本文選題：LOC339524蛋白 + 大鼠心臟發(fā)育��； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：目的:隨著人口老齡化、冠心病和高血壓等手術(shù)患者比例升高,圍術(shù)期心肌發(fā)生缺血/再灌注損傷的風險也在上升。人體心肌細胞在出生后很快離開細胞分裂周期,幾乎不再分裂。在遭受缺血、缺氧等損傷時,壞死的心肌只能依靠疤痕組織來修復(fù),致使心肌細胞數(shù)量減少,心臟功能減退甚至引起嚴重的心力衰竭。因此,如何實現(xiàn)壞死心肌組織的再生修復(fù)仍是醫(yī)學(xué)上有待攻克的難點。最近的研究成果顯示,成年心肌細胞存在零星的、緩慢的增殖和更新[1][2],同時發(fā)現(xiàn)了一些能誘導(dǎo)成年心肌細胞分裂、增殖的信號通路和方法[3][4][5]。然而,阻止成年心肌細胞重回細胞周期的分子機制仍不清楚。一旦能調(diào)控成年心肌細胞進行分裂增殖,將對心肌梗死、心衰等心臟疾病的心肌修復(fù)提供最有效的治療方法,同時也對心臟疾病和心臟手術(shù)患者圍術(shù)期心肌缺血/再灌注損傷的防治提供有效手段。課題組首次在人心臟早期停跳灌流液中分離、鑒定出了人LOC339524蛋白[6],并對其所在的染色體位點以及對應(yīng)的核苷酸和氨基酸序列等做了初步分析,同時制備了單克隆抗體。本研究分析了LOC339524蛋白在大鼠心臟發(fā)育過程中的表達規(guī)律;同時采用LOC339524基因重組腺病毒(pAd-LOC339524)載體和RNA干擾片段(si-hLOC339524),通過基因轉(zhuǎn)染在大鼠H9C2心肌細胞中過表達和沉默LOC339524基因,研究了LOC339524蛋白表達對H9C2心肌細胞分裂、增殖的調(diào)控作用與機制,為開發(fā)以LOC339524蛋白為靶點的心肌細胞增殖、再生誘導(dǎo)藥物提供理論依據(jù)。方法:1、取胚胎13天至21天(E13~E21),出生后2h至7天(P0~P7),以及成年(2個月)和老年(18個月)的大鼠心肌,采用RT-qPCR、Western Blot和組織免疫熒光染色法等分析大鼠心肌發(fā)育過程中LOC339524基因和蛋白的表達變化。2、構(gòu)建LOC339524基因重組腺病毒(pAd-LOC339524)載體,并轉(zhuǎn)染至H9C2細胞,通過Western Blot和RT-PCR等方法檢測LOC339524的表達水平和最佳表達時間。3、合成三條針對不同靶序列的RNA干擾片段(si-h-LOC339524),以脂質(zhì)體為載體,將其分別轉(zhuǎn)染至H9C2細胞,采用RT-PCR和Western Blot等方法檢測不同si-h-LOC339524、不同濃度和不同時間對LOC339524基因表達的影響,篩選出沉默能力最強的si-h-LOC339524,及其最佳濃度和最佳時間。4、采用構(gòu)建的重組腺病毒載體和篩選的si-h-LOC339524,過表達和沉默H9C2心肌細胞LOC339524基因的表達后,Cell counting kit-8(CCK-8法)檢測細胞增殖活性、EdU檢測細胞DNA合成期的改變情況;Western Blot法檢測p21和cyclin D1蛋白的表達水平。結(jié)果:1、在胚胎期,LOC339524基因總體低表達,但在心房分隔完成(E15)和房室瓣及冠狀動脈重塑完成(E19)2個胚胎晚期心臟發(fā)育的關(guān)鍵時間點表達明顯增高,并且主要在胞漿表達;出生后2h(P0)至7天(P7),隨著心肌細胞分裂增殖能力的逐漸喪失,LOC339524蛋白表達逐漸增加,且主要表達在胞漿,少量胞核表達;而在分裂增殖能力很低的成年和老年大鼠心肌細胞,LOC339524蛋白集中表達在細胞核,胞漿表達顯著減少。2、成功構(gòu)建LOC339524基因重組腺病毒(pAd-LOC339524)載體,并在H9C2心肌細胞實現(xiàn)LOC339524的過表達。3、si-h-LOC339524-002(靶序列為GCAAGTCCATCTCATCGCT)以200nmol/L濃度轉(zhuǎn)染、作用24h時,沉默LOC339524基因的表達效果最強。4、LOC339524基因過表達的H9C2心肌細胞增殖明顯減弱,同時處于DNA合成期的細胞數(shù)量明顯減少,細胞周期依賴性激酶抑制因子(cyclin-dependent-kinase inhibitor,CKI)p21的表達增加,細胞周期蛋白cyclin D1的表達下降;而沉默LOC339524表達后,心肌細胞增殖顯著增強,并且處于DNA合成期的細胞數(shù)量顯著增加,p21的表達下降,cyclin D1的表達增加。結(jié)論:1、LOC339524可能參與了大鼠心臟發(fā)育過程中心肌細胞的分裂、增殖的調(diào)控,細胞核內(nèi)LOC339524蛋白的表達可能對成年大鼠心肌細胞的分裂、增殖起抑制作用。2、LOC339524蛋白表達對H9C2心肌細胞的分裂、增殖有調(diào)控作用,可能通過介導(dǎo)p21上調(diào)表達和cyclin D1下調(diào)表達而抑制心肌細胞的分裂、增殖。3、LOC339524蛋白可能是阻止成年心肌細胞重回細胞周期的重要分子,這為研發(fā)誘導(dǎo)心肌細胞分裂、增殖藥物提供新靶點。
[Abstract]:Objective: with the aging of the population, the increase in the proportion of patients with coronary heart disease and hypertension, the risk of myocardial ischemia / reperfusion injury in the perioperative period is also rising. The human cardiac myocytes will soon leave the cell division cycle and almost no longer split. The necrotic myocardium can only depend on the scar tissue when it suffers from ischemia, hypoxia and so on. To repair, the number of cardiac myocytes, the loss of heart function and even severe heart failure. Therefore, how to regenerate and repair necrotic myocardium