PHB对高糖环境下的心肌细胞中活性氧及细胞凋亡的影响及机制研究

发布时间：2018-03-29 11:41

本文选题：PHB　切入点：心肌细胞　出处：《中国免疫学杂志》2017年12期

【摘要】：目的:探讨抗增殖蛋白(PHB)对高糖诱导的H9C2心肌细胞凋亡的影响以及机制研究。方法:选取p CDNA3-PHB、p CDNA3-NC重组质粒转染和高糖干预心肌细胞,实验分为4组:正常对照组(Control组)、高糖刺激组(HG组)、高糖+空载体组(HG+p CDNA3-NC组)、高糖加p CDNA3-PHB重组质粒组(HG+p CDNA3-PHB);Western blot检测细胞转染后各组细胞中PHB蛋白、B细胞淋巴瘤/白血病-2相关X蛋白(Bax)/B细胞淋巴瘤/白血病-2(Bcl-2)、剪切的半胱氨酸的天冬氨酸蛋白水解酶-3(c-caspase3)、蛋白激酶B(AKt)、磷酸化蛋白激酶B(p-AKt)蛋白的表达量,二氢乙啶(DHE)染色法检测高糖环境下心肌细胞内的活性氧(ROS)水平,实时荧光定量PCR(qRT-PCR)检测细胞中TNF-αmRNA和IL-6 mRNA含量的测定,流式细胞仪检测细胞的凋亡率。结果:p CDNA3-PHB重组质粒可使PHB表达量增加;与Control组相比,HG组、HG+p CDNA3-NC组、HG+p CDNA3-PHB组细胞凋亡率显著增高(P0.05);与HG组相比,HG+p CDNA3-NC组细胞凋亡率变化不显著(P0.05);HG+p CDNA3-PHB组细胞凋亡率明显降低(P0.05)。与Control组相比,HG组、HG+p CDNA3-NC组、HG+p CDNA3-PHB组细胞内ROS的水平、TNF-αmRNA、IL-6 mRNA、Bax/Bcl-2的相对表达量、c-caspase3(P0.05)蛋白相对表达量显著增高;与HG组相比,HG+p CDNA3-NC组细胞内ROS的水平(P0.05)、TNF-αmRNA(P0.05)、IL-6 mRNA(P0.05)、Bax/Bcl-2的相对表达量(P0.05)、c-caspase3蛋白相对表达量(P0.05)无明显差异;但p CDNA3-PHB组细胞中ROS的水平(P0.05)、TNF-αmRNA(P0.05)、IL-6 mRNA(P0.05)、Bax/Bcl-2的相对表达量(P0.05)、c-caspase3(P0.05)蛋白相对表达量明显降低。经统计分析显示,各组细胞中AKt蛋白的相对表达量无显著差异(P0.05)。经比较后,HG组、HG+p CDNA3-NC组、HG+p CDNA3-PHB组心肌细胞中p-AKt蛋白的相对表达量显著低于Control组(P0.05);HG+p CDNA3-NC组心肌细胞中p-AKt蛋白的相对表达量与HG组间无显著差异(P0.05);但HG+p CDNA3-PHB组心肌细胞中p-AKt蛋白的相对表达量明显高于HG组(P0.05)。结论:PHB过表达可抑制高糖环境下H9C2心肌细胞的凋亡和炎症反应,主要是通过调控PI3K/Akt信号通路、ROS、Bax/Bcl-2、c-caspase3蛋白的水平,为糖尿病心肌病的治疗提供新的思路与方法。
[Abstract]:Objective: to investigate the effect and mechanism of anti-proliferating protein (CDNA3-NC) on apoptosis of H9C2 cardiomyocytes induced by high glucose. Methods: pCDNA3-PHBP CDNA3-NC recombinant plasmid was used to transfect cardiomyocytes. The experiment was divided into four groups: control group of normal control group, HG group of high glucose stimulation group, HP CDNA3-NC group of high sugar empty vector group, and high sugar plus p CDNA3-PHB recombinant plasmid group. The PHB protein of B cell lymphoma was detected by Western blot after transfection. / Leukemia 2 associated X protein B cell lymphoma / Leukemia 2 Bcl-2N, cut cysteine aspartic protein hydrolase -3caspase3, protein kinase BKB, phosphorylated protein kinase (PPRK) BPK-AKT, The levels of reactive oxygen species (Ros) in cardiac myocytes were detected by dihydroethidime diethidime (DHEH) staining, and the contents of TNF- 伪 mRNA and IL-6 mRNA in cardiac myocytes were detected by real-time fluorescence quantitation of PCR- 伪 mRNA and IL-6 mRNA. The apoptotic rate of the cells was detected by flow cytometry. Results the PHB expression was increased by the recombinant plasmid of 1: P CDNA3-PHB. Compared with Control group, the apoptosis rate in HG p CDNA3-NC group was significantly higher than that in HG p CDNA3-PHB group, and the apoptosis rate in HG p CDNA3-NC group was not significantly different from that in HG group. The apoptosis rate in HG p CDNA3-PHB group was significantly lower than that in HG p CDNA3-PHB group. Compared with Control group, HG p CDNA3-PHB group was significantly lower than that in Control group. In p CDNA3-PHB group, the relative expression of TNF- 伪 mRNA-IL-6 mRNA-IL-6 mRNA-Bax / Bcl-2 was significantly higher than that of c-caspase3 (P0.05) protein. Compared with HG group, there was no significant difference in the level of ROS in HGP CDNA3-NC group (P0.05TNF- 伪 mRNA-P0.05TNF- 伪 mRNA-P0.05TNF- 伪 mRNA-P0.05 Bax-Bax / Bcl-2 relative expression level and P0.05- caspase3 protein relative expression level (P0.05). However, the relative expression of ROS in the cells of p CDNA3-PHB group was significantly lower than that in the control group. Statistical analysis showed that the relative expression level of ROS was significantly lower than that of P0.05- 伪 mRNA-TNF- 伪 mRNA-TNF- 伪 mRNA-TNF- 伪 mRNA-TNF- 伪 mRNA-TNF- 伪 -mRNA-TNF- 伪 mRNA. There was no significant difference in the relative expression of AKt protein among the three groups. The relative expression of p-AKt protein in the cardiomyocytes of HG p CDNA3-NC group was significantly lower than that of Control group (P 0.05 HG p CDNA3-NC group), and the relative surface of p-AKt protein in HG p CDNA3-NC group was significantly lower than that in Control group (P 0.05 HG p CDNA3-NC group). There was no significant difference between HG group and HG group (P 0.05), but the relative expression of p-AKt protein in HG p CDNA3-PHB group was significantly higher than that in HG group (P 0 05). Conclusion the overexpression of p-AKt B can inhibit the apoptosis and inflammatory response of H9C2 cardiomyocytes in high glucose environment. It is mainly by regulating the PI3K/Akt signaling pathway that the Bax-bcl-2ccaspase3 protein level is regulated, which provides a new idea and method for the treatment of diabetic cardiomyopathy.
【作者单位】：西安外事学院医学院;
【基金】：陕西省科技厅社会发展公关项目(2016SF-220)资助
【分类号】：R587.2

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