循环外泌体相关microRNA在干细胞介导的心血管修复中的作用机制研究

发布时间：2018-06-18 10:37

本文选题：心肌源性microRNA(Myo-miRs) + 外泌体(exosome)　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分循环外泌体相关心肌源性microRNAs通过作用于CXCR4调节急性心肌梗死后骨髓干/祖细胞的动员 [目的]心肌源性microRNAs (myocardial microRNAs, myo-miRs)在急性心肌梗死(AMl)患者的外周循环中显著升高,但其效应靶器官及功能学作用尚不清楚。循环中的外泌体是一类由细胞主动性分泌的具有细胞膜结构的小型运输体,能介导不同细胞或组织间mRNA、microRNA等遗传物质的交换。目前已知急性心梗能介导骨髓干细胞的动员。因此,本研究拟探讨急性心肌梗死后循环中升高的myo-miRs是否通过外泌体远程传输到骨髓,从而发挥对骨髓中干/祖细胞的动员作用。 [方法与结果]在8～10周龄雄性C57BJ/L小鼠体内构建急性心肌梗死模型,6小时后处死,使用实时定量PCR技术检测四种myo-miRs (miR-1a、miR-208a、miR-133a以及miR-499-5p)在血清、血清外泌体、非外泌体血清成份、骨髓单核细胞以及其它组织中的表达水平。结果显示急性心肌梗死6小时后myo-miRs在外泌体和非外泌体血清成份中均显著升高；且miR-1a, miR-208a和miR-499-5p主要在外泌体中升高,而miR-133a则主要在非外泌体血清成份中升高。为了研究myo-miRs在外泌体中增高的作用,从急性心肌梗死模型小鼠的血清中分离外泌体并用PKH67标记,通过尾静脉注射到正常小鼠体内；12小时后分离股骨,荧光显微镜检测骨组织切片PKH67阳性的细胞,发现PKH67阳性的细胞存在于骨髓干骺端,提示循环中的外泌体被转运到了骨髓中且存在于骨髓干细胞区域。同时比较急性心肌梗死后不同组织心肌源性myo-miRs的表达水平,发现骨髓单核细胞中myo-miRs表达量显著高于肝,肾,脾,说明循环中的myo-miRs主要被转运到骨髓中。下一步,分别将急性心梗分离的外泌体和非外泌体血清成份跟骨髓单核细胞共培养,发现只有外泌体共培养的骨髓单核细胞中myo-miRs的表达增高,说明主要是由外泌体介导了循环中myo-miRs向骨髓单核细胞的转运oSDF-1/CXCR4信号通路在骨髓干细胞动员中起重要作用。在体外转染myo-miRs的特异性化学模拟物(microRNA mimics)到骨髓单核细胞中,发现这四种myo-miR均对CXCR4有明显抑制作用。而后,将急性心梗小鼠体内分离的外泌体与骨髓单核细胞共培养,证明了急性心梗相关外泌体能够显著抑制骨髓单核细胞内CXCR4的表达;而当myo-miRs被抑制后,急性心梗相关外泌体对CXCR4的调节作用明显减弱。为了进一步验证急性心梗分泌的外泌体是否介导了骨髓干细胞的动员,在小鼠体内静脉注射急性心肌梗外泌体及正常对照外泌体,结果发现在急性心肌梗死外泌体处理的小鼠外周血中Lin-、c-kit+及c-kit+Lin-的细胞显著增多,提示急性心梗分泌的外泌体能够介导骨髓干细胞的动员。 [结论]综上所述,急性心肌梗死外周循环中升高的myo-miRs,通过外泌体转运骨髓单核细胞中,抑制骨髓干细胞中CXCR4表达,从而介导骨髓干/祖细胞动员。本研究为干细胞治疗提供了新的潜在靶点。第二部分急性心肌梗死相关血清外泌体miR-1通过CX3CR1调节骨髓间充质干细胞的迁移 [目的]急性心肌梗死后,缺血心肌分泌大量心肌特异性的microRNA到外周循环中。文献报道,急性心梗后循环中miR-1显著增高,但是循环中的miR-1是如何被转运及其生物作用目前还不清楚。外泌体是一类由细胞主动性分泌的具有细胞膜结构的小型运输体,能介导不同细胞或组织间mRNA以及micRNA等遗传物质的交换。CX3CR1是一个在骨髓间充质干细胞中丰富表达的趋化因子受体,CX3CR1/Fractalkine信号通路对于骨髓间充质干细胞的向缺血损伤组织趋化具有重要调节作用。本研究旨在探索急性心肌梗死后血清中的心肌源性miR-1是否通过外泌体形式被释放,以及其对骨髓间充质干细胞中CX3CR1的潜在调节作用。 [方法与结果]在雄性SD大鼠体内构建急性心肌梗死模型,收集对照组以及急性心梗组的大鼠外周血清并从中提取出血清外泌体,使用实时定量PCR技术检测各组血清以及血清外泌体中miR-1的表达水平。结果发现,急性心肌梗死组中miR-1在血清中显著增高,这跟文献报道一致。同时还发现,miR-1在血清外泌体中的增高水平显著高于非外泌体血清成份,提示循环中miR-1主要存在于外泌体中。为了进一步研究血清外泌体miR-1对骨髓间充质干细胞的调节作用,在体外培养诱导SD大鼠的骨髓间充质干细胞,并将急性心梗的血清外泌体进行荧光标记后与骨髓间充质干细胞进行共培养,荧光显微镜的观察发现骨髓间充质干细胞能够有效摄取血清外泌体。此外,还将急性心肌梗死后6小时分离的血清外泌体与骨髓间充质干细胞共培养,通过实时定量PCR技术证明急性心梗相关血清外泌体能够显著上调骨髓间充质干细胞内的miR-1的表达水平。为探讨血清外泌体miR-1转运到骨髓间充质干细胞内潜在的生物学作用,在骨髓间充质干细胞中转染miR-1的特异性化学模拟物,实时定量PCR技术和蛋白印迹技术检测表明miR-1可以明显下调骨髓间充质干细胞内CX3CR1的mRNA和蛋白质的表达水平,同时,还通过体外迁移实验证明miR-1显著抑制了骨髓间充质干细胞向fractalkine的趋化作用。 [结论]综上所述,大鼠体内急性心肌梗死相关血清外泌体miR-1具有下调骨髓间充质干细胞内CX3CR1表达水平并抑制骨髓间充质干细胞趋化能力的潜能,这一现象将有可能成为骨髓间充质干细胞治疗心脏缺血性损伤的新靶点。
[Abstract]:Part 1 circulating exocrine related cardiac derived microRNAs modulates the mobilization of bone marrow stem / progenitor cells after CXCR4 in acute myocardial infarction.
[Objective] myocardial derived microRNAs (myocardial microRNAs, myo-miRs) is significantly elevated in the peripheral circulation of patients with acute myocardial infarction (AMl), but the effect of its target organ and function is not clear. The exocrine in the circulation is a small transporter, which is secreted by cells, and can mediate different cells or different cells. It is known that acute myocardial infarction can mediate the mobilization of bone marrow stem cells. Therefore, this study intends to explore whether or not the elevated myo-miRs in the post infarction cycle is transmitted to the bone marrow through the exocrine after acute myocardial infarction, so as to mobilize the stem / progenitor cells in the bone marrow.