remains a difficult medical problem. Recent research shows that adult cardiomyocytes have sporadic, slow proliferation and renewal of [1][2], and some can be found. The molecular mechanism of preventing adult cardiomyocytes from returning to cell cycle is still unclear, however, the molecular mechanism of preventing adult cardiomyocytes to return to cell cycle is still unclear. Once the adult cardiomyocytes are regulated and proliferated, the most effective treatment for cardiac muscle repair, such as myocardial infarction and heart failure, will be provided, and it will also be used for the treatment of cardiac muscle repair, such as myocardial infarction and heart failure. The prevention and treatment of myocardial ischemia / reperfusion injury during perioperative period in patients with heart disease and cardiac surgery is effective. The group was first isolated from human cardiac arrest perfusion fluid for the first time. The human LOC339524 protein [6] was identified, and its chromosomal loci and corresponding nucleotide and amino acid sequences were preliminarily analyzed and prepared at the same time. In this study, the expression of LOC339524 protein in rat heart development was analyzed, and LOC339524 gene recombinant adenovirus (pAd-LOC339524) vector and RNA interference fragment (si-hLOC339524) were used to express and silence the LOC339524 gene in rat H9C2 cardiomyocytes by gene transfection, and the LOC339524 protein was studied. The regulation and regulation of H9C2 myocardial cell division and proliferation can provide theoretical basis for the development of LOC339524 protein as the target of the proliferation of cardiomyocytes and regenerative drugs. Methods: 1, from 13 to 21 days (E13~E21), from 2H to 7 days (P0~P7) after birth, and in adult (2 months) and old (18 months) rat myocardium, using RT-qPCR, West Ern Blot and tissue immunofluorescence staining were used to analyze the expression of LOC339524 gene and protein during the development of rat myocardium,.2, the recombinant adenovirus (pAd-LOC339524) vector of the LOC339524 gene was constructed and transfected into H9C2 cells. The expression level of LOC339524 and the best expression time.3 were detected by Western Blot and RT-PCR, and three pieces were synthesized. The RNA interference fragment (si-h-LOC339524) of different target sequences (si-h-LOC339524) was transfected into H9C2 cells by liposomes. The effects of different si-h-LOC339524, concentration and time on the expression of LOC339524 gene were detected by RT-PCR and Western Blot, and the best concentration of si-h-LOC339524 and its optimum concentration were screened. And at the best time.4, the recombinant adenovirus vector and the screened si-h-LOC339524 were used to express and silence the expression of LOC339524 gene in H9C2 cardiomyocytes. Cell counting kit-8 (CCK-8 method) was used to detect the cell proliferation activity, EdU to