本文選題：Wip1 + 心功能　； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：研究一:Wip1基因缺失對小鼠心功能的影響目的:野生型 p53 誘導(dǎo)的磷酸酶 1(Wildtypep53-induced phosphatase1,Wip1)在很多疾病和生理過程中起著重要作用,但其在心臟中的作用尚未研究報道。本研究探討敲除Wip1基因?qū)π∈笮墓δ艿挠绊懠靶呐K組織中基因和蛋白表達(dá)水平的變化。方法:隨機選取野生型(Wildtype,WT)小鼠和Wip1基因敲除(Wip1 knockout,Wip1-KO)小鼠各10只,分別檢測兩組小鼠的心功能和心臟重量/體重比,應(yīng)用蘇木精伊紅(Hematoxylin and eosin,HE)染色觀察小鼠心肌組織的病理形態(tài),應(yīng)用實時定量反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(Real-time Polymerase Chain Reaction,RT-PCR)檢測心肌組織中 Wip1、心房利鈉肽(Atrial natriuretic peptide,ANP)、腦鈉肽(Brain natriuretic peptide,BNP)、單核細(xì)胞趨化蛋白1(Monocyte chemoattractant protein,MCP-1)和 α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)的基因表達(dá)水平,并應(yīng)用蛋白免疫印跡實驗(Western Blot)檢測凋亡相關(guān)蛋白包括B淋巴細(xì)胞瘤-2(B-cell lymphoma,Bcl-2)、B淋巴細(xì)胞瘤-2相關(guān)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)表達(dá)水平。結(jié)果:與WT小鼠相比,Wip1-KO小鼠心肌組織中Wip1 mRNA表達(dá)明顯降低,左室射血分?jǐn)?shù)和縮短分?jǐn)?shù)降低,左心室收縮末期內(nèi)徑增大。雖然Wip1-KO小鼠的心臟重量與WT小鼠之間沒有差異,但Wip1-KO小鼠體重較WT小鼠減輕,心重/體重比增加。HE染色結(jié)果顯示W(wǎng)ip1-KO小鼠的心肌形態(tài)與WT小鼠之間沒有明顯差異。心肌組織中ANP、BNP、MCP-1和α-SMA基因表達(dá)水平在兩組小鼠之間沒有統(tǒng)計學(xué)差異。Wip1-KO小鼠心肌組織中凋亡相關(guān)蛋白Bax/Bcl-2和c-caspase3表達(dá)水平與WT小鼠之間沒有明顯差異。結(jié)論:敲除Wip1基因可以損害小鼠心功能,但相關(guān)的機制還有待進一步闡明。研究二:敲除Wip1基因加重心肌梗死引起的缺血性損傷目的:既往有很多關(guān)于Wip1在腫瘤和不同類型干細(xì)胞中調(diào)節(jié)作用的報道,但Wip1在心肌梗死中的作用尚未有研究。本研究擬探討Wip1在心肌梗死中的作用以及可能的分子機制。方法:通過永久性結(jié)扎小鼠左前降支冠狀動脈構(gòu)建心肌梗死模型。心肌梗死模型構(gòu)建成功后,持續(xù)觀察小鼠4周,統(tǒng)計小鼠生存率。應(yīng)用小鼠心臟超聲檢測系統(tǒng)評估小鼠心功能,通過測量小鼠心臟重量/體重比和麥胚凝集素(Wheat germ agglutinin,WGA)染色檢測小鼠心臟肥厚情況,心肌組織切片后行HE染色評估小鼠心肌梗死面積,行Masson染色檢測小鼠心肌組織纖維增生情況。通過RT-PCR方法檢測小鼠心肌組織中白細(xì)胞介素-6(Interleukin-6,IL-6)、腫瘤壞死因子-α(Tumor necrosis factor-α,TNF-α)、白細(xì)胞介素-1 β(Interleukin-1 β,IL-1 β)和膠原纖維Collagen I的基因表達(dá)水平,通過Western blot檢測小鼠心肌組織中信號轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活蛋白 3(Signal transducers and activators of transcription 3,stat3)、磷酸化信號轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活蛋白3(Phosphor-stat3,p-stat3)以及雷帕霉素靶蛋白(Mechanistic target of rapamycin,mTOR)底物激酶pS6的表達(dá)水平。結(jié)果:與WT小鼠相比,心肌梗死后Wip1-KO小鼠死亡率較高,心肌肥厚程度較重,心功能降低較明顯。