本文選題：缺血性心肌損傷　切入點：細胞凋亡　出處：《新疆醫(yī)科大學》2017年博士論文　論文類型：學位論文

【摘要】：目的:本研究旨在探尋小鼠急性心肌梗死(AMI)后心室重塑早期蛋白質組學的變化,從蛋白質組層面研究心肌梗死后參與心室重塑的分子事件,為心肌梗死后早期心肌保護提供新的思路:1)采用相對和絕對定量的等量異位標簽(iTRAQ)技術,高通量、高效率地鑒定及篩選小鼠AMI后差異蛋白的表達;2)在動物模型水平,采用分子生物學手段進行候選靶蛋白的表達驗證;3)在細胞模型水平,應用基因轉染技術進行靶蛋白的功能驗證,探討靶蛋白的分子生物學作用。方法:1)將雄性實驗小鼠隨機分為4組,分別為假手術-3天組(Sham-3d)、假手術-7天組(Sham-7d)、心肌梗死-3天組(MI-3d)以及心肌梗死-7天組(MI-7d)組。采用結扎小鼠左冠狀動脈的方法建立AMI模型。建模后通過超聲心動圖、血流動力學以及病理組織學檢測方法評估小鼠AMI后心功能及心室重塑情況。提取蛋白質,采用iTRAQ技術進行AMI后3天和7天差異蛋白質的鑒定及篩選,并通過生物信息學分析,對差異表達蛋白的生物學功能進行初步分析;2)在動物組織水平,采用病理組織學檢測,評價AMI后早期心肌纖維化導致心室重塑的情況,并進一步采用qRT-PCR及Western Blot方法對小鼠心肌組織進行靶蛋白的表達驗證,選擇需要進一步功能驗證的靶蛋白;3)在細胞水平,建立缺氧/復氧心肌細胞損傷模型,觀察維生素D結合蛋白(VDBP)在心肌細胞損傷0h,6h,12h,24h,48h以及72h的表達變化。進一步構建質粒瞬時轉染H9C2心肌細胞以過表達VDBP,并采用MTS法、Tunel法及Annexin V/PI法檢測細胞凋亡與壞死,采用qRT-PCR及Western Blot方法檢測VDBP相關因子的mRNA轉錄水平及蛋白表達水平變化,探討VDBP在心肌損傷早期的分子作用。結果:1)成功建立小鼠AMI模型,MI-3d組、MI-7d組心肌梗死面積分別為45.29±7.52%、44.83±7.36%,MI-3d組與MI-7d組左心室重量/脛骨長度均較Sham組增高(P0.001);MI-7d組肺臟重量/脛骨長度較Sham-7d組增高(P0.001)。超聲心動圖檢測顯示,與Sham-3d組相比,MI-3d組左心室內徑及舒張期外徑顯著增大(P0.01),左心室前壁及后壁厚度變薄(P0.001),左室短軸縮短率顯著下降(P0.001);與Sham-7d組相比,MI-7d組左心室內徑及舒張期外徑顯著增大,左心室前壁及后壁厚度變薄,左室短軸縮短率顯著下降(P0.001)。血流動力學測定顯示,與Sham-3d組相比,MI-3d組收縮壓、平均主動脈壓、左心室壓及左心室壓力變化速率下降(P0.05);與Sham-7d組相比,MI-7d組收縮壓、左心室舒張末壓及左心室壓力變化速率下降下降(P0.01)。HE染色顯示,MI-3d組可見大量心肌細胞壞死,可見炎性細胞浸潤;MI-7組可見梗死區(qū)出現(xiàn)肉芽組織增生,心肌纖維化表現(xiàn)。采用i TRAQ蛋白質組學技術總共鑒定了2540個蛋白,篩選了AMI后表達顯著變化的776個差異蛋白。通過對差異蛋白的聚類分析、GO富集及KEGG通路分析,發(fā)現(xiàn)wnt、內質網應激(ERS)、維生素D結合蛋白(VDBP)以及肌動蛋白(actin)相關信號通路可能參與AMI后心室重塑的分子生物學過程,其中VDBP可能參與其余3條信號通路的分子生物過程,可能在AMI后心室重塑分子機制中發(fā)揮重要作用;2)采用Masson染色發(fā)現(xiàn)AMI后心肌膠原含量增加,進一步行免疫組化染色檢測I型和III型膠原含量,發(fā)現(xiàn)兩種類型的膠原在AMI后均增高(P0.01)。通過qRT-PCR檢測AMI后心肌損傷因子,與Sham組相比,MI組LDH、cTNT、BNP的mRNA轉錄水平上調(P0.05)。對靶蛋白信號通路中的關鍵因子進行qRT-PCR檢測,發(fā)現(xiàn)在AMI后3天、7天wnt、ERS、VDBP以及actin相關信號通路活化。采用Western blot檢測MI組建模后心梗區(qū)和Sham組小鼠左心室組織,結果可見VDBP,維生素D受體(VDR)和凝溶膠蛋白(GSN)的蛋白表達水平較假手術組明顯上調(P0.001),證實VDBP在AMI后出現(xiàn)蛋白表達的差異性變化;3)成功構建缺氧/復氧H9C2心肌細胞損傷模型,發(fā)現(xiàn)VDBP在缺氧/復氧處理后6h、12h的H9C2中表達上調(P0.001)。構建過表達VDBP質粒及對照質粒,并采用脂質體介導的方法瞬時轉染H9C2,采用qRT-PCR及Western Blot方法檢測VDBP的表達顯示,過表達VDBP轉染H9C2后,VDBP的mRNA轉錄水平及蛋白表達水平明顯上調(P0.001)。過表達VDBP組經缺氧/復氧6h處理后,細胞凋亡和壞死增加(P0.001)。過表達VDBP處理組的VDR的轉錄水平及蛋白表達水平明顯下調(P0.01),GSN,Caspase3的轉錄水平及蛋白表達水平上調(P0.001)。結論:1)本研究采用iTRAQ蛋白質組學技術高通量鑒定差異蛋白并篩選,通過分子生物學技術驗證,發(fā)現(xiàn)wnt、ERS、VDBP以及actin相關信號通路參與AMI后心室重塑的分子生物學過程;2)采用過表達VDBP轉染H9C2心肌細胞的方法,在缺氧/復氧心肌細胞損傷模型中進行功能驗證,證明VDBP加重心肌細胞損傷的作用,并發(fā)現(xiàn)過表達VDBP后抑制VDR、上調GSN活性的分子現(xiàn)象;3)在缺氧/復氧心肌細胞損傷模型中,過表達VDBP后caspase 3的蛋白表達水平上調,證明VDBP可能通過caspase 3介導的細胞凋亡參與AMI后早期心肌細胞損傷的病理生理機制,為靶向干預AMI后心室重塑提供了新的思路。
[Abstract]:Objective: the purpose of this study is to explore the mouse acute myocardial infarction (AMI) on ventricular remodeling after early proteomics changes from the proteome level study of molecular events after myocardial infarction in the early ventricular remodeling, myocardial protection and provide a new way of thinking after myocardial infarction: 1) using isobaric tags for relative and absolute quantitation (iTRAQ) technology, high throughput, high efficiency of the differential expression of protein identification and screening of mice after AMI; 2) in animal model level, to confirm the expression of candidate target proteins by molecular biology; 3) in the cell model, functional verification of target proteins using gene transfection technique, to investigate the molecular biological function of target protein. Methods: 1) male