LOC339524蛋白对心肌细胞分裂、增殖的调控作用与机制研究

发布时间：2018-05-07 11:56

本文选题：LOC339524蛋白 + 大鼠心脏发育　；参考：《第三军医大学》2015年硕士论文

【摘要】：目的:随着人口老龄化、冠心病和高血压等手术患者比例升高,围术期心肌发生缺血/再灌注损伤的风险也在上升。人体心肌细胞在出生后很快离开细胞分裂周期,几乎不再分裂。在遭受缺血、缺氧等损伤时,坏死的心肌只能依靠疤痕组织来修复,致使心肌细胞数量减少,心脏功能减退甚至引起严重的心力衰竭。因此,如何实现坏死心肌组织的再生修复仍是医学上有待攻克的难点。最近的研究成果显示,成年心肌细胞存在零星的、缓慢的增殖和更新[1][2],同时发现了一些能诱导成年心肌细胞分裂、增殖的信号通路和方法[3][4][5]。然而,阻止成年心肌细胞重回细胞周期的分子机制仍不清楚。一旦能调控成年心肌细胞进行分裂增殖,将对心肌梗死、心衰等心脏疾病的心肌修复提供最有效的治疗方法,同时也对心脏疾病和心脏手术患者围术期心肌缺血/再灌注损伤的防治提供有效手段。课题组首次在人心脏早期停跳灌流液中分离、鉴定出了人LOC339524蛋白[6],并对其所在的染色体位点以及对应的核苷酸和氨基酸序列等做了初步分析,同时制备了单克隆抗体。本研究分析了LOC339524蛋白在大鼠心脏发育过程中的表达规律;同时采用LOC339524基因重组腺病毒(pAd-LOC339524)载体和RNA干扰片段(si-hLOC339524),通过基因转染在大鼠H9C2心肌细胞中过表达和沉默LOC339524基因,研究了LOC339524蛋白表达对H9C2心肌细胞分裂、增殖的调控作用与机制,为开发以LOC339524蛋白为靶点的心肌细胞增殖、再生诱导药物提供理论依据。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个月)和老年(18个月)的大鼠心肌,采用RT-qPCR、Western Blot和组织免疫荧光染色法等分析大鼠心肌发育过程中LOC339524基因和蛋白的表达变化。2、构建LOC339524基因重组腺病毒(pAd-LOC339524)载体,并转染至H9C2细胞,通过Western Blot和RT-PCR等方法检测LOC339524的表达水平和最佳表达时间。3、合成三条针对不同靶序列的RNA干扰片段(si-h-LOC339524),以脂质体为载体,将其分别转染至H9C2细胞,采用RT-PCR和Western Blot等方法检测不同si-h-LOC339524、不同浓度和不同时间对LOC339524基因表达的影响,筛选出沉默能力最强的si-h-LOC339524,及其最佳浓度和最佳时间。4、采用构建的重组腺病毒载体和筛选的si-h-LOC339524,过表达和沉默H9C2心肌细胞LOC339524基因的表达后,Cell counting kit-8(CCK-8法)检测细胞增殖活性、EdU检测细胞DNA合成期的改变情况;Western Blot法检测p21和cyclin D1蛋白的表达水平。结果:1、在胚胎期,LOC339524基因总体低表达,但在心房分隔完成(E15)和房室瓣及冠状动脉重塑完成(E19)2个胚胎晚期心脏发育的关键时间点表达明显增高,并且主要在胞浆表达;出生后2h(P0)至7天(P7),随着心肌细胞分裂增殖能力的逐渐丧失,LOC339524蛋白表达逐渐增加,且主要表达在胞浆,少量胞核表达;而在分裂增殖能力很低的成年和老年大鼠心肌细胞,LOC339524蛋白集中表达在细胞核,胞浆表达显著减少。2、成功构建LOC339524基因重组腺病毒(pAd-LOC339524)载体,并在H9C2心肌细胞实现LOC339524的过表达。3、si-h-LOC339524-002(靶序列为GCAAGTCCATCTCATCGCT)以200nmol/L浓度转染、作用24h时,沉默LOC339524基因的表达效果最强。4、LOC339524基因过表达的H9C2心肌细胞增殖明显减弱,同时处于DNA合成期的细胞数量明显减少,细胞周期依赖性激酶抑制因子(cyclin-dependent-kinase inhibitor,CKI)p21的表达增加,细胞周期蛋白cyclin D1的表达下降;而沉默LOC339524表达后,心肌细胞增殖显著增强,并且处于DNA合成期的细胞数量显著增加,p21的表达下降,cyclin D1的表达增加。结论:1、LOC339524可能参与了大鼠心脏发育过程中心肌细胞的分裂、增殖的调控,细胞核内LOC339524蛋白的表达可能对成年大鼠心肌细胞的分裂、增殖起抑制作用。2、LOC339524蛋白表达对H9C2心肌细胞的分裂、增殖有调控作用,可能通过介导p21上调表达和cyclin D1下调表达而抑制心肌细胞的分裂、增殖。3、LOC339524蛋白可能是阻止成年心肌细胞重回细胞周期的重要分子,这为研发诱导心肌细胞分裂、增殖药物提供新靶点。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614

【参考文献】