【作者】 刘佳；

【导师】 赵家军；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 研究背景：缺血性心脏病(IHD)是一类由于冠状动脉循环障碍而导致心肌供血缺乏的疾病,已成为当今世界威胁人类健康的重大疾病之一,其防治是全球性首要卫生健康问题。其表现渐进性的心功能的下降,从而导致患者生活质量的显著性降低。当前开展的内外科治疗手段,可以改善冠状动脉供血以及挽救缺血心肌,但对梗死心肌或无功能心肌,尚无良好治疗措施。研究结果表明,当疾病进展到心肌梗死后,产生了一系列心室重塑过程的细胞表型及功能的改变,心室重塑将进展为失代偿的缺血性心力衰竭,其病理特征是心肌细胞数量减少和心脏功能降低。几十年来,学者们一直试图寻找有效治疗方法来逆转其上述缺血性的心肌病变,细胞移植研究为缺血性心脏病的治疗带来了新的希望。近年来研究发现,人体存在多器官多来源的干细胞,这些干细胞具有多向分化潜能,可以直接定向分化成熟心肌细胞或间接促进心肌细胞再生。目前,胚胎干细胞、骨髓间充质干细胞、骨骼肌卫星细胞、心肌干细胞以及最近发现的诱导性多能干细胞等多种细胞,已被用于体外实验和缺血性心肌病动物模型的研究。其中,心肌干细胞由于其确定的可以提交分化成成熟的心肌细胞、血管内皮细胞和血管平滑肌细胞的特性,被认为是干细胞移植治疗缺血性心肌病的一种理想细胞来源。目前,不同的研究组已经利用不用的分离方法,独立得分离出不同表面标志或特性的心肌干细胞,例如side population心肌干细胞,Sca-1+心肌干细胞,lin-c-kit+心肌干细胞,Islet-1+心肌干细胞等。由于细胞分离方法以及细胞特性的差异,到目前为止,这些干细胞是否其实是同一个细胞群体还不清楚。但已有多项研究表明,心肌干细胞的移植可以减小心梗动物的梗死面积,抑制心脏收缩末期和舒张末期容积增加,防止心功能恶化,促进心肌的再生。但很多研究者发现在细胞移植后的48小时之内,只有大约1%-10%的移植细胞可以存活下来。因此,如何提高移植细胞存活率是促进干细胞治疗效果的关键。微小RNA是一种内源性表达的、大约20个核苷酸组成的非编码RNA,其参与生理、病理,细胞的增殖、分化、存活等多个生物过程。由于其只是瞬时过表达于被转导的细胞,并不广泛影响细胞本身的特性,近年来已经被越来越多的研究者作为一种分子生物学工具应用于实验室甚至于临床前期实验的研究。不同的研究组有研究报道,在几种不同的原代细胞中,几种不同的microRNA可以促进细胞的存活。这些都为我们应用微小RNA提高心肌干细胞移植后存活率提供了理论基础。有意义的是,microRNA-155被多次报道在巨噬细胞、胰腺细胞等多种细胞系中可以抑制细胞凋亡,而且我们microRNA微阵列新芯片的检测发现miR-155又在Sca-1+心肌干细胞中内源性表达,因此我们将其作为促进心肌干细胞存活的一个候选微小RNA。本研究的主旨在于应用分子生物学或者化学药物的方法,研究如何改善心肌干细胞移植治疗心肌梗死,分为以下两个部分：一、过表达小RNA155抑制过氧化环境下心肌干细胞坏死及其作用机理的研究。二、应用心梗免疫缺陷小鼠(SCID-NOD)体内过表达小RNA155提高心肌干细胞移植后存活率的研究。一、微小RNA155下调RIP1,抑制过氧化氢诱导的心肌干细胞坏死目的利用微小RNA为平台、过氧化环境模拟缺血性心肌病的细胞环境,寻找一种可以促进心肌干细胞存活的微小RNA及其作用机理,为进一步体内研究奠定基础。方法1、应用anti mouse-Sca-1 antibody MACS,从human fetal heart或adult heart autricle分离并体外培养心肌干细胞2、微小RNA位列阵检测内源性微小RNA在心肌干细胞的表达3、Taqman microRNA assay确认微小RNA155在心肌干细胞内源性表达4、转染寡核苷酸小RNA155前体Pre-mir-155、.小RNA155抑制剂Anti-mir-155和negative mir control,并用3’UTR luciferase assay检测转染的有效性。5、流式细胞仪分析：应用AnnexinV/7-ADD staining,检测在过氧化氢的刺激下,过表达小RNA155对细胞存活、凋亡和坏死的影响。6、时间梯度实验检测过表达小RNA155在不同时间点对细胞凋亡、坏死的影响；细胞活性实验确认小RNA155的细胞保护作用；Propidium iodide staining确认坏死细胞百分率。7、PCR和western检测RIP1是否在心肌干细胞中内源性表达,及过表达microRNA-155能否在蛋白和／或基因水平下调RIP1的表达,8、检测应用小干扰RNA(siRIPl)或RIP1抑制剂Nec-1,能否抑制过氧化环境下细胞的坏死。9、PCR微矩阵芯片检测过表达microRNA-155分别对参与细胞凋亡和Akt细胞存活通路的84个基因表达的影响。结果1. microRNA微距阵检测表明小RNA155在Sca-1+心肌干细胞中内源性表达,这一结果也被microRNA RT-PCR所确认。Sca-1+心肌干细胞在过氧化氢刺激下,小RNA155表达水平上调。2.过表达小RNA155能抑制过氧化氢刺激下Sca-1+心肌干细胞的坏死,但不影响细胞凋亡。时间梯度实验和caspase活性实验表明细胞坏死是本刺激下主要的一种死亡方式。过表达小RNA155不影响Sca-1+心肌干细胞的分化潜能。3.RIP1是小RNA155的靶基因,过表达小RNA155同时在基因和蛋白水平下调RIP1的表达,小RNA155能够特异性靶向RIP1从而抑制细胞的坏死。5.siRIP1或RIP1抑制剂Necrostatin-1也能有效的抑制细胞坏死,不影响细胞凋亡。6.过表达小RNA155不影响细胞生存通路(Akt信号通路)和细胞凋亡通路通路的相关基因表达。结论1、小RNA155在心肌干细胞中内源性表达,在过氧化环境下表达上调。2、过表达小RNA155通过下调RIP1,抑制过氧化环境下心肌干细胞坏死,但不影响细胞凋亡。且过表达小RNA155不影响细胞的分化潜能。3、siRIP1或Necrostatin-1同样抑制过氧化环境下细胞坏死,但不涉及细胞凋亡,证实了小RNA155通过下调RIP1抑制细胞坏死。