Wip1、αB-crystallin和HspB2在心肌损伤中的作用研究

发布时间：2018-06-04 04:11

本文选题：Wip1 + 心功能　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究一:Wip1基因缺失对小鼠心功能的影响目的:野生型 p53 诱导的磷酸酶 1(Wildtypep53-induced phosphatase1,Wip1)在很多疾病和生理过程中起着重要作用,但其在心脏中的作用尚未研究报道。本研究探讨敲除Wip1基因对小鼠心功能的影响及心脏组织中基因和蛋白表达水平的变化。方法:随机选取野生型(Wildtype,WT)小鼠和Wip1基因敲除(Wip1 knockout,Wip1-KO)小鼠各10只,分别检测两组小鼠的心功能和心脏重量/体重比,应用苏木精伊红(Hematoxylin and eosin,HE)染色观察小鼠心肌组织的病理形态,应用实时定量反转录聚合酶链式反应(Real-time Polymerase Chain Reaction,RT-PCR)检测心肌组织中 Wip1、心房利钠肽(Atrial natriuretic peptide,ANP)、脑钠肽(Brain natriuretic peptide,BNP)、单核细胞趋化蛋白1(Monocyte chemoattractant protein,MCP-1)和 α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的基因表达水平,并应用蛋白免疫印迹实验(Western Blot)检测凋亡相关蛋白包括B淋巴细胞瘤-2(B-cell lymphoma,Bcl-2)、B淋巴细胞瘤-2相关X蛋白(Bcl-2-associated X protein,Bax)和活化半胱氨酸天冬氨酸蛋白酶-3(Cleaved caspase-3,c-caspase3)表达水平。结果:与WT小鼠相比,Wip1-KO小鼠心肌组织中Wip1 mRNA表达明显降低,左室射血分数和缩短分数降低,左心室收缩末期内径增大。虽然Wip1-KO小鼠的心脏重量与WT小鼠之间没有差异,但Wip1-KO小鼠体重较WT小鼠减轻,心重/体重比增加。HE染色结果显示Wip1-KO小鼠的心肌形态与WT小鼠之间没有明显差异。心肌组织中ANP、BNP、MCP-1和α-SMA基因表达水平在两组小鼠之间没有统计学差异。Wip1-KO小鼠心肌组织中凋亡相关蛋白Bax/Bcl-2和c-caspase3表达水平与WT小鼠之间没有明显差异。结论:敲除Wip1基因可以损害小鼠心功能,但相关的机制还有待进一步阐明。研究二:敲除Wip1基因加重心肌梗死引起的缺血性损伤目的:既往有很多关于Wip1在肿瘤和不同类型干细胞中调节作用的报道,但Wip1在心肌梗死中的作用尚未有研究。本研究拟探讨Wip1在心肌梗死中的作用以及可能的分子机制。方法:通过永久性结扎小鼠左前降支冠状动脉构建心肌梗死模型。心肌梗死模型构建成功后,持续观察小鼠4周,统计小鼠生存率。应用小鼠心脏超声检测系统评估小鼠心功能,通过测量小鼠心脏重量/体重比和麦胚凝集素(Wheat germ agglutinin,WGA)染色检测小鼠心脏肥厚情况,心肌组织切片后行HE染色评估小鼠心肌梗死面积,行Masson染色检测小鼠心肌组织纤维增生情况。通过RT-PCR方法检测小鼠心肌组织中白细胞介素-6(Interleukin-6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素-1 β(Interleukin-1 β,IL-1 β)和胶原纤维Collagen I的基因表达水平,通过Western blot检测小鼠心肌组织中信号转导及转录激活蛋白 3(Signal transducers and activators of transcription 3,stat3)、磷酸化信号转导及转录激活蛋白3(Phosphor-stat3,p-stat3)以及雷帕霉素靶蛋白(Mechanistic target of rapamycin,mTOR)底物激酶pS6的表达水平。结果:与WT小鼠相比,心肌梗死后Wip1-KO小鼠死亡率较高,心肌肥厚程度较重,心功能降低较明显。HE染色结果显示Wip1-KO小鼠心肌梗死面积较大,Masson染色显示两组小鼠的心肌纤维增生在术后均有增加,但两组之间没有统计学差异,Collagen I的基因表达水平在两组小鼠之间也没有差异。RT-PCR结果表明Wip1-KO小鼠中IL-6 mRNA表达较低,而TNF-α mRNA和IL-1 β mRNA的表达在两组小鼠之间没有统计学差异。虽然心肌梗死后两组小鼠的stat3磷酸化均有增加,但Wip1-KO小鼠中p-stat3表达较低,两组小鼠之间具有统计学差异。两组小鼠之间pS6表达水平没有统计学差异。结论:敲除Wip1基因可以加重心肌梗死引起的缺血损伤。研究三:α B-crystallin和HspB2在Tsc1基因敲除引起的心肌损伤中的作用目的:既往研究报道αB晶体蛋白(αB-crystallin)在雷帕霉素靶蛋白(Mechanistic target of rapamycin,mTOR)活化诱导的细胞和动物模型中起着促进肿瘤发生发展的作用。具有分子伴侣功能的αB-crystallin和HspB2都是小分子热休克蛋白,可以辅助维持心脏稳态。本实验室前期研究发现敲除Tsc1基因可以引起心脏增大等心肌损伤,而Tsc1基因敲除小鼠中αB-crystallin和HspB2表达上调。但α B-crystallin和HspB2在mTOR活化诱导的心肌损伤中有着怎样的作用并不清楚。方法:本课题共构建心脏特异性敲除Tsc1基因小鼠(Tsc1 heart conditional-KO,T1-hKO)、αB-crystallin 和 HspB2 双基因敲除小鼠(Double knockout,dKO)、Tsc1,α B-crystallin和HspB2三基因敲除小鼠(Triple knockout,tKO)和对照小鼠(Control,CTLs)四种基因敲除小鼠。观察四种基因型小鼠的生存率,应用超声评估小鼠的心功能,称量小鼠体重和心脏重量,计算小鼠心脏重量/体重比,通过组织病理染色分析小鼠病理形态改变,应用RT-PCR方法评估小鼠心肌组织中胚胎基因心房利钠肽(Atrial natriuretic peptide,ANP)、脑钠肽(Brain natriuretic peptide,BNP)、α-骨骼肌肌动蛋白(α-skeletal actin,α-SKA)和β-肌球重链蛋白(β-myosin heavy chain,β-MHC)的表达水平,应用HE染色评估小鼠心肌形态、WGA染色检测小鼠心肌细胞面积、Masson染色观察小鼠心脏纤维化程度,应用电镜观察小鼠心肌超微结构改变以及通过Western blot检测小鼠心肌组织中蛋白表达水平。结果:敲除αB-crystallin和HspB2基因可以减少T1-hKO小鼠的死亡率。超声结果显示,T1-hKO小鼠心功能明显下降,但敲除αB-crystallin和HspB2基因可以改善T1-hKO小鼠心功能。T1-hKO组小鼠心重/体重比增加,心肌细胞面积增大,tKO组小鼠的心肌细胞面积较T1-hKO组小鼠减小。组织学染色分析发现T1-hKO组小鼠出现心肌细胞损伤,心脏纤维化增加,而tKO组小鼠心肌细胞损伤减轻,心脏纤维化明显缓解。心肌组织细胞超微结构结果显示T1-hKO组小鼠心肌细胞中线粒体结构明显异常,体积肿大,线粒体嵴数量减少、肌纤维变性、肌节连接异常,而tKO组小鼠中线粒体、肌纤维等结构基本恢复到正常状态。T1-hKO组小鼠心肌组织中抗增殖细胞核抗原抗体(Proliferating cell nuclear antigen,PCNA)染色阳性部分强度明显增加,说明细胞处于旺盛的增殖状态,而tKO组小鼠中PCNA染色较其减弱,增殖程度减轻。进一步探究可能的分子机制发现,敲除αB-crystallin和HspB2基因可以抑制mTOR信号通路,即α B-crystallin和HspB2基因可能通过mTOR信号通路调节心肌损伤。结论:α B-crystallin和HspB2在mTOR活化诱导的心肌损伤中发挥着重要作用,α B-crystal 1 in和HspB2可能通过mTOR信号通路调节小鼠心功能和心肌细胞存活。
