胞外HSP60诱导心肌表达炎症因子的作用与机制研究
发布时间：2018-10-26 07:45
【摘要】：热休克蛋白60(heat shock protein 60,HSP60)在生理状态下位于细胞内,对维持细胞的正常功能不可或缺,并对细胞应激反应具有保护作用。在细胞受到缺血等刺激因素作用下,HSP60可“主动”分泌出胞或者由坏死细胞释放到细胞外。有研究表明,胞外HSP60能够激活免疫细胞胞膜中的toll样受体(toll-like recepors, TLRs)-4而引起炎症反应。心肌细胞中也有TLRs分布,但是胞外HSP60是否可能激活心肌细胞TLRs而直接引起心肌炎症反应尚不明确。本课题在消化分离的成年大鼠心肌细胞和培养的大鼠H9C2心肌细胞模型,研究了外源性H9C2刺激炎症因子表达的作用,并探讨了其作用机制。 研究目的: 明确胞外HSP60直接刺激心肌炎症因子表达的作用,并探讨心肌细胞TLRs受体在其中的介导作用及其信号通路。在心肌缺血等病理生理状态下,HSP60异常出胞,可能成为HSP60引起无菌性炎症反应的内源性因子,本课题研究有助于为认识缺血心肌发生无菌性炎症反应的机制提供线索。 研究方法: 1、通过结扎成年雄性SD大鼠冠状动脉左前降支(left anterior descending coronary artery ,LAD),建立心脏缺血模型。取假手术或者冠脉结扎所致心肌缺血的大鼠心脏,通过在Langendorff系统上进行离体心脏左心室逆行灌流,胶原酶消化分离获得心肌细胞。给予HSP60刺激,通过实时荧光定量PCR法检测TNF、IL-6、TLR2/TLR4 mRNA的表达量变化;ELISA法检测细胞培液上清中的炎症因子TNF、IL-6的含量;比色法检测细胞培液上清中的LDH活性以分析心肌细胞损伤情况。 2、按常规培养H9C2大鼠心肌细胞系,给予HSP60刺激,通过实时荧光定量PCR法检测TNF-а、IL-6、TLR2、TLR4mRNA的表达量变化;ELISA法检测细胞培液上清中的炎症因子TNF-а、IL-6的含量;western blot法检测TLR2和TLR4受体蛋白表达量。应用TLR2/TLR4特异性抗体或者转染siRNA来阻断TLR2/TLR4,观察如何影响HSP60的致炎效应。并且,通过细胞免疫荧光法检测HSP60是否引起P65发生核转位,从而判断HSP60是否激活了致炎转录因子NF-kB。 结果 1、以外源性HSP60孵育酶解分离获得的成年大鼠心肌细胞,炎性细胞因子TNF-a和IL-6的表达以及释放均明显上调。当给予HSP60 1ug/ml孵育3h后,假手术组心肌细胞TNF-а和IL-6mRNA表达量分别上调1.76±0.13和1.89±0.09倍;LAD结扎组心肌细胞TNF-а和IL-6mRNA表达量分别上调3.07±0.11和3.35±0.18倍。给予HSP60 5ug/ml孵育3h后,假手术组心肌细胞TNF-а和IL-6mRNA表达量分别上调2.04±0.12和2.46±0.13倍;LAD结扎组TNF-а和IL-6mRNA表达量分别上调3.97±0.13和4.28±0.17倍;HSP60引起的TNF-а和IL-6mRNA表达上调均有统计学意义。并且,HSP60引起TNF-а和IL-6释放量显著增加。HSP60 1ug/ml孵育3h,假手术组TNF-а和IL-6释放量分别是84.5±6.8和72.7±3.7 pg/ml,显著高于未给药组(63.1±5.2和53.6±4.5pg/ml,P0.05);LAD结扎组TNF-а和IL-6释放量分别是98.6±7.9和90.6±7.4pg/ml,显著高未给药组(90.8±6.5和78.6±4.4pg/ml,P0.05)。当给予高剂量HSP60(5ug/ml),假手术组TNF-а和IL-6释放量进一步升高,分别是112.7±6.2和95.7±4.7pg/ml, LAD结扎组TNF-а和IL-6释放量也进一步升高,分别是137.6±8.0和132.4±6.8pg/ml。 2、以外源性HSP60孵育培养的H9C2心肌细胞,细胞因子TNF-а和IL-6的表达以及释放也明显上调。HSP60 1ug/ml处理组炎症因子TNF-а和IL-6mRNA表达量分别上调1.42±0.06和1.69±0.11倍;HSP60 5ug/ml处理组TNF-а和IL-6mRNA表达量分别上调1.87±0.13和2.25±0.12倍,与未给药组相比均有统计学差异(P0.05)。TLR2和TLR4mRNA表达显著上调,有统计学差异。Western blot结果显示TLR4蛋白表达量在HSP60处理后3h显著升高,12h达到最好,24h仍然高。此外,对细胞培液上清中LDH活性检测的结果显示,LDH的释放量在HSP60处理后24h内呈递增趋势,有统计学差异(P0.05)。 3、用TLR4特异性抗体阻断TLR4受体后,H9C2细胞培液上清中炎症因子TNF和IL-6释放量分别为25.8±4.9和33.9±4.5pg/ml,两者均显著低于HSP60处理组的释放量(54.1±4.7和60.7±4.1pg/ml,P0.05)。该结果提示TLR4参与介导了HSP60的致炎效应。 4、在培养的H9C2心肌细胞,细胞免疫荧光法观察到炎性转录因子NF-кB的亚单位P65在外源性HSP60处理后从细胞浆转位进入细胞核(NF-кB是TLRs受体下游的关键信号分子)。表明HSP60激活了NF-кB。 结论 1、胞外的HSP60能够直接诱导心肌细胞产生炎症因子TNF-a和IL-6。 2、HSP60引起TLR2和TLR4受体表达上调。 3、TLR4抗体能够有效抑制HSP60的致炎效应,提示TLR4参与介导了HSP60的致炎效应。TLR4可能作为受体与HSP60相结合,从而直接介导HSP60的作用;也可能通过其表达上调而间接介导HSP60的作用。 4、HSP60引起心肌细胞P65发生核转位,表明HSP60激活了炎性转录因子NF-кB,提示TLR4/NF-кB通路参与介导了HSP60的致炎效应。
[Abstract]:Heat shock protein 60 (HSP60) is located in cells under physiological condition, which is indispensable for maintaining normal function of cells and has protective effect on cell stress response. HSP60 may "Active" secreted out of the cell or released by necrotic cells to the outside of the cell. Studies have shown that extracellular HSP60 is capable of activating toll-like receptors (TLRs)-4 in immune cell membranes to induce inflammatory responses. There are also TLRs distribution in cardiomyocytes, but it is not clear whether extracellular HSP60 