow cytometry evaluated apoptosis rate.Result:1.miR-21was down-regulated in the ischemic area after I/R compared with the Sham group (P<0.05).but miR-21expression in the non-ischemia area was significantly increased compared with the Sham group (P<0.01). MiR-21expression and the level of Bcl-2/Bax was decreased, the level of Caspase-3was increased in the I/R group than the Sham group at2h,4h and6h after I/R (P<0.05).2.The level of PTEN protein decreased when miR-21over-expressed (P<0.01), and the level of PTEN mRNA was not affected (P>0.05). Compared with I/R group, the expression of miR-21, p-AKT, Bcl-2/Bax was up-regulated in miR-21over-expressed rat suffered from H/R, meanwhile the expression of PTEN, Caspase-3was down-regulated (P<0.05).3. Compared with VC, the protein level of Bcl-2/Bax was up-regulated, which of Caspase-3was down-regulated when cells were dealt with H/R, and cell apoptosis was increased (P<0.05). Cell apoptosis were found to be aggravated after inhibition of miR-21(P<0.05), otherwise it was improved after over-expression of miR-21(P<0.05).4. Rats were treated with TMZ before H/R exhibit increases in Bcl-2/Bax, and decreases Caspase-3protein levels compared H/R rats, and cell apoptosis decreased (P<0.05).When the expression of miR-21was down-regulated after transfected inhibitors, Bcl-2/Bax was decreased, the level of Caspase-3and cell apoptosis rate was increased (P<0.05)Conclusion:1.The expression of miR-21was down-regulated and cell apoptosis was increased in ischemic area at the early phase of I/R, and it was aggravated with the prolonged reperfusion period.2. AAV9was an ideal gene vector who can stably and efficiently expressed in rat myocardium without affecting cardiac function.3. Cell apoptosis can be improved by miR-21over-expressed, otherwise it can be exacerbated when miR-21was inhibited.4.miR-21mediated cell apoptosis induced by I/R via modulation of PTEN/AKT pathway.4. The protective effects of TMZ was partly mediated by miR-21.
【Key words】 miR-21; ischemia-reperfusion; cell apoptosis; PTEN; AKT; trimetazidine;