Mechanism of protective effects of tumor necrosis factor receptor associated protein 1 on hypoxic cardiomyocytes of rats
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摘要: 目的 探讨肿瘤坏死因子受体相关蛋白1(TRAP1)对大鼠缺氧心肌细胞保护作用的机制。 方法 取1~3 d龄SD大鼠乳鼠,分离培养心肌细胞,用于以下实验。(1)取细胞,按随机数字表法(分组方法下同)分为TRAP1组和对照组,提取细胞总蛋白。TRAP1组细胞总蛋白加入小鼠抗大鼠TRAP1单克隆一抗,对照组细胞总蛋白加入小鼠来源的同型IgG,免疫共沉淀法和蛋白质谱分析检测TRAP1可能作用的蛋白。(2)取细胞,分为常氧空白对照组、常氧+TRAP1干扰对照组、常氧+TRAP1干扰组、常氧+TRAP1过表达对照组、常氧+TRAP1过表达组,每组1孔。常氧空白对照组细胞常规培养,后4组细胞分别加入TRAP1 RNA干扰空病毒载体、TRAP1 RNA干扰腺病毒载体、TRAP1过表达空病毒载体、TRAP1过表达腺病毒载体。另取细胞,分为常氧空白对照组、缺氧空白对照组、缺氧+TRAP1干扰对照组、缺氧+TRAP1干扰组、缺氧+TRAP1过表达对照组、缺氧+TRAP1过表达组,每组1孔。各缺氧组细胞同前对应的常氧组细胞处理后,缺氧6 h。实时荧光定量RT-PCR检测各组细胞中细胞色素C氧化酶亚基Ⅱ(COXⅡ)mRNA表达。本实验重复3次。(3)取细胞,分为常氧空白对照组、缺氧空白对照组、缺氧+TRAP1过表达对照组、缺氧+TRAP1过表达组、缺氧+TRAP1过表达+COXⅡ干扰对照组、缺氧+TRAP1过表达+COXⅡ干扰组,每组3孔。前4组细胞的处理同(2),后2组细胞分别加入COXⅡ RNA干扰空病毒载体、COXⅡ RNA干扰腺病毒载体转染后,再分别加入TRAP1过表达腺病毒载体。细胞计数试剂盒8及酶标仪检测细胞增殖活性,碘化丙啶和Hoechst 33342染色检测细胞死亡率。另取细胞,分为常氧空白对照组、缺氧空白对照组、缺氧+TRAP1干扰对照组、缺氧+TRAP1干扰组、缺氧+TRAP1干扰+COXⅡ过表达对照组、缺氧+TRAP1干扰+COXⅡ过表达组,每组3孔,前4组细胞的处理同(2),后2组细胞均加入TRAP1 RNA干扰腺病毒载体转染后,再分别加入COXⅡ过表达空病毒载体、COXⅡ过表达腺病毒载体,同前检测细胞增殖活性和死亡率。本实验重复3次。对数据行单因素方差分析、LSD检验。 结果 (1)TRAP1组细胞有TRAP1表达,对照组细胞未见TRAP1表达。TRAP1可能作用的3个蛋白是角蛋白、COX Ⅱ和预测分子量为13×103的未知蛋白。(2)与常氧空白对照组比较,常氧+TRAP1干扰对照组、常氧+TRAP1过表达对照组细胞COXⅡ mRNA表达量无明显变化(
P 值均大于0.05),常氧+TRAP1干扰组细胞COXⅡ mRNA表达量明显降低(P <0.01),常氧+TRAP1过表达组细胞COXⅡ mRNA表达量明显升高(P <0.01)。缺氧空白对照组细胞COXⅡ mRNA表达量较常氧空白对照组明显降低(P <0.01)。与缺氧空白对照组比较,缺氧+TRAP1干扰对照组、缺氧+TRAP1过表达对照组细胞COXⅡ mRNA表达量无明显变化(P 值均大于0.05),缺氧+TRAP1干扰组细胞COXⅡ mRNA表达量明显下降(P <0.01),缺氧+TRAP1过表达组细胞COXⅡ mRNA表达量明显升高(P <0.01)。(3)常氧空白对照组、缺氧空白对照组、缺氧+TRAP1过表达对照组、缺氧+TRAP1过表达组、缺氧+TRAP1过表达+COXⅡ干扰对照组、缺氧+TRAP1过表达+COXⅡ干扰组细胞增殖活性分别为0.498±0.022、0.303±0.018、0.313±0.032、0.456±0.031、0.448±0.034、0.335±0.026,细胞死亡率分别为(4.7±1.5)%、(24.7±3.1)%、(26.0±2.7)%、(13.3±2.5)%、(12.7±2.1)%、(21.0±1.7)%。与常氧空白对照组比较,缺氧空白对照组细胞增殖活性降低、细胞死亡率增加(P 值均小于0.01)。与缺氧空白对照组比较,缺氧+TRAP1过表达对照组细胞增殖活性、细胞死亡率均无明显变化(P 值均大于0.05),缺氧+TRAP1过表达组细胞增殖活性升高、细胞死亡率降低(P 值均小于0.01)。与缺氧+TRAP1过表达组比较,缺氧+TRAP1过表达+COXⅡ干扰对照组细胞增殖活性、细胞死亡率均无明显变化(P 值均大于0.05),缺氧+TRAP1过表达+COXⅡ干扰组细胞增殖活性降低、细胞死亡率增加(P 值均小于0.01)。(4)常氧空白对照组、缺氧空白对照组、缺氧+TRAP1干扰对照组、缺氧+TRAP1干扰组、缺氧+TRAP1干扰+COXⅡ过表达对照组、缺氧+TRAP1干扰+COX Ⅱ过表达组细胞增殖活性分别为0.444±0.025、0.275±0.016、0.283±0.021、0.150±0.009、0.135±0.011、0.237±0.017,细胞死亡率分别为(3.7±0.6)%、(21.0±2.7)%、(20.3±3.1)%、(31.7±2.5)%、(33.3±3.2)%、(19.3±1.5)%。与缺氧空白对照组比较,缺氧+TRAP1干扰对照组细胞增殖活性、细胞死亡率均无明显变化(P 值均大于0.05)。与缺氧空白对照组和缺氧+TRAP1干扰对照组比较,缺氧+TRAP1干扰组细胞增殖活性降低、细胞死亡率增加(P 值均小于0.01)。与缺氧+TRAP1干扰组比较,缺氧+TRAP1干扰+COXⅡ过表达对照组细胞增殖活性、细胞死亡率均无明显变化(P 值均大于0.05),缺氧+TRAP1干扰+COXⅡ过表达组细胞增殖活性升高、细胞死亡率降低(P 值均小于0.01)。 结论 TRAP1能够正向调节COXⅡ mRNA表达, COXⅡ是TRAP1保护缺氧心肌细胞的下游效应分子。-
关键词:
- 肌细胞,心脏 /
- 缺氧 /
- 细胞增殖 /
- 细胞死亡 /
- 肿瘤坏死因子受体相关蛋白1 /
- 细胞色素c氧化酶亚基Ⅱ
Abstract: Objective To investigate the mechanism of protective effects of tumor necrosis factor receptor associated protein 1 (TRAP1) on hypoxic cardiomyocytes of rats. Methods Primary cultured cardiomyocytes were obtained from neonatal Sprague-Dawley rats (aged 1 to 3 days) and then used in the following experiments. (1) Cells were divided into group TRAP1 and control group according to the random number table (the same grouping method below), and then the total protein of cells was