【摘要】：熱休克蛋白60(heat shock protein 60,HSP60)在生理狀態(tài)下位于細(xì)胞內(nèi),對(duì)維持細(xì)胞的正常功能不可或缺,并對(duì)細(xì)胞應(yīng)激反應(yīng)具有保護(hù)作用。在細(xì)胞受到缺血等刺激因素作用下,HSP60可“主動(dòng)”分泌出胞或者由壞死細(xì)胞釋放到細(xì)胞外。有研究表明,胞外HSP60能夠激活免疫細(xì)胞胞膜中的toll樣受體(toll-like recepors, TLRs)-4而引起炎癥反應(yīng)。心肌細(xì)胞中也有TLRs分布,但是胞外HSP60是否可能激活心肌細(xì)胞TLRs而直接引起心肌炎癥反應(yīng)尚不明確。本課題在消化分離的成年大鼠心肌細(xì)胞和培養(yǎng)的大鼠H9C2心肌細(xì)胞模型,研究了外源性H9C2刺激炎癥因子表達(dá)的作用,并探討了其作用機(jī)制。 研究目的: 明確胞外HSP60直接刺激心肌炎癥因子表達(dá)的作用,并探討心肌細(xì)胞TLRs受體在其中的介導(dǎo)作用及其信號(hào)通路。在心肌缺血等病理生理狀態(tài)下,HSP60異常出胞,可能成為HSP60引起無(wú)菌性炎癥反應(yīng)的內(nèi)源性因子,本課題研究有助于為認(rèn)識(shí)缺血心肌發(fā)生無(wú)菌性炎癥反應(yīng)的機(jī)制提供線索。 研究方法: 1、通過(guò)結(jié)扎成年雄性SD大鼠冠狀動(dòng)脈左前降支(left anterior descending coronary artery ,LAD),建立心臟缺血模型。取假手術(shù)或者冠脈結(jié)扎所致心肌缺血的大鼠心臟,通過(guò)在Langendorff系統(tǒng)上進(jìn)行離體心臟左心室逆行灌流,膠原酶消化分離獲得心肌細(xì)胞。給予HSP60刺激,通過(guò)實(shí)時(shí)熒光定量PCR法檢測(cè)TNF、IL-6、TLR2/TLR4 mRNA的表達(dá)量變化;ELISA法檢測(cè)細(xì)胞培液上清中的炎癥因子TNF、IL-6的含量;比色法檢測(cè)細(xì)胞培液上清中的LDH活性以分析心肌細(xì)胞損傷情況。 2、按常規(guī)培養(yǎng)H9C2大鼠心肌細(xì)胞系,給予HSP60刺激,通過(guò)實(shí)時(shí)熒光定量PCR法檢測(cè)TNF-а、IL-6、TLR2、TLR4mRNA的表達(dá)量變化;ELISA法檢測(cè)細(xì)胞培液上清中的炎癥因子TNF-а、IL-6的含量;western blot法檢測(cè)TLR2和TLR4受體蛋白表達(dá)量。應(yīng)用TLR2/TLR4特異性抗體或者轉(zhuǎn)染siRNA來(lái)阻斷TLR2/TLR4,觀察如何影響HSP60的致炎效應(yīng)。并且,通過(guò)細(xì)胞免疫熒光法檢測(cè)HSP60是否引起P65發(fā)生核轉(zhuǎn)位,從而判斷HSP60是否激活了致炎轉(zhuǎn)錄因子NF-kB。 結(jié)果 1、以外源性HSP60孵育酶解分離獲得的成年大鼠心肌細(xì)胞,炎性細(xì)胞因子TNF-a和IL-6的表達(dá)以及釋放均明顯上調(diào)。當(dāng)給予HSP60 1ug/ml孵育3h后,假手術(shù)組心肌細(xì)胞TNF-а和IL-6mRNA表達(dá)量分別上調(diào)1.76±0.13和1.89±0.09倍;LAD結(jié)扎組心肌細(xì)胞TNF-а和IL-6mRNA表達(dá)量分別上調(diào)3.07±0.11和3.35±0.18倍。給予HSP60 5ug/ml孵育3h后,假手術(shù)組心肌細(xì)胞TNF-а和IL-6mRNA表達(dá)量分別上調(diào)2.04±0.12和2.46±0.13倍;LAD結(jié)扎組TNF-а和IL-6mRNA表達(dá)量分別上調(diào)3.97±0.13和4.28±0.17倍;HSP60引起的TNF-а和IL-6mRNA表達(dá)上調(diào)均有統(tǒng)計(jì)學(xué)意義。