[Abstract]:Study 1: the effect of Wip1 gene deletion on the cardiac function of mice Objective: the wild type p53 induced phosphatase 1 (Wildtypep53-induced phosphatase1, Wip1) plays an important role in many diseases and physiological processes, but its role in the heart has not been reported. This study explored the effect of the knockout of the Wip1 gene on the cardiac function of mice and the heart. Methods: the changes in the gene and protein expression level in the tissue. Methods: 10 mice were randomly selected from Wildtype (WT) mice and Wip1 gene knockout (Wip1 knockout, Wip1-KO) mice. The cardiac function and heart weight / weight ratio of the two groups were detected respectively. The pathology of the myocardium of mice was observed by hematoxylin eosin (Hematoxylin and eosin, HE) staining. Morphologic, Real-time Polymerase Chain Reaction (RT-PCR) was used to detect Wip1 in cardiac tissue, atrial natriuretic peptide (Atrial natriuretic peptide, ANP), brain natriuretic peptide (Brain natriuretic), monocyte chemoattractant protein 1 and alpha smooth muscle muscle. The gene expression level of active protein (alpha -smooth muscle actin, alpha -SMA), and the application of protein immunoblotting test (Western Blot) to detect apoptosis related proteins including B lymphocytoma -2 (B-cell lymphoma, Bcl-2), B lymphocyte tumor and activated cysteine aspartic proteinase Spase-3, c-caspase3) expression level. Results: compared with WT mice, the expression of Wip1 mRNA in the myocardium of Wip1-KO mice decreased significantly, the left ventricular ejection fraction and shortening fraction decreased and the left ventricular end systolic diameter increased. Although the cardiac weight of the Wip1-KO mice was not different from that of the WT mice, the weight of the Wip1-KO mice was less than the WT mice, and the heart weight / heart weight was reduced. The weight ratio increased.HE staining results showed no significant difference between Wip1-KO mice and WT mice. There was no statistical difference between the ANP, BNP, MCP-1 and alpha -SMA gene expression levels in the two groups of mice. The expression level of Bax/ Bcl-2 and c-caspase3 of apoptosis related proteins in the myocardium of.Wip1-KO mice was not between the mice and WT mice. There are obvious differences. Conclusion: knockout Wip1 gene can damage the cardiac function of mice, but the related mechanism remains to be further clarified. Study two: the aim of the Wip1 gene to aggravate the ischemic injury caused by myocardial infarction: there are many reports about the regulation of Wip1 in the tumor and different types of stem cells, but the Wip1 is in the myocardial infarction. The purpose of this study is to explore the role and possible molecular