4.小RNA155促进细胞存活不依赖于Akt prosurvival及apoptosis信号传导通路。二、小RNA155促进心肌干细胞移植后存活的体内研究目的小RNA155能促进体外过氧化环境下心肌干细胞的存活,本实验研究小RNA155转染心肌干细胞后,能否在体内促进其移植后的存活。方法1、构建能够在体外易被筛选,在体内易被追踪的心肌干细胞：在心肌干细胞中转染同时表达荧光素酶和绿色荧光蛋白的慢转录病毒2、应用流式细胞计数检测和筛选成功转染慢转录病毒的心肌干细胞3、在成功转染慢转录病毒的心肌干细胞中过表达小RNA155,检测过氧化氢刺激下,过表达小RNA155能否抑制转染慢转录病毒的心肌细胞的坏死4、心肌内注射表达荧光素酶和绿色荧光蛋白的心肌干细胞,检测移植细胞数量与荧光信号强弱的相关性5、建立心梗的免疫缺陷小鼠(SCID-NOD)模型6、心肌内注射过表达miR-155及荧光素酶和绿色荧光蛋白的心肌干细胞,检测移植后不同时间点的荧光素信号,以检测细胞的存活情况。结果1、慢转录病毒感染后的心肌干细胞稳定地表达荧光素酶和绿色荧光蛋白。2、过氧化氢刺激下,小RNA155抑制表达荧光素酶和绿色荧光蛋白的心肌干细胞死亡。3、过表达小RNA155有促进心肌干细胞移植后生存的趋势。结论1、过表达小RNA155有增强心肌干细胞移植后的存活的趋势2、瞬时过表达小RNA155可能可以作为促进心肌干细胞移植疗效的一种分子生物学手段,从而促进缺血性心肌病的细胞治疗。总之,本研究试图应用分子生物学技术来改善心肌干细胞移植治疗：在体外,小RNA155通过下调参与细胞坏死通路的基因RIP1,抑制过氧化氢诱导的心肌干细胞坏死；免疫缺陷心梗小鼠细胞移植的初步研究结果发现,小RNA155具有很强的促进心肌干细胞移植后存活的趋势,从而为改善干细胞移植治疗的疗效提供了一种更广泛的可能性。更多还原

【Abstract】 BackgroundMyocardial infarction is a life-threatening disease, characterized by massive cardiomyocytes loss and consequently deterioration of cardiac function. Enormous effort has been put to investigate alternatives to restore better cardiac function after myocardial infarction. Until recently, the paradigm that heart is a post-mitotic organ is challenged by the accumulating evidence that the heart habors cardiac progenitor cells. The existence of cardiac progenitor cells provides an opportunity to regenerate cardiomyocytes, compensating the loss of cells after damage. Different research group identified different progenitor cell populations with different characteristics, respectively, using various isolation methods. Hierlihy and colleagues demonstrated for the first time that a putative cardiac progenitor cell population exists in the adult heart, the side population (SP), which have the ability to efflux Hoescht or Rhodamine dyes through ATP-binding transporters and to differentiate into cardiac lineage. Further, Beltrami et al. reported the existence of c-kit+CPCs, which possess cardiac potential and express neither cardiac lineage transcription factors such as Nkx2.5, GATA-4 nor membrane and cytoplasimc proteins[2]. Mouse Sca-1+CPCs [3,4] and Isl-1+cells [5]were identified by different groups, showing the capability for cardiomyocytes regeneration.CPCs are potentially an ideal candidate due to their capability of differentiating into all cell types required for cardiac repair; functional cardiomyocytes, endothelial cells and smooth muscle cells, all required for cardiac repair. Previously, we reported the isolation of cardiomyocyte progenitor cells (CMPCs) from the human heart that are able to proliferate and efficiently differentiate into functional