extracted. Total protein of cells in group TRAP1 was added with mouse anti-rat TRAP1 monoclonal antibody, while that in control group was added with the same type of IgG from mouse. Co-immunoprecipitation and protein mass spectrography analysis were used to determine the possible proteins interacted with TRAP1. (2) Cells were divided into normoxia blank control group (NBC), normoxia+ TRAP1 interference control group (NTIC), normoxia+ TRAP1 interference group (NTI), normoxia+ TRAP1 over-expression control group (NTOC), and normoxia+ TRAP1 over-expression group (NTO), with 1 well in each group. Cells in group NBC were routinely cultured, while cells in the latter four groups were respectively added with TRAP1 RNA interference empty virus vector, TRAP1 RNA interference adenovirus vector, TRAP1 over-expression empty virus vector, and TRAP1 over-expression adenovirus vector. Another batch of cells were divided into group NBC, hypoxic blank control group (HBC), hypoxic+ TRAP1 interference control group (HTIC), hypoxic+ TRAP1 interference group (HTI), hypoxic+ TRAP1 over-expression control group (HTOC), and hypoxic+ TRAP1 over-expression group (HTO), with 1 well in each group. Cells in hypoxic groups were under hypoxic condition for 6 hours after being treated as those in the corresponding normoxia groups, respectively. The mRNA expression of cytochrome c oxidase subunit Ⅱ (COXⅡ) of cells in each group was detected by real time fluorescent quantitive reverse transcription polymerase chain reaction. Experiments were repeated for three times. (3) Cells were divided into group NBC, group HBC, group HTOC, group HTO, hypoxic+ TRAP1 over-expression+ COXⅡinterference control group (HTOCIC), and hypoxic+ TRAP1 over-expression+ COXⅡinterference group (HTOCI), with 3 wells in each group. Cells in the previous 4 groups were treated as those in experiment (2). Cells in group HTOCIC and HTOCI were respectively transfected with COXⅡ RNA interference empty virus vector and COXⅡ RNA interference adenovirus vector, and then both added with TRAP1 over-expression adenovirus vector. The proliferation activity of cells was determined by cell counting kit 8 and microplate reader, and the ratio of death cells was measured by propidium lodide and Hoechst 33342 staining. Another batch of cells were divided into group NBC, group HBC, group HTIC, group HTI, hypoxic+ TRAP1 interference+ COXⅡover-expression control group (HTICOC), and hypoxic+ TRAP1 interference+ COXⅡ over-expression group (HTICO), with 3 wells in each group. Cells in the previous 4 groups were treated as those in experiment (2). Cells in group HTICOC and HTICO were both transfected with TRAP1 RNA interference adenovirus vector, and then respectively added with COXⅡ over-expression empty virus vector and COXⅡ over-expression adenovirus vector. The proliferation activity of cells and the ratio of death cells were detected as before. Experiments were repeated for three times. Data were processed with one-way analysis of variance and LSD test. Results (1) The expression of TRAP1 was found in cells of group TRAP1, while that was not found in cells of control group. The possible proteins