mechanism of Wip1 in myocardial infarction. Methods: a model of myocardial infarction was constructed by permanent ligation of the left anterior descending coronary artery in mice. After the successful construction of the model of myocardial infarction, the mice were continuously observed for 4 weeks, and the survival rate of mice was counted. The cardiac hypertrophy of mice was measured by measuring the heart weight / weight ratio of mice and Wheat germ agglutinin (WGA). The myocardial infarction area was assessed by HE staining and Masson staining was used to detect the fibrous hyperplasia in the myocardium of mice by Masson staining. The myocardium of mice was detected by RT-PCR method. The gene expression level of interleukin -6 (Interleukin-6, IL-6), tumor necrosis factor - alpha (Tumor necrosis factor- alpha, TNF- a), interleukin -1 beta (Interleukin-1 beta, IL-1 beta) and collagen fiber Collagen I were detected, and the signal transduction and transcription activator protein 3 in the murine myocardium was detected. Activators of transcription 3, STAT3), phosphorylated signal transduction and transcriptional activator 3 (Phosphor-stat3, p-STAT3), and the expression level of rapamycin target protein (Mechanistic target of rapamycin, mTOR) substrate kinase pS6. The results of.HE staining showed that the infarct area of Wip1-KO mice was larger. Masson staining showed that the proliferation of myocardial fibers in the two groups increased after the operation, but there was no statistical difference between the two groups. There was no difference between the Collagen I gene expression level between the two groups of mice and the.RT-PCR results showed IL-6 mR in the Wip1-KO mice. The expression of NA was low, while the expression of TNF- alpha mRNA and IL-1 beta mRNA had no statistical difference between the two groups. Although the STAT3 phosphorylation of the two groups of mice increased after myocardial infarction, the p-STAT3 expression in the Wip1-KO mice was lower and the two groups had statistical differences. There was no statistical difference in the pS6 expression level between the two groups. Wip1 gene knockout can aggravate ischemic injury caused by myocardial infarction. Study three: the role of alpha B-crystallin and HspB2 in myocardial injury induced by Tsc1 gene knockout: Previous studies reported that the alpha B crystal protein (alpha B-crystallin) is activated by the activated cell and animal models of the rapamycin target protein (Mechanistic target of rapamycin, mTOR). It plays a role in promoting the development of tumor. Alpha B-crystallin and HspB2 with molecular chaperone function are small molecular heat shock proteins and can assist in maintaining cardiac homeostasis. Earlier studies in this laboratory found that knockout of Tsc1 gene could cause cardiac damage, such as cardiac enlargement, and the expression of alpha B-crystallin and HspB2 in Tsc1 knockout mice However, the role of alpha B-crystallin and HspB2 in mTOR activation induced myocardial injury is not clear. Methods: the subject was to construct a total of cardiac specific knockout Tsc1 gene mice (Tsc1 heart