cardiomyocytes without requiring co-culture with neonatal myocytes[6,7]. Three months after transplantation of CMPCs in a mouse model of myocardial infarction, we observed less outward remodeling and improved cardiac function as compared to control injections[8].Although high numbers of cells were injected in several studies, few implanted cells survived, limiting their potential contribution for myocardial repair. Most of the engrafted cells died in the first 48h after transplantation, partially due to the hostile microenvironment of the ischemic myocardium[9-11]. Therefore there is a lot to gain if we can find a strategy to improve cell survival after implantation.Since the last decade, microRNAs have been identified and considered as important gene-regulaitng molecules. They are 20-22 nucleotide length, endogenously expressed, non-coding small RNAs. MicroRNAs mostly downregulate genes expression by binding to the 3’UTR of the targets, thereby blocking the translation of target proteins or directly degrading target mRNAs. They are involved in organ development, cell proliferation, differentiation etc. Accumulating evidence has suggested that miRNAs also play an important role in cell survival. MicroRNA-155 (miR-155) is demonstrated to be involved in cell death in several cell lines, e.g. marcrophages and pancreatic cells. Recently, we observed that miR-155 is expressed in growing CMPCs, and demonstrated that miR-155 is not involved in cellular proliferation. In this study, we to investigate:1)whether mir-155 could improve cardiomyocyte progenitor cells survival after oxidative stress and the underlined mechanism.2)whether mir-155 could enhance cell survival after transplantation in a MI mouse model, using bioluminescence imaging (BLI).Part I MicroRNA-155 blocks necrosis induced by oxidative stress in cardiomyocyte progenitor cells (CMPCs) via targeting RIP1Objective:To investigate whether a candidate microRNA, microRNA-155 could improve cell survival after oxidative stress and the possible mechanism.Methods:1. cardiomyocyte progenitor cells isolation from human fetal heart using mouse anti-Sca-1 antibody MACS2. Taqman microRNA PCR to detect microRNA-155 expression level upon oxidative stress3. microRNA tranfection:pre-miR155/anti-miR155/scr-miR to overexpress miR-155 in CMPCs.3’UTR luciferase activity assay confirms the effectiveness of small miR molecules. 