detect the change of the cell DNA period, and the Western Blot method was used to detect the expression level of the protein. Results: 1, the expression of LOC339524 gene was low in the embryonic stage, but the expression of E15 and atrioventricular valve and coronary artery remodeling completed (E19) were significantly increased at the critical time point of the 2 embryonic development of the embryo, and mainly in the cytoplasm; after birth, 2h (P0) to 7 days (P7), with the gradual loss of proliferation ability of myocardial cells, L The expression of OC339524 protein increased gradually and expressed mainly in the cytoplasm and a small number of nuclei, while in the adult and elderly rat cardiomyocytes with low proliferation ability, the LOC339524 protein was concentrated in the nucleus, the cytoplasm expression was significantly reduced by.2, and the LOC339524 gene regroup adenovirus (pAd-LOC339524) vector was successfully constructed, and the H9C2 myocardial cells were successfully constructed. The expression of LOC339524 over expression.3, si-h-LOC339524-002 (target sequence GCAAGTCCATCTCATCGCT) transfected with 200nmol/L concentration, the expression of silent LOC339524 gene is the strongest.4, LOC339524 gene overexpressed H9C2 cardiomyocyte proliferation obviously weakened, and the number of cells in DNA synthesis period decreased significantly, cell cycle dependence. The expression of kinase inhibitory factor (cyclin-dependent-kinase inhibitor, CKI) p21 increased, and the expression of cyclin cyclin D1 decreased; while silent LOC339524 expression, the proliferation of cardiomyocytes increased significantly, and the number of cells in the DNA synthesis period increased significantly, the p21 table decreased, and the cyclin D1 increased. Conclusion: 1, LOC339524 may be increased. It participates in the division of the central myocytes of the rat heart development and the regulation of proliferation. The expression of LOC339524 protein in the nucleus may divide the adult rat cardiomyocytes and inhibit the proliferation of.2. The expression of LOC339524 protein can regulate the division of H9C2 cardiomyocytes, and the proliferation of the cells may be regulated by mediating the expression of p21 and under the cyclin D1. The expression can inhibit the mitosis of cardiac myocytes and proliferate.3, and LOC339524 protein may be an important molecule to prevent the cell cycle of adult cardiomyocytes to return to cell cycle. This provides a new target for the development of cardiomyocyte division and proliferation of drugs.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R614

【參考文獻】

相關(guān)期刊論文 前3條

1 易婷婷;李洪;吳瀟瀟;楊天德;;UQCRC1對H9c2心肌細胞耐受缺氧/復(fù)氧損傷的影響[J];重慶醫(yī)學(xué);2014年11期

2 李曉靜;劉敏;王妍;戶義;尹士男;母義明;;血管緊張素Ⅱ2型受體重組腺病毒的擴增及其在INS-1細胞中的轉(zhuǎn)染[J];軍事醫(yī)學(xué);2014年12期

3 吳瀟瀟;李洪;易婷婷;楊天德;;Uqcrc1的RNA干擾片段篩選及其對H9C2心肌細胞耐受缺氧/復(fù)氧損傷的影響[J];中華臨床醫(yī)師雜志(電子版);2013年24期

，

本文編號：1856822