内皮祖细胞对肾脏纤维化的保护作用及其机制研究

发布时间：2018-05-31 04:14

本文选题：内皮祖细胞 + 肾脏纤维化　；参考：《华中科技大学》2016年博士论文

【摘要】：肾间质纤维化是慢性肾脏病进展至终末期肾脏病的共同病理途径,一旦发生难以逆转。目前临床上尚无治疗肾间质纤维化的有效途径。肾血管网的稀疏导致的慢性肾脏缺血缺氧是肾间质纤维化发生和进展的关键因素,针对改善肾脏微血管网的修复,进而改善纤维化这一研究方向,相关认识不足,研究较少。既往报道显示内皮祖细胞(Endothelial Progenitor Cells, EPCs)是一种血管内皮细胞的前体细胞,在病理情况下,能够从骨髓和外周血归巢至缺血缺氧的器官,促进血管修复和血管新生,改善氧供,减轻组织器官损伤。因此,本课题旨在研究EPCs在肾脏纤维化中的作用及可能机制。通过小鼠单侧输尿管结扎(Unilateral Ureteral Occlusion, UUO)建立了肾间质纤维化的模型,一方面体外培养骨髓来源的EPCs,将其输注到UUO小鼠模型中,观察其对慢性肾纤维化的保护作用。另一方面,使用小分子的CXCR4 [Chemokine (C-X-C motif) Receptor 4