conditional-KO, T1-hKO), alpha B-crystallin and HspB2 double gene knockout mice. Three gene knockout mice (Triple knockout, tKO) and control mice (Control, CTLs) were knocked out of four gene knockout mice. The survival rate of four genotypes of mice was observed, the cardiac function of mice was evaluated by ultrasound, the weight and weight of the mice were weighed and the heart weight / weight ratio of the mice was calculated, and the pathological changes of mice were analyzed by histopathological staining. RT-PCR method was used to evaluate the expression of Atrial natriuretic peptide (ANP), brain natriuretic peptide (Brain natriuretic peptide, BNP), alpha skeletal muscle actin (alpha -skeletal actin, alpha -SKA) and beta muscle mass chain protein. Morphology, WGA staining was used to detect the area of murine cardiac myocytes, Masson staining was used to observe the degree of cardiac fibrosis in mice. The ultrastructural changes in the myocardium of mice were observed by electron microscopy and the protein expression level in the myocardium of mice was detected by Western blot. Results: the death rate of T1-hKO mice was reduced by knockout of the alpha B-crystallin and HspB2 genes. The results showed that the cardiac function of T1-hKO mice decreased significantly, but the knockout of the alpha B-crystallin and HspB2 genes could improve the heart weight / weight ratio of T1-hKO mice in.T1-hKO group, increase the area of cardiac myocytes, and decrease the area of myocardial cells in the tKO group than that of the T1-hKO group. The histological staining analysis found that the loss of cardiac myocytes in the T1-hKO mice was found in the T1-hKO mice. The injury and cardiac fibrosis were increased, and the myocardial cells in the tKO group were relieved and the cardiac fibrosis was relieved. The ultrastructural results of the myocardial cells showed that the mitochondria structure in the T1-hKO group was obviously abnormal, the volume was enlarged, the number of mitochondrial crista decreased, the muscle fiber degeneration, and the muscle joint abnormal, and the mitochondria and muscle in the tKO group mice. The structure of fiber and other structures was basically restored to the normal state of the.T1-hKO group. The positive part of the anti proliferating cell nuclear antigen antibody (Proliferating cell nuclear antigen, PCNA) increased significantly, indicating that the cell was in a vigorous proliferation state, while the PCNA staining in the tKO group was weaker and the proliferation degree was reduced. Further exploration could be made. It is found that the knockout of the alpha B-crystallin and HspB2 genes can inhibit the mTOR signaling pathway, that is, the alpha B-crystallin and HspB2 genes may regulate myocardial damage through the mTOR signaling pathway. Conclusion: alpha B-crystallin and HspB2 play an important role in the myocardial injury induced by mTOR activation, and alpha B-crystal 1 in and HspB2 may pass through Signal pathway regulates cardiac function and cardiomyocyte survival in mice.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54

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