4. flow cytometry analysis:AnnexinV/7AAD staining to detect viable, apoptotic and necrotic cells5. Homogeneous caspases activity assay detects the endogenous active caspases activity upon PI staining to confirm the necrotic cells6. PCR and western blot detect mRNA and protein expression of RIP1 in miR-155 transfected CMPCs 7. RIP1 inhibitor Nec-1 or small RNA interference (siRIP1) mediated knockdown CMPCs8. PCR of 84 Akt-prosurvival pathway related genes, apoptosis related genes PCR arrayResults:1. Using quantitative PCR, we observed a four-fold increase of miR-155 when CMPCs were exposed to hydrogen-peroxide stimulation2. Flow cytometric analysis of cell viability, apoptosis, and necrosis showed that necrosis is the main cause of cell death. Overexpressing miR-155 in CMPCs attenuated necrotic cell death by 40±2.3% via targeting RIP1, receptor interacting protein 1.3. inhibiting RIP1, either by pre-incubating the cells with a RIP1 specific inhibitor, necrostatin-1 (Nec-1) or siRNA mediated knockdown, reduced necrosis by 38±2.5% and 33±1.9%, respectively.4. analyzing gene expression using a PCR-array showed that increased miR-155 levels did not change cell survival and apoptotic related gene expression.Conclusion:By targeting RIP1, miR-155 repressed necrotic cell death of CMPCs, independent of activation of Akt pro-survival pathway. MiR-155 provides the opportunity to block necrosis, a conventionally thought non-regulated process, and might be a potential novel approach to improve cell engraftment for cell therapy.PartⅡMicroRNA-155 improves CMPCs survival after transplantation in a murine myocardial infarction modelObjective:To investigate whether microRNA-155 could promote CMPCs survival after implantation using bioluminescence imaging.Methods:1. transduce fetal CMPCs with pLV-CMV-Luc2-GFP vector2. FACS analysis confirms and detect the effective transduction by detecting GFP positive cells 48h after transduction3. culture transduced CMPCs and compare transduced cells with untransduced cells in phenotype, proliferation speed aspects.4. transfect luc-GFP-CMPCs with miR-155 and stimulate with hydrogen perioxide, followed by cell counting for viable cells and dead cells5. miR-155 transfected luc-GFP-CMPC transplantation in MI SCID-NOD mice6. detect oxyluciferin signal by bioluminescence imaging 2days and 4days post transplantation.Results: 1. Transduced CMPCs stably express luciferase and GFP2. miR-155 protects luc-GFP-CMPCs from death induced by oxidative stress3. Overexpression miR-155 in CMPCs shows a strong trend to improve cell survival after transplantationConclusions:1. miR-155 inhibits cell death induced by H2O2 in luc-GFP-CMPCs in vitro.2. miR-155 might offer a molecular strategy to improve cell survival after transplantation, thereby promoting the effect of cell-based therapy for ischemic heart disease.更多还原