并且,HSP60引起TNF-а和IL-6釋放量顯著增加。HSP60 1ug/ml孵育3h,假手術(shù)組TNF-а和IL-6釋放量分別是84.5±6.8和72.7±3.7 pg/ml,顯著高于未給藥組(63.1±5.2和53.6±4.5pg/ml,P0.05);LAD結(jié)扎組TNF-а和IL-6釋放量分別是98.6±7.9和90.6±7.4pg/ml,顯著高未給藥組(90.8±6.5和78.6±4.4pg/ml,P0.05)。當(dāng)給予高劑量HSP60(5ug/ml),假手術(shù)組TNF-а和IL-6釋放量進(jìn)一步升高,分別是112.7±6.2和95.7±4.7pg/ml, LAD結(jié)扎組TNF-а和IL-6釋放量也進(jìn)一步升高,分別是137.6±8.0和132.4±6.8pg/ml。 2、以外源性HSP60孵育培養(yǎng)的H9C2心肌細(xì)胞,細(xì)胞因子TNF-а和IL-6的表達(dá)以及釋放也明顯上調(diào)。HSP60 1ug/ml處理組炎癥因子TNF-а和IL-6mRNA表達(dá)量分別上調(diào)1.42±0.06和1.69±0.11倍;HSP60 5ug/ml處理組TNF-а和IL-6mRNA表達(dá)量分別上調(diào)1.87±0.13和2.25±0.12倍,與未給藥組相比均有統(tǒng)計(jì)學(xué)差異(P0.05)。TLR2和TLR4mRNA表達(dá)顯著上調(diào),有統(tǒng)計(jì)學(xué)差異。Western blot結(jié)果顯示TLR4蛋白表達(dá)量在HSP60處理后3h顯著升高,12h達(dá)到最好,24h仍然高。此外,對(duì)細(xì)胞培液上清中LDH活性檢測(cè)的結(jié)果顯示,LDH的釋放量在HSP60處理后24h內(nèi)呈遞增趨勢(shì),有統(tǒng)計(jì)學(xué)差異(P0.05)。 3、用TLR4特異性抗體阻斷TLR4受體后,H9C2細(xì)胞培液上清中炎癥因子TNF和IL-6釋放量分別為25.8±4.9和33.9±4.5pg/ml,兩者均顯著低于HSP60處理組的釋放量(54.1±4.7和60.7±4.1pg/ml,P0.05)。該結(jié)果提示TLR4參與介導(dǎo)了HSP60的致炎效應(yīng)。 4、在培養(yǎng)的H9C2心肌細(xì)胞,細(xì)胞免疫熒光法觀察到炎性轉(zhuǎn)錄因子NF-кB的亞單位P65在外源性HSP60處理后從細(xì)胞漿轉(zhuǎn)位進(jìn)入細(xì)胞核(NF-кB是TLRs受體下游的關(guān)鍵信號(hào)分子)。表明HSP60激活了NF-кB。 結(jié)論 1、胞外的HSP60能夠直接誘導(dǎo)心肌細(xì)胞產(chǎn)生炎癥因子TNF-a和IL-6。 2、HSP60引起TLR2和TLR4受體表達(dá)上調(diào)。 3、TLR4抗體能夠有效抑制HSP60的致炎效應(yīng),提示TLR4參與介導(dǎo)了HSP60的致炎效應(yīng)。TLR4可能作為受體與HSP60相結(jié)合,從而直接介導(dǎo)HSP60的作用;也可能通過(guò)其表達(dá)上調(diào)而間接介導(dǎo)HSP60的作用。 4、HSP60引起心肌細(xì)胞P65發(fā)生核轉(zhuǎn)位,表明HSP60激活了炎性轉(zhuǎn)錄因子NF-кB,提示TLR4/NF-кB通路參與介導(dǎo)了HSP60的致炎效應(yīng)。
[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鹵4.7鍜，

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