小鼠成纤维细胞向内皮细胞转分化的体外研究
发布时间：2019-06-28 20:25
【摘要】：背景:干细胞治疗是一种新兴的心肌缺血损伤后修复的再生医学治疗模式。然而,干细胞在体外扩增后移植到缺血心脏,面临移植所致心律失常、多能干细胞在体定向分化不可控、致瘤风险以及干细胞的远期存活率低等问题。近年来发现并完善的转分化策略,有望从一定程度上解决上述问题。转分化依赖于特异性的转录因子或化学诱导实现体细胞谱系转变,绕过了多能性状态,相对高效和安全。但该方法本身无法避免病毒载体转染引起的基因修饰,限制了该方法获得期望的细胞类型及其产品的治疗性应用。而小分子因为其快速、方便使用、无外源基因整合、效应可逆等特点在转分化中是一种替代谱系特异性转录因子的较好的选择。但由于小分子在重编程中的应用是一个新兴的、发展的话题,迄今为止,心血管领域相关小分子策略的研究都是孤立的、前驱性的,其在转分化中的作用机制及分子路线图有待于未来进一步阐明,更多的研究需要去验证目前的发现和评估只使用小分子的完全化学转分化来产生不同谱系细胞类型的可能性。因此,本实验研究旨在模拟急性心肌梗死环境,调查血清剥夺刺激是否可以激活心肌成纤维细胞向内皮细胞转分化。方法:我们取C57/BL新生小鼠(出生1-3天)新鲜分离心脏组织,获取成纤维细胞进行细胞培养,用淋巴细胞抑制因子(LIF)促进成纤维细胞自我更新、长程维持干性和抑制其向终末分化。将第3代的成纤维细胞分为5组:对照组(DMEM+10%FBS)血清饥饿24h组(DMEM+0%FBS)、血清饥饿48h组(DMEM+0%FBS)、血清饥饿72h组(DMEM+0%FBS)、血清饥饿48小时且不添加LIF(DMEM+0%FBS)。各组细胞在不同处理后,分别采用qPCR法、体外血管生成试验和Dil标记-乙酰化低密度脂蛋白(Dil-Ac-LDL)吞噬功能试验来检测细胞谱系特异性基因变化、体外血管新生能力和内皮细胞标记性的功能,采用ELISA的方法检测血清饥饿后成纤维细胞分泌VEGF的情况。结果:血清饥饿组内皮细胞特异性基因CD31阳性表达的细胞是对照组的22倍,ve-herin阳性表达的细胞是对照组的50倍,P0.05。经历3个不同的时间的血清饥饿刺激后,血清饥饿48小时组内皮基因提高最明显,是血清饥饿72小时组的7.7倍,P0.05。和对照组比较,血清饥饿24小时组ve-herin表达无显著提高,P0.05。此外,Fbs获得了一定的内皮细胞的功能,和对照组相比,倒置荧光显微镜下观察血清剥夺组Fbs形成微血管结构和分泌VEGF的能力显著提高。结论:血清剥夺刺激可以激活心肌成纤维细胞向内皮细胞转分化。
[Abstract]:BACKGROUND: Stem cell therapy is a new type of regenerative medicine for the post-injury of myocardial ischemia. However, after in vitro amplification of stem cells, the stem cells are transplanted to the ischemic heart, which faces the problems of the arrhythmia caused by the transplantation, the incontrollable directional differentiation of the pluripotent stem cells in the body, the tumor risk and the long-term survival rate of the stem cells, and the like. In recent years, it is expected to solve the above-mentioned problems to some extent. The transdifferentiation depends on specific transcription factors or chemical-induced transformation of the somatic lineage, bypassing the pluripotent state, relatively high efficiency and safety. However, the method itself cannot avoid the gene modification caused by the transfection of the viral vector, limiting the method to obtain the desired cell type and therapeutic application of the product. And the small molecule is a better choice for the alternative lineage specific transcription factor in the transdifferentiation because of the characteristics of rapid, convenient use, no foreign gene integration, reversible effect and the like. But because the application of small molecule in reprogramming is a new and developing topic, so far, the research of small molecular strategy in the field of cardiovascular field is isolated, the mechanism of its role in transdifferentiation and the molecular road map need to be further clarified in the future, More research has to be done to verify that the present finding and evaluation only uses the complete chemical transformation of small molecules to produce the possibility of different lineage cell types. Therefore, the purpose of this study is to simulate the acute myocardial infarction environment and investigate whether the serum deprivation stimulation can activate the differentiation of the myocardial fibroblasts to the endothelial cells. Methods: We used C57/ BL neonatal mice (1-3 days of birth) to separate the heart tissue, to get the fibroblasts for cell culture, to promote the self-renewal of fibroblasts with a lymphocyte inhibitory factor (LIF), to maintain the long-range maintenance and to inhibit its terminal differentiation. The fibroblasts of the third generation were divided into 5 groups: control group (DMEM + 10% FBS), serum starvation 24 h (DMEM + 0% FBS), serum starvation of 48 h (DMEM + 0% FBS), serum starvation 72h (DMEM + 0% FBS), serum starvation for 48 hours, and no LIF (DMEM + 0% FBS). after different treatment, each group of cells respectively adopts the qPCR method, the in vitro blood vessel generation test and the Dil-labeled-B-type low-density lipoprotein (Dil-Ac-LDL) phagocytic function test to detect the function of the cell lineage specific gene change, the in vitro angiogenesis capacity and the endothelial cell labeling property, The expression of VEGF was detected by ELISA. Results: The expression of CD31-positive cells in the serum-hungry group was 22-fold in the control group, and the expression of ve-herin was 50-fold in the control group, P 0.05. After 3 different time of serum hunger stimulation, the increase of the endothelial gene in the 48-hour group was the most obvious, which was 7.7 times that of the 72-hour group of serum starvation, P0.05. Compared with the control group, the expression of live-herin was not significantly increased in 24 hours of serum starvation, and P0.05. In addition, Fbs obtained a certain function of endothelial cells, and compared with the control group, the ability of the Fbs to form a microvessel structure and to secrete VEGF was significantly improved under an inverted fluorescence microscope. Conclusion: The serum deprivation stimulation can activate the differentiation of the cardiac fibroblasts to the endothelial cells.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
