论文部分内容阅读
目的探讨缺氧条件下线粒体动态学变化及Sirt1在其中的作用。方法 1收集2014年于新桥医院心血管外科行手术治疗的先心病患儿29例,其中非紫绀型先心病14例,紫绀型先心病15例,取术中所切除的右室流出道心肌组织为心肌标本,Western blot检测促线粒体分裂蛋白Drp1与Fis1表达情况。2将H9c2心肌细胞置入缺氧培养箱(94%N2,5%CO2,1%O2)中进行培养,检测缺氧0、2、4、8、12 h后促线粒体分裂蛋白Drp1与Fis1的表达水平。3将线粒体靶向质粒分别同空载质粒、Sirt1过表达质粒和Sirt1干扰质粒共转染H9c2细胞,常氧或缺氧培养箱中培养12 h后,激光共聚焦显微镜下观察线粒体融合、分裂情况;4运用腺病毒Ad-Sirt1和慢病毒Lv-Sh-Sirt1分别在H9c2细胞中过表达或干扰Sirt1后,将细胞置于缺氧培养箱中培养12 h,检测Drp1与Fis1表达水平;5分别转染空载质粒、Sirt1过表达质粒和Sirt1干扰质粒至H9c2细胞后,缺氧培养12 h,LDH试剂盒检测细胞毒性。结果 1同非紫绀组相比,紫绀组心肌中Drp1与Fis1表达显著增高,Drp1相对灰度值:[(0.47±0.11)vs(0.76±0.12)、Fis1相对灰度值(0.53±0.02)vs(0.83±0.03),P<0.05];2细胞水平研究结果显示Drp1与Fis1的蛋白表达随缺氧的时间的延长呈递增趋势;3激光共聚焦结果表明,Sirt1过表达后能够一定程度地缓解缺氧诱导的线粒体分裂(P<0.05),而当Sirt1受到干扰后,缺氧所诱导的线粒体分裂水平明显提高(P<0.05);4在缺氧条件下,过表达Sirt1能够抑制Drp1与Fis1蛋白表达水平(P<0.05),而Sirt1被抑制后,Drp1与Fis1蛋白表达水平增加(对照组,过表达组,低表达组Drp1相对灰度值依次为[(0.47±0.02)vs(0.36±0.02)vs(0.78±0.04);Fis1相对灰度值依次为(0.64±0.02)vs(0.42±0.02)vs(0.80±0.04),P<0.05];5Sirt1过表达能够降低缺氧刺激后H9c2心肌细胞的死亡率。结论 Sirt1可能通过调控促线粒体分裂蛋白Drp1与Fis1表达水平促进心肌细胞在缺氧条件下的适应。
Objective To investigate the dynamic change of mitochondria under hypoxia and its role in Sirt1. Twenty-nine children with congenital heart disease underwent surgery in Xinqiao Hospital in 2014. Among them, 14 cases were non-cyanotic congenital heart disease and 15 cases were cyanotic congenital heart disease. The right ventricular outflow tract myocardium Tissues were obtained from myocardial samples. Western blot was used to detect the expressions of mitochondrial protein Drp1 and Fis1. 2 H9c2 cardiomyocytes were cultured in hypoxia incubator (94% N2, 5% CO2, 1% O2) to detect the expression of mitochondrial protein Drp1 and Fis1 after 0,2,4,8 and 12 h hypoxia The expression level. The mitochondria-targeting plasmids were co-transfected into H9c2 cells with empty plasmid, Sirt1 overexpression plasmid and Sirt1 interference plasmid respectively. After cultured in normoxia or hypoxia incubator for 12 h, the mitochondrial fusion and mitosis were observed under confocal laser scanning microscope ; 4 After overexpression or interference with Sirt1 in adenovirus Ad-Sirt1 and lentivirus Lv-Sh-Sirt1 respectively in H9c2 cells, the cells were cultured in hypoxia incubator for 12 h, and the expression of Drp1 and Fis1 were detected; 5 After transfected with empty plasmid, Sirt1 overexpression plasmid and Sirt1 interference plasmid into H9c2 cells, the cells were cultured in hypoxia for 12 h, and the cytotoxicity was detected by LDH kit. Results 1 Compared with non-cyanotic group, the expressions of Drp1 and Fis1 in cyanotic group were significantly higher than those in non-cyanotic group (P <0.05) (0.83 ± 0.03), P <0.05]. The results of 2 cell level showed that the expression of Drp1 and Fis1 protein increased with the prolongation of hypoxia time. 3 Laser confocal results showed that Sirt1 could be alleviated to a certain degree after overexpression Hypoxia-induced mitochondrial cleavage (P <0.05), but when Sirt1 was disturbed, hypoxia-induced mitochondrial cleavage was significantly increased (P <0.05); 4 Overexpression of Sirt1 in hypoxia inhibited the expression of Drp1 and Fis1 (P <0.05). However, the expression of Drp1 and Fis1 increased after Sirt1 was inhibited (compared with control group, overexpression group and low expression group, the relative gray values of Drp1 were [(0.47 ± 0.02) vs 0.36 ± 0.02) vs 0.78 ± 0.04; Fis1 relative gray values were (0.64 ± 0.02) vs (0.42 ± 0.02) vs (0.80 ± 0.04), P <0.05, respectively; 5Sirt1 overexpression decreased H9c2 myocardium Cell death.Conclusion Sirt1 may promote the adaptation of cardiomyocytes under hypoxic conditions by regulating the expression of mitochondrial cleavage proteins Drp1 and Fis1