interacted with TRAP1 were keratin, COXⅡ, and an unknown protein with predicted molecular weight 13×103. (2) Compared with that in group NBC, the mRNA expression of COXⅡof cells had no significant change in group NTIC and group NTOC (withP values above 0.05), but significantly decreased in group NTI (P <0.01), and significantly increased in group NTO (P <0.01). Compared with that in group NBC, the mRNA expression of COXⅡof cells in group HBC was significantly decreased (P <0.01). Compared with that in group HBC, the mRNA expression of COXⅡof cells had no significant change in group HTIC and group HTOC (withP values above 0.05), but significantly decreased in group HTI (P <0.01), and significantly increased in group HTO (P <0.01). (3) The proliferation activity of cells in group NBC, group HBC, group HTOC, group HTO, group HTOCIC, and group HTOCI was respectively 0.498±0.022, 0.303±0.018, 0.313±0.032, 0.456±0.031, 0.448±0.034, and 0.335±0.026, and the ratios of death cells in above groups were respectively (4.7±1.5)%, (24.7±3.1)%, (26.0±2.7)%, (13.3±2.5)%, (12.7±2.1)%, and (21.0±1.7)%. Compared with those in group NBC, the proliferation activity of cells in HBC was decreased, while the ratio of death cells was increased (withP values below 0.01). Compared with those in group HBC, the proliferation activity of cells and the ratio of death cells in group HTOC had no significant change (withP values above 0.05), while the proliferation activity of cells was increased and the ratio of death cells was decreased in group HTO (withP values below 0.01). Compared with those in group HTO, the proliferation activity of cells and the ratio of death cells in group HTOCIC had no significant change (withP values above 0.05), while the proliferation activity of cells was decreased and the ratio of death cells was increased in group HTOCI (withP values below 0.01). (4) The proliferation activity of cells in group NBC, group HBC, group HTIC, group HTI, group HTICOC, and group HTICO was respectively 0.444±0.025, 0.275±0.016, 0.283±0.021, 0.150±0.009, 0.135±0.011, and 0.237±0.017, and the ratios of death cells in above groups were respectively (3.7±0.6)%, (21.0±2.7)%, (20.3±3.1)%, (31.7±2.5)%, (33.3±3.2)%, and (19.3±1.5)%. Compared with those in group HBC, the proliferation activity of cells and the ratio of death cells in group HTIC had no significant change (withP values above 0.05). Compared with those in group HBC and group HTIC, the proliferation activity of cells was decreased and the ratio of death cells was significantly increased in group HTI (withP values below 0.01). Compared with those in group HTI, the proliferation activity of cells and the ratio of death cells in group HTICOC had no significant change (withP values above 0.05), while the proliferation activity of cells was increased and the ratio of death cells was significantly decreased in group HTICO (withP values below 0.01). Conclusions TRAP1 can up-regulate the expression of COXⅡ mRNA, and COXⅡ is one of the downstream effector molecules that TRAP1 mediates its protective effects on hypoxic cardiomyocytes.
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