[methods and results] an acute myocardial infarction model was constructed in 8~10 weeks old male C57BJ/L mice and executed after 6 hours. Real-time quantitative PCR was used to detect the expression of four kinds of myo-miRs (miR-1a, miR-208a, miR-133a and miR-499-5p) in serum, serum exocrine, non exocrine serum components, bone marrow mononuclear cells and other tissues. The results showed that myo-miRs increased significantly in both exocrine and non exocrine serum levels after 6 hours of acute myocardial infarction, and miR-1a, miR-208a and miR-499-5p increased mainly in the exocrine, while miR-133a increased mainly in the non exocrine serum components. In order to study the increase of myo-miRs in the exocrine, acute myocardial infarction was from acute myocardial infarction. The sera of the dead model mice were separated and labeled with PKH67, and the femur was injected into the normal mice by the tail vein. After 12 hours, the femur was separated and the PKH67 positive cells were detected by the fluorescence microscope. It was found that the PKH67 positive cells existed in the metaphysis of the bone marrow, suggesting that the exocrine in the circle was transported to the bone marrow and existed in the bone marrow. At the same time, the expression level of cardiac myo-miRs in different tissues after acute myocardial infarction was compared. The expression of myo-miRs in bone marrow mononuclear cells was significantly higher than that of liver, kidney and spleen, indicating that the myo-miRs in the circulation was mainly transported to the bone marrow. The next step was to separate the exocrine and non exocrine serum from the acute myocardial infarction. It was found that the expression of myo-miRs in bone marrow mononuclear cells increased in only exocrine co cultured mononuclear cells, indicating that the exocrine mediated transport of myo-miRs to bone marrow mononuclear cells in the circulation plays an important role in the mobilization of bone marrow stem cells. The specific transfection of myo-miRs in vitro is specific. In microRNA mimics to bone marrow mononuclear cells, the four myo-miR were found to have a significant inhibitory effect on CXCR4. Then, the exosbodies isolated from the acute myocardial infarction mice and bone marrow mononuclear cells were co cultured, which proved that the acute myocardial infarction related exocrine could significantly inhibit the expression of CXCR4 in the mononuclear cells of the bone marrow; and when myo-m In order to further verify whether the exocrine secreted by acute myocardial infarction mediates the mobilization of bone marrow stem cells in order to further verify that the exocrine secreted by acute myocardial infarction mediates the mobilization of the bone marrow stem cells, the acute myocardial infarction is administered by intravenous injection of acute myocardial infarction and normal exocrine in mice, and the fruit is now treated with acute myocardial infarction outside the iRs. The Lin-, c-kit+ and c-kit+Lin- cells in the peripheral blood of mice increased significantly, suggesting that exocrine secreted by acute myocardial infarction can mediate the mobilization of bone marrow stem cells.