mice were randomly divided into 4 groups, sham operation group were -3 days (Sham-3d), sham operation group (Sham-7d) -7 days -3 days, myocardial infarction group (MI-3d) and myocardial infarction group (MI-7d group) -7 days Methods by ligation of the left coronary artery of mice to establish AMI model. After modeling by echocardiography, hemodynamics and pathological evaluation of detection methods in AMI mice after cardiac function and ventricular remodeling. The extraction of protein was AMI after 3 days and 7 days difference of protein identification and screening by iTRAQ technology, and through the analysis of bioinformatics the biological function of protein expression, preliminary analysis of the difference; 2) in animal tissues, using pathological detection, evaluation of AMI early after myocardial fibrosis leads to ventricular remodeling, and the expression of Western and qRT-PCR by one step Blot method for target protein on myocardial tissue of mice to select target protein for further verification, functional verification 3); at the cellular level, the establishment of reoxygenation myocardial cell injury model of hypoxia / observation of vitamin D binding protein (VDBP) in myocardial injury of 0h cells, 6h, 12h 24h, 48h and 72h, changes of expression. Further constructed plasmids were transiently transfected into H9C2 cells the expression of VDBP in myocardium, and using the method of MTS, apoptosis and necrosis of Tunel method and Annexin V/PI assay, the expression of mRNA mRNA and protein by qRT-PCR and Western Blot VDBP method for the detection of related factors, to explore the VDBP in the early the molecular mechanism of myocardial injury. Results: 1) successfully established a rat model of AMI, MI-3d group, MI-7d group of myocardial infarction area were 45.29 + 7.52%, 44.83 + 7.36%, MI-3d group and MI-7d group, left ventricular weight / tibia length were higher than those in group Sham (P0.001); group MI-7d lung weight / tibia length compared with the Sham-7d group increased (P0.001). Echocardiography showed that compared with the Sham-3d group, MI-3d group, left ventricular diameter and diastolic diameter increased significantly (P0.01), left ventricular anterior wall and posterior wall thickness (P0.001), left ventricular fractional shortening decreased significantly (P0.001); compared with Sham-7d group, MI-7d group, left ventricular diameter and diastolic diameter increased significantly, left ventricular anterior wall and posterior wall thickness, left ventricular fractional shortening decreased significantly (P0.001). Hemodynamic measurements showed that compared with the Sham-3d group, MI-3d group, systolic blood pressure, mean arterial pressure, left changes of ventricular pressure and left ventricular pressure drop rate (P0.05); compared with Sham-7d group, MI-7d group, systolic blood pressure, left ventricular end diastolic pressure and left ventricular pressure changing rate of decline (P0.01).HE staining showed that MI-3d group showed a lot of myocardial cell necrosis, inflammatory cells infiltration; MI-7 group showed granulation appeared in infarction area tissue, myocardial fibrosis. Using I TRAQ proteomics technology in identification of 2540 proteins, 776 differentially expressed proteins were significantly changed after AMI. By clustering the differential protein analysis, pathway