HE染色結(jié)果顯示W(wǎng)ip1-KO小鼠心肌梗死面積較大,Masson染色顯示兩組小鼠的心肌纖維增生在術(shù)后均有增加,但兩組之間沒有統(tǒng)計學(xué)差異,Collagen I的基因表達(dá)水平在兩組小鼠之間也沒有差異。RT-PCR結(jié)果表明Wip1-KO小鼠中IL-6 mRNA表達(dá)較低,而TNF-α mRNA和IL-1 β mRNA的表達(dá)在兩組小鼠之間沒有統(tǒng)計學(xué)差異。雖然心肌梗死后兩組小鼠的stat3磷酸化均有增加,但Wip1-KO小鼠中p-stat3表達(dá)較低,兩組小鼠之間具有統(tǒng)計學(xué)差異。兩組小鼠之間pS6表達(dá)水平?jīng)]有統(tǒng)計學(xué)差異。結(jié)論:敲除Wip1基因可以加重心肌梗死引起的缺血損傷。研究三:α B-crystallin和HspB2在Tsc1基因敲除引起的心肌損傷中的作用目的:既往研究報道αB晶體蛋白(αB-crystallin)在雷帕霉素靶蛋白(Mechanistic target of rapamycin,mTOR)活化誘導(dǎo)的細(xì)胞和動物模型中起著促進腫瘤發(fā)生發(fā)展的作用。具有分子伴侶功能的αB-crystallin和HspB2都是小分子熱休克蛋白,可以輔助維持心臟穩(wěn)態(tài)。本實驗室前期研究發(fā)現(xiàn)敲除Tsc1基因可以引起心臟增大等心肌損傷,而Tsc1基因敲除小鼠中αB-crystallin和HspB2表達(dá)上調(diào)。但α B-crystallin和HspB2在mTOR活化誘導(dǎo)的心肌損傷中有著怎樣的作用并不清楚。方法:本課題共構(gòu)建心臟特異性敲除Tsc1基因小鼠(Tsc1 heart conditional-KO,T1-hKO)、αB-crystallin 和 HspB2 雙基因敲除小鼠(Double knockout,dKO)、Tsc1,α B-crystallin和HspB2三基因敲除小鼠(Triple knockout,tKO)和對照小鼠(Control,CTLs)四種基因敲除小鼠。觀察四種基因型小鼠的生存率,應(yīng)用超聲評估小鼠的心功能,稱量小鼠體重和心臟重量,計算小鼠心臟重量/體重比,通過組織病理染色分析小鼠病理形態(tài)改變,應(yīng)用RT-PCR方法評估小鼠心肌組織中胚胎基因心房利鈉肽(Atrial natriuretic peptide,ANP)、腦鈉肽(Brain natriuretic peptide,BNP)、α-骨骼肌肌動蛋白(α-skeletal actin,α-SKA)和β-肌球重鏈蛋白(β-myosin heavy chain,β-MHC)的表達(dá)水平,應(yīng)用HE染色評估小鼠心肌形態(tài)、WGA染色檢測小鼠心肌細(xì)胞面積、Masson染色觀察小鼠心臟纖維化程度,應(yīng)用電鏡觀察小鼠心肌超微結(jié)構(gòu)改變以及通過Western blot檢測小鼠心肌組織中蛋白表達(dá)水平。結(jié)果:敲除αB-crystallin和HspB2基因可以減少T1-hKO小鼠的死亡率。超聲結(jié)果顯示,T1-hKO小鼠心功能明顯下降,但敲除αB-crystallin和HspB2基因可以改善T1-hKO小鼠心功能。T1-hKO組小鼠心重/體重比增加,心肌細(xì)胞面積增大,tKO組小鼠的心肌細(xì)胞面積較T1-hKO組小鼠減小。組織學(xué)染色分析發(fā)現(xiàn)T1-hKO組小鼠出現(xiàn)心肌細(xì)胞損傷,心臟纖維化增加,而tKO組小鼠心肌細(xì)胞損傷減輕,心臟纖維化明顯緩解。心肌組織細(xì)胞超微結(jié)構(gòu)結(jié)果顯示T1-hKO組小鼠心肌細(xì)胞中線粒體結(jié)構(gòu)明顯異常,體積腫大,線粒體嵴數(shù)量減少、肌纖維變性、肌節(jié)連接異常,而tKO組小鼠中線粒體、肌纖維等結(jié)構(gòu)基本恢復(fù)到正常狀態(tài)。T1-hKO組小鼠心肌組織中抗增殖細(xì)胞核抗原抗體(Proliferating cell nuclear antigen,PCNA)染色陽性部分強度明顯增加,說明細(xì)胞處于旺盛的增殖狀態(tài),而tKO組小鼠中PCNA染色較其減弱,增殖程度減輕。進一步探究可能的分子機制發(fā)現(xiàn),敲除αB-crystallin和HspB2基因可以抑制mTOR信號通路,即α B-crystallin和HspB2基因可能通過mTOR信號通路調(diào)節(jié)心肌損傷。結(jié)論:α B-crystallin和HspB2在mTOR活化誘導(dǎo)的心肌損傷中發(fā)揮著重要作用,α B-crystal 1 in和HspB2可能通過mTOR信號通路調(diào)節(jié)小鼠心功能和心肌細(xì)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R54

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