[Conclusion] to sum up, myo-miRs, which is elevated in the peripheral circulation of acute myocardial infarction, can inhibit the expression of CXCR4 in bone marrow stem cells through exosomatic transport of bone marrow mononuclear cells and mediate the mobilization of bone marrow stem / progenitor cells. This study provides a new potential target for stem cell therapy.
The second part of the acute myocardial infarction related serum exocrine miR-1 regulates the migration of bone marrow mesenchymal stem cells through CX3CR1.
[Objective] after acute myocardial infarction, the ischemic myocardium secretes a large number of myocardium specific microRNA to peripheral circulation. It is reported that miR-1 is significantly increased in the posterior circulation of acute myocardial infarction, but it is not clear how the miR-1 in the circulation is transported and its biological effect is still unclear. The exocrine is a cell membrane structure secreted by cell activity. The small transporter, which mediates the exchange of genetic material such as mRNA and micRNA between different cells or tissues, is a chemokine receptor rich in bone marrow mesenchymal stem cells. The CX3CR1/Fractalkine signaling pathway plays an important role in the chemotaxis of bone marrow mesenchymal stem cells to ischemic tissue. The purpose of this study is to investigate the effect of.CX3CR1 on the chemotaxis of bone marrow mesenchymal stem cells. In the exploration of acute myocardial infarction, the release of cardiac miR-1 in the serum through the form of exocrine and its potential regulation of CX3CR1 in bone marrow mesenchymal stem cells.
[methods and results] the acute myocardial infarction model was constructed in the male SD rats. The peripheral serum of the rats in the control group and the acute myocardial infarction group were collected and the haemorrhagic exocrine was extracted from the rats. The expression of miR-1 in the serum and the serum exocrine was detected by real-time quantitative PCR technique. The results showed that the miR-1 in the acute myocardial infarction group was in the acute myocardial infarction group. The serum levels were significantly higher, which was consistent with the literature. At the same time, it was found that the increase of miR-1 in the serum exocrine was significantly higher than that of the non exocrine serum, suggesting that miR-1 mainly existed in the exocrine. In order to further study the regulation of serum exocrine miR-1 on bone marrow mesenchymal stem cells, the induction of SD in vitro was large. The rat bone marrow mesenchymal stem cells were cultured with bone marrow mesenchymal stem cells after fluorescence labeling of acute myocardial infarction. The fluorescence microscopy showed that bone marrow mesenchymal stem cells could effectively absorb the serum exocrine. In addition, the serum exocrine and bone marrow were separated 6 hours after acute myocardial infarction. Coculture of stromal cells, through real-time quantitative PCR technique, demonstrated that acute myocardial infarction related serum exocrine could significantly increase the level of miR-1 expression in bone marrow mesenchymal stem cells, and to explore the potential biological role of serum exocrine miR-1 transport to bone marrow mesenchymal stem cells, and the specific transfection of miR-1 in bone marrow mesenchymal stem cells. The chemical mimics, real-time quantitative PCR and Western blot assays showed that miR-1 could obviously reduce the level of mRNA and protein expression of CX3CR1 in bone marrow mesenchymal stem cells. At the same time, in vitro migration experiments showed that miR-1 significantly inhibited the chemotaxis of bone marrow mesenchymal stem cells to fractalkine.
[Conclusion] to sum up, the acute myocardial infarction related serum exosomatic miR-1 in rats has the potential to downregulate the expression of CX3CR1 in bone marrow mesenchymal stem cells and inhibit the chemotaxis of bone marrow mesenchymal stem cells. This phenomenon may be a new target for the treatment of cardiac ischemic injury by bone marrow mesenchymal stem cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R542.2

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