enrichment analysis and KEGG GO, found w NT, endoplasmic reticulum stress (ERS), vitamin D binding protein (VDBP) and actin (actin) signaling pathway may be involved in the molecular biology of ventricular remodeling after AMI process, VDBP may be involved in the rest of the 3 signal pathways and molecular biological processes which may play an important role in the molecular mechanism of myocardial remodeling after AMI in 2;) using Masson staining indicated that AMI after myocardial collagen content increased further, immunohistochemical staining for detection of type I and type III collagen content, found two types of collagen were increased after AMI (P0.01). The heart muscle damage factor qRT-PCR detection after AMI, compared with Sham group, MI group, LDH, cTNT, BNP the transcription level of mRNA increased (P0.05). QRT-PCR was detected in the key factor of target protein signaling, found in AMI after 3 days and 7 days of Wnt, ERS, VDBP and actin related signaling pathway by Western blot detection of MI and Sham after establishing the model of myocardial infarction Mice left ventricular tissue, the results showed VDBP, vitamin D receptor (VDR) and gelsolin (GSN) protein expression level was significantly increased compared with the sham operation group (P0.001), confirmed that the VDBP difference of the protein expression changes after AMI; 3) the successful construction of H9C2 reoxygenation injury heart muscle cell model of hypoxia / VDBP, found in hypoxia / reoxygenation after 6h, upregulating the expression of 12h H9C2 (P0.001). Construction of VDBP over expression plasmid and control plasmid, and using the method of liposome mediated transient transfection of H9C2 expression by qRT-PCR and Western Blot method for detection of VDBP showed that over expression of VDBP after transfection of H9C2 expression. The transcription level of mRNA VDBP and protein levels were significantly up-regulated (P0.001). The expression of VDBP after hypoxia / reoxygenation after 6h treatment, cell apoptosis and necrosis increased (P0.001). The expression levels of mRNA and protein expression in VDBP group VDR was significantly reduced (P0.01), GSN, Caspase3 The expression of mRNA and protein level increased (P0.001). Conclusion: 1) this research uses iTRAQ technologies for high-throughput proteomics screening and identification of differential proteins, through molecular biology technology verification, found that Wnt, ERS, VDBP and molecular biology actin related signal pathway in AMI after ventricular reshaping process; 2) by over expression methods H9C2 myocardial cells transfected with VDBP, functional verification on reoxygenation myocardial cell injury model in hypoxia / VDBP, increase the myocardial cell injury, and have been found to inhibit the expression of VDR VDBP, part of the increase of GSN activity of the phenomenon; 3) in hypoxia reoxygenation myocardial cell injury model / overexpression of VDBP. After the expression of caspase 3 protein level increased, prove the pathophysiological mechanism of early myocardial cell damage in VDBP cells apoptosis through caspase mediated by AMI in 3, targeting the ventricular remodeling after the intervention of AMI provides a new Thinking.

【學位授予單位】：新疆醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R542.22

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