[Abstract]:BACKGROUND: Stem cell therapy is a new type of regenerative medicine for the post-injury of myocardial ischemia. However, after in vitro amplification of stem cells, the stem cells are transplanted to the ischemic heart, which faces the problems of the arrhythmia caused by the transplantation, the incontrollable directional differentiation of the pluripotent stem cells in the body, the tumor risk and the long-term survival rate of the stem cells, and the like. In recent years, it is expected to solve the above-mentioned problems to some extent. The transdifferentiation depends on specific transcription factors or chemical-induced transformation of the somatic lineage, bypassing the pluripotent state, relatively high efficiency and safety. However, the method itself cannot avoid the gene modification caused by the transfection of the viral vector, limiting the method to obtain the desired cell type and therapeutic application of the product. And the small molecule is a better choice for the alternative lineage specific transcription factor in the transdifferentiation because of the characteristics of rapid, convenient use, no foreign gene integration, reversible effect and the like. But because the application of small molecule in reprogramming is a new and developing topic, so far, the research of small molecular strategy in the field of cardiovascular field is isolated, the mechanism of its role in transdifferentiation and the molecular road map need to be further clarified in the future, More research has to be done to verify that the present finding and evaluation only uses the complete chemical transformation of small molecules to produce the possibility of different lineage cell types. Therefore, the purpose of this study is to simulate the acute myocardial infarction environment and investigate whether the serum deprivation stimulation can activate the differentiation of the myocardial fibroblasts to the endothelial cells. Methods: We used C57/ BL neonatal mice (1-3 days of birth) to separate the heart tissue, to get the fibroblasts for cell culture, to promote the self-renewal of fibroblasts with a lymphocyte inhibitory factor (LIF), to maintain the long-range maintenance and to inhibit its terminal differentiation. The fibroblasts of the third generation were divided into 5 groups: control group (DMEM + 10% FBS), serum starvation 24 h (DMEM + 0% FBS), serum starvation of 48 h (DMEM + 0% FBS), serum starvation 72h (DMEM + 0% FBS), serum starvation for 48 hours, and no LIF (DMEM + 0% FBS). after different treatment, each group of cells respectively adopts the qPCR method, the in vitro blood vessel generation test and the Dil-labeled-B-type low-density lipoprotein (Dil-Ac-LDL) phagocytic function test to detect the function of the cell lineage specific gene change, the in vitro angiogenesis capacity and the endothelial cell labeling property, The expression of VEGF was detected by ELISA. Results: The expression of CD31-positive cells in the serum-hungry group was 22-fold in the control group, and the expression of ve-herin was 50-fold in the control group, P 0.05. After 3 different time of serum hunger stimulation, the increase of the endothelial gene in the 48-hour group was the most obvious, which was 7.7 times that of the 72-hour group of serum starvation, P0.05. Compared with the control group, the expression of live-herin was not significantly increased in 24 hours of serum starvation, and P0.05. In addition, Fbs obtained a certain function of endothelial cells, and compared with the control group, the ability of the Fbs to form a microvessel structure and to secrete VEGF was significantly improved under an inverted fluorescence microscope. Conclusion: The serum deprivation stimulation can activate the differentiation of the cardiac fibroblasts to the endothelial cells.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
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