【摘要】：背景:干細(xì)胞治療是一種新興的心肌缺血損傷后修復(fù)的再生醫(yī)學(xué)治療模式。然而,干細(xì)胞在體外擴(kuò)增后移植到缺血心臟,面臨移植所致心律失常、多能干細(xì)胞在體定向分化不可控、致瘤風(fēng)險(xiǎn)以及干細(xì)胞的遠(yuǎn)期存活率低等問(wèn)題。近年來(lái)發(fā)現(xiàn)并完善的轉(zhuǎn)分化策略,有望從一定程度上解決上述問(wèn)題。轉(zhuǎn)分化依賴于特異性的轉(zhuǎn)錄因子或化學(xué)誘導(dǎo)實(shí)現(xiàn)體細(xì)胞譜系轉(zhuǎn)變,繞過(guò)了多能性狀態(tài),相對(duì)高效和安全。但該方法本身無(wú)法避免病毒載體轉(zhuǎn)染引起的基因修飾,限制了該方法獲得期望的細(xì)胞類型及其產(chǎn)品的治療性應(yīng)用。而小分子因?yàn)槠淇焖佟⒎奖闶褂�、無(wú)外源基因整合、效應(yīng)可逆等特點(diǎn)在轉(zhuǎn)分化中是一種替代譜系特異性轉(zhuǎn)錄因子的較好的選擇。但由于小分子在重編程中的應(yīng)用是一個(gè)新興的、發(fā)展的話題,迄今為止,心血管領(lǐng)域相關(guān)小分子策略的研究都是孤立的、前驅(qū)性的,其在轉(zhuǎn)分化中的作用機(jī)制及分子路線圖有待于未來(lái)進(jìn)一步闡明,更多的研究需要去驗(yàn)證目前的發(fā)現(xiàn)和評(píng)估只使用小分子的完全化學(xué)轉(zhuǎn)分化來(lái)產(chǎn)生不同譜系細(xì)胞類型的可能性。因此,本實(shí)驗(yàn)研究旨在模擬急性心肌梗死環(huán)境,調(diào)查血清剝奪刺激是否可以激活心肌成纖維細(xì)胞向內(nèi)皮細(xì)胞轉(zhuǎn)分化。方法:我們?nèi)57/BL新生小鼠(出生1-3天)新鮮分離心臟組織,獲取成纖維細(xì)胞進(jìn)行細(xì)胞培養(yǎng),用淋巴細(xì)胞抑制因子(LIF)促進(jìn)成纖維細(xì)胞自我更新、長(zhǎng)程維持干性和抑制其向終末分化。將第3代的成纖維細(xì)胞分為5組:對(duì)照組(DMEM+10%FBS)血清饑餓24h組(DMEM+0%FBS)、血清饑餓48h組(DMEM+0%FBS)、血清饑餓72h組(DMEM+0%FBS)、血清饑餓48小時(shí)且不添加LIF(DMEM+0%FBS)。各組細(xì)胞在不同處理后,分別采用qPCR法、體外血管生成試驗(yàn)和Dil標(biāo)記-乙�；兔芏戎鞍�(Dil-Ac-LDL)吞噬功能試驗(yàn)來(lái)檢測(cè)細(xì)胞譜系特異性基因變化、體外血管新生能力和內(nèi)皮細(xì)胞標(biāo)記性的功能,采用ELISA的方法檢測(cè)血清饑餓后成纖維細(xì)胞分泌VEGF的情況。結(jié)果:血清饑餓組內(nèi)皮細(xì)胞特異性基因CD31陽(yáng)性表達(dá)的細(xì)胞是對(duì)照組的22倍,ve-herin陽(yáng)性表達(dá)的細(xì)胞是對(duì)照組的50倍,P0.05。經(jīng)歷3個(gè)不同的時(shí)間的血清饑餓刺激后,血清饑餓48小時(shí)組內(nèi)皮基因提高最明顯,是血清饑餓72小時(shí)組的7.7倍,P0.05。和對(duì)照組比較,血清饑餓24小時(shí)組ve-herin表達(dá)無(wú)顯著提高,P0.05。此外,Fbs獲得了一定的內(nèi)皮細(xì)胞的功能,和對(duì)照組相比,倒置熒光顯微鏡下觀察血清剝奪組Fbs形成微血管結(jié)構(gòu)和分泌VEGF的能力顯著提高。結(jié)論:血清剝奪刺激可以激活心肌成纖維細(xì)胞向內(nèi)皮細(xì)胞轉(zhuǎn)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R54

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本文編號(hào)：2507588