【作者】 刘学彬；

【导师】 陈曦； 丛祥凤； 韩变梅；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 干细胞移植治疗缺血性心肌病无论是动物实验还是临床实验均获得了较大的进展。然而,细胞移植后数小时内大量的供体细胞死亡严重制约了细胞治疗的疗效。心肌缺血微环境诱导的凋亡性死亡被认为是导致供体细胞大量丢失的一个主要因素。迄今为止,许多策略已经被探索用于提高移植细胞的存活,然而,这些策略或因其自身不足不能很好的提高供体细胞存活或因使受体产生一定的毒副作用而限制了其临床上的使用。溶血磷脂(Lysophospholipids,LPs)是简单的磷脂类,最初作为细胞膜生物合成中的组分而被识别。研究表明,溶血磷脂酸(Lysophosphatidic acid,LPA)和1-磷酸鞘氨醇(Sphingosine 1-phosphate,S1P)作为溶血磷脂家族中两个主要成员分别通过各自特异的G蛋白偶联受体参与调节多种重要的生理以及病理生理学过程。我们前期的研究表明,LPA能够拮抗缺氧无血清诱导的骨髓间充质干细胞(mesenchymal stem cells,MSCs)凋亡,而LPA是否体内也有保护作用以及S1P作为LPA的同一个家族成员是否也能抗缺血诱导的MSC凋亡均不清楚。MicroRNA(miRNA)是近年来发现的进化上保守的非编码的小RNA,其以一种翻译抑制或mRNA降解的方式转录后调节基因表达。miRNA在生物体发育、细胞的增殖与凋亡、细胞分化、肿瘤形成等一系列的生命活动中均扮演着不可替代的角色。究竟miRNA在缺血诱导的MSCs凋亡的过程中扮演了怎样的角色,其通过何种机制参与MSCs的凋亡均未可知。本论文首先致力于LPA对移植的MSCs的保护作用体内实验的探讨;同时对另一种更有临床应用前景的脂质分子S1P对MSC的抗凋亡作用进行体内体外实验的详细研究。另外,我们对miRNA在缺血诱导的MSCs凋亡中的作用进行了初步研究。这些研究将有助于产生促进供体细胞存活的治疗新策略。本研究包括以下3部分内容:1、雌性SD大鼠左冠状动脉前降支结扎建立心梗模型,然后被随机分成3组,包括单纯DMEM注射组(Group1),雄性MSCs注射组(Group2)和LPA预处理的雄性MSCs注射组(Group3),细胞注射量为2x10~6。通过Real time-PCR技术检测SRY基因和TUNEL的方法评价移植细胞存活,结果表明LPA预处理提高了移植MSCs存活;而且免疫组化染色血小板内皮细胞粘附分子-1表明移植LPA预处理的MSCs促进了毛细血管的发生,同样体外实验也表明LPA促进了缺氧无血清条件下MSCs的VEGF分泌。超声心动图评价心功能揭示了LPA预处理的MSCs移植没有获得心功能的改善。以上数据表明,LPA预处理能够提高移植MSCs存活,增强其旁分泌作用,促进血管的发生,是一种有效地提高移植的MSC存活的策略。2、通过建立的缺氧无血清诱导的大鼠MSCs凋亡模型来评价S1P的抗凋亡作用。结果显示,S1P通过Gi蛋白偶联S1P1受体以及下游的Akt和ERK1/2信号通路浓度依赖性的拮抗了缺氧无血清诱导的MSC凋亡。体内实验方法同LPA一致,结果表明:在细胞移植1小时、1天和1周后,S1P处理组存活的MSCs明显多于无S1P处理组存活的MSCs;移植S1P处理的MSCs在1周后促进了心肌梗死区和周边区血管的发生。另外,S1P促进了缺氧无血清条件下VEGF的分泌,证明了其旁分泌作用的存在。以上数据表明,S1P处理MSCs是一种新的改善MSCs存活和促进血管发生的治疗策略。3、对0小时、1.5小时、6小时缺氧无血清处理的MSC做miRNA芯片分析,结果表明,随着时间进程,miR-503等4个miRNA表达逐渐升高;而miR-185等3个miRNA表达先升高再降低;miR-195等4个miRNA先降低再升高;miR-483等2个miRNA逐渐降低。Real-time PCR的方法验证上述芯片结果表明miR-503等4个miRNA表达升高,其中miR-337是我们芯片结果没有发现而我们验证时发现了其表达的异常升高;miR-122a等3个miRNA表达降低。统计学表明,miR-503和miR-337的上调以及miR-483的下调均具显著性差异。另外,在miRNA靶点寻找中,我们通过Western blot发现了miR-337的可能靶点为API5;VEGF的分泌受到了的miRNA调控。综上所述,本研究成果不仅探讨了抗缺血诱导的移植MSC凋亡的新的治疗策略而且还首次在miRNA水平上探索了MSCs发生凋亡的机制,为MSCs移植治疗缺血性心肌病提供了一个全新的思路和理论依据。更多还原

【Abstract】 Remarkable advances have been made in animal and clinical studies of stem cell-based therapy for ischemic cardiomyopathy.However,majority of engrafted cells die within hours that severely limits the effectiveness of transplantation therapy.The ischemic environment induced apoptotic death is believed as a major factor responsible for massive cell death.Several strategies have been explored to enhance graft cells survival.But several disadvantages hamper their clinical implications.The lysophospholipids(LPs) are simple phospholipids that have been recognized for decades as components in the biosynthesis of cell membranes.Lysophosphatidic acid (LPA) and sphingosine 1-phosphate(S1P),as two of the best characterized LPs have been revealed to participate in the regulation of many important physiological and pathophysiological processes via specific G protein-coupled receptors termed LPA1-5 and S1P1-5.We have previously shown that LPA antagonized the apoptosis of mesenchymal stem cells(MSCs) induced by hypoxia and serum deprivation(hypoxia/SD) mimicking ischemic myocardium microenvironment.Whether LPA has the same potentially beneficial effect on MSCs in vivo is unknown.In addition,it is unclear whether S1P could protect ischemia induced apoptosis of MSCs.MicroRNAs(miRNAs) are a recently discovered class of small,evolutionarily conserved,non-protein-coding RNA molecules that negatively regulate gene expression by inhibiting protein translation or by destabilizing target transcripts at the post-transcriptional level.MicroRNAs have been implicated in the control of many fundamental cellular and physiological processes