may activate myocardial cells TLRs. In this study, the effects of exogenous H9C2 on the expression of inflammatory factors were studied in isolated adult rat cardiomyocytes and cultured rat H9C2 cardiomyocytes, and its mechanism of action was discussed. Study Objective: To clarify the effect of extracellular HSP60 on the expression of myocardial inflammatory factors and to explore the role of TLRs receptors in myocardial cells. In the pathophysiological state of myocardial ischemia and the like, HSP60 is abnormal out of cell and can be an endogenous factor causing aseptic inflammation reaction by HSP60, and the research of this project can help to understand the machine of aseptic inflammation reaction in ischemic myocardium. To provide a clue. Methods: 1. Left anterior descending coronary artery (LA) was ligated by ligation of left anterior descending artery (LA) of adult male SD rats. D) establishing a heart ischemia model, taking a sham operation or coronary artery ligation for myocardial ischemia in a rat heart, performing retrograde perfusion on the left ventricle of the body from the left ventricle of the body on a Langerdorff system, collagen, The expression of TNF, IL-6, TLR2/ TLR4 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). The expression of TNF, IL-6, TLR2/ TLR4 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). In order to analyze the damage of myocardial cells, H9C2 rat cardiomyocytes were cultured according to routine culture, HSP60 was stimulated, TNF-, IL-6, TLR2 and TLR4 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). The content of sub-TNF-gamma and IL-6; western blot method for detection of TL R2 and TLR4 receptor protein expression. TLR2/ TLR4 specific antibody or siRNA transfected siRNA to block TLR2/ TLR4, observe How to influence the inflammatory effect of HSP60, and whether HSP60 caused P65 nuclear translocation was detected by cell immunofluorescence to determine whether HSP60 activation Results 1. The expression of NF-kB in adult rats was isolated by exogenous HSP60 incubation. The expression and release of F-a and IL-6 were increased significantly. After incubation for 3 h with HSP60 1ug/ ml, the expression levels of TNF-CoV and IL-6mRNA in the myocardium of sham operation group were increased by 1. 76, 0. 13 and 1. 89, respectively. After incubation for 3 h with HSP60 5ug/ ml, the expression levels of TNF-CoV and IL-6mRNA in the myocardium of sham operation group were up-regulated by 2.04, 0.012 and 2.46, respectively. The expression levels of TNF-CoV and IL-6mRNA in the sham-operated group were up-regulated by 3.97, 0.013 and 4.28, respectively. and the expression of IL-6mRNA was up-regulated. The release of TNF-and IL-6 was significantly increased in the sham operation group. The release of TNF-and IL-6 in sham operation group was 84.5, 6.8 and 72. 7 pg/ ml, respectively, which was significantly higher than that in the untreated group (63. 1, 5. 2 and 53. 6, 4. 5 pg/ ml, P0.05). The release amounts of TNF-and IL-6 in the sham-operated group were 98. 6, 7. 9 and 90. 6, 7. 4pg/ ml, respectively.. 8, 6. 5 and The release of TNF-PAF and IL-6 in sham operation group increased further with high dose of HSP60 (5ug/ ml), and the release of TNF-and IL-6 in sham operation group increased further, which was 137 respectively. The cultured H9C2 cardiomyocytes were incubated with exogenous HSP60, and the cells were cultured with exogenous HSP60. The expression and release of TNF-and IL-6 in HSP60 1ug/ ml treatment group were up-regulated respectively. The expression levels of TNF-and IL-6mRNA in HSP60 ug/ ml treatment group were increased by 1. 42, 0. 06 and 1. 69 to 0. 11 times, respectively. The expression levels of TNF-and IL-6mRNA in HSP60 5ug/ ml treatment group were increased by 1. 87% 0. 13 and 2.25 g 0. 12 times, respectively (P0.05). TLR The expression of TLR4 protein in HSP60 was detected by Western blot. In addition, the results of LDH activity detection in the supernatant of cell culture showed that the release of LDH was after treatment with HSP60. After the TLR4 receptor was blocked by TLR4 specific antibody, the release of TNF and IL-6 in the supernatant of H9C2 cell culture was 25. 8, 4.9 and 33. 9, 4.5pg/ ml, respectively. Both of them were significantly lower than that of HSP60 treatment group (54. 05). 1卤4.7鍜，
本文编号：2295037
[Abstract]:Heat shock protein 60 (HSP60) is located in cells under physiological condition, which is indispensable for maintaining normal function of cells and has protective effect on cell stress response. HSP60 may "Active" secreted out of the cell or released by necrotic cells to the outside of the cell. Studies have shown that extracellular HSP60 is capable of activating toll-like receptors (TLRs)-4 in immune cell membranes to induce inflammatory responses. There are also TLRs distribution in cardiomyocytes, but it is not clear whether extracellular HSP60 may activate myocardial cells TLRs. In this study, the effects of exogenous H9C2 on the expression of inflammatory factors were studied in isolated adult rat cardiomyocytes and cultured rat H9C2 cardiomyocytes, and its mechanism of action was discussed. Study Objective: To clarify the effect of extracellular HSP60 on the expression of myocardial inflammatory factors and to explore the role of TLRs receptors in myocardial cells. In the pathophysiological state of myocardial ischemia and the like, HSP60 is abnormal out of cell and can be an endogenous factor causing aseptic inflammation reaction by HSP60, and the research of this project can help to understand the machine of aseptic inflammation reaction in ischemic myocardium. To provide a clue. Methods: 1. Left anterior descending coronary artery (LA) was ligated by ligation of left anterior descending artery (LA) of adult male SD rats. D) establishing a heart ischemia model, taking a sham operation or coronary artery ligation for myocardial ischemia in a rat heart, performing retrograde perfusion on the left ventricle of the body from the left ventricle of the body on a Langerdorff system, collagen, The expression of TNF, IL-6, TLR2/ TLR4 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). The expression of TNF, IL-6, TLR2/ TLR4 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). In order to analyze the damage of myocardial cells, H9C2 rat cardiomyocytes were cultured according to routine culture, HSP60 was stimulated, TNF-, IL-6, TLR2 and TLR4 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). The content of sub-TNF-gamma and IL-6; western blot method for detection of TL R2 and TLR4 receptor protein expression. TLR2/ TLR4 specific antibody or siRNA transfected siRNA to block TLR2/ TLR4, observe How to influence the inflammatory effect of HSP60, and whether HSP60 caused P65 nuclear translocation was detected by cell immunofluorescence to determine whether HSP60 activation Results 1. The expression of NF-kB in adult rats was isolated by exogenous HSP60 incubation. The expression and release of F-a and IL-6 were increased significantly. After incubation for 3 h with HSP60 1ug/ ml, the expression levels of TNF-CoV and IL-6mRNA in the myocardium of sham operation group were increased by 1. 76, 0. 13 and 1. 89, respectively. After incubation for 3 h with HSP60 5ug/ ml, the expression levels of TNF-CoV and IL-6mRNA in the myocardium of sham operation group were up-regulated by 2.04, 0.012 and 2.46, respectively. The expression levels of TNF-CoV and IL-6mRNA in the sham-operated group were up-regulated by 3.97, 0.013 and 4.28, respectively. and the expression of IL-6mRNA was up-regulated. The release of TNF-and IL-6 was significantly increased in the sham operation group. The release of TNF-and IL-6 in sham operation group was 84.5, 6.8 and 72. 7 pg/ ml, respectively, which was significantly higher than that in the untreated group (63. 1, 5. 2 and 53. 6, 4. 5 pg/ ml, P0.05). The release amounts of TNF-and IL-6 in the sham-operated group were 98. 6, 7. 9 and 90. 6, 7. 4pg/ ml, respectively.. 8, 6. 5 and The release of TNF-PAF and IL-6 in sham operation group increased further with high dose of HSP60 (5ug/ ml), and the release of TNF-and IL-6 in sham operation group increased further, which was 137 respectively. The cultured H9C2 cardiomyocytes were incubated with exogenous HSP60, and the cells were cultured with exogenous HSP60. The expression and release of TNF-and IL-6 in HSP60 1ug/ ml treatment group were up-regulated respectively. The expression levels of TNF-and IL-6mRNA in HSP60 ug/ ml treatment group were increased by 1. 42, 0. 06 and 1. 69 to 0. 11 times, respectively. The expression levels of TNF-and IL-6mRNA in HSP60 5ug/ ml treatment group were increased by 1. 87% 0. 13 and 2.25 g 0. 12 times, respectively (P0.05). TLR The expression of TLR4 protein in HSP60 was detected by Western blot. In addition, the results of LDH activity detection in the supernatant of cell culture showed that the release of LDH was after treatment with HSP60. After the TLR4 receptor was blocked by TLR4 specific antibody, the release of TNF and IL-6 in the supernatant of H9C2 cell culture was 25. 8, 4.9 and 33. 9, 4.5pg/ ml, respectively. Both of them were significantly lower than that of HSP60 treatment group (54. 05). 1卤4.7鍜，
本文编号：2295037
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