such as tissue development,cellular proliferation and apoptosis,oncogenesis.Whether miRNA plays important roles in the process of ischemia induced apopotosis of MSCs and the mechanisms it involves are unkown.The present study explored the protective actions of LPA on transplanted MSCs in vivo and the antiapoptotic effects of S1P on MSCs in vitro and in vivo.In addition,we studied the mechanisms of miRNA mediated the apoptosis of implantated MSCs.All of these studies will contribute to the new strategies of promoting the survival of donor cells.1.Female rats were operated to induce coronary artery occlusion.Animals were then grouped to receive intramyocardial injection with DMEM(30μL,Groupl),male MSCs(2×10~6 per rat,Group2) or male MSCs pretreated with LPA(2×10~6 per rat, Group3),respectively.The results demonstrated that LPA treatment improved graft MSC survival in ischemic myocardium assessed in a gender-mismatched transplantation model by real time-PCR,as well as by TUNEL assay.Moreover, transplantation of LPA treated MSCs enhanced capillary density determined by immmunostaining for platelet endothelial cell adhesion molecule(PECAM)-1,and it is also found that LPA enhanced vascular endothelial growth factor(VEGF) release from MSCs under hypoxia/SD in vitro.We did not get an improvement in Left ventricular(LV) function at 1 weak after transplantation of LPA treated MSCs.These data suggest that LPA exerts both protective actions on MSC survival and enhancement on MSC paracine in vivo and may represent a novel and effective treatment strategy in cell transplantation.2.Rat MSCs were prepared and subjected to hypoxia and serum deprivation (hypoxia/SD for 6 hours with apoptotic cell death determined by flow cytometry. Addition of S1P significantly decreased the percentage of early apoptosis via Gi-coupled S1P1 receptor and activation of downstream Akt and ERK1/2 signaling pathways.In the in vivo study,the same experimental system was used as LPA.The results showed that S1P treatment significantly enhanced the survival of transplanted MSCs at 1 hour,1 day and 1 week after being injected into ischemic heart; transplantation of S1P treated MSCs enhanced angiogenesis 1 week after transplantation in ischemic myocardium.In addition,S1P increased the secretion of VEGF from MSCs under hypoxia and serum deprivation.All of these data suggest that S1P treatment may be a novel strategy to improve MSC survival and promote angiogenesis in ischemic hearts.3.To identify microRNAs that are differentially expressed during MSC apoptosis,we used miRNA microarray analysis of RNA from the MSCs subjected to hypoxia/SD at 3 different time points(Oh,1.5h,and 6h).We were excited to find that,compared with the control group,4 miRNAs were upregulated,3 miRNAs were upregulated first and then downregulated,4 miRNAs were downregulated first and then upregulated and only 2 was downregulated.Further studies were done to validate some microarray results by Realtime-PCR assay and we found that a new miR-337 was upregulated.The final statistical analysis revealed that the differential change of miR-337,miR-503 and miR-483 were statistically significant.Furthermore,we extended our studies to identify the target proteins and we found that API5 maybe a target ofmiR-337 and VEGF maybe regulated by miRNA.Our present study not only explored a new and effective strategy for enhancing graft cells survival but also a new probable mechanism about the apoptosis of transplanted MSCs mediated by microRNA,providing a new idea and theoretical basis for actual application of MSC-based therapy for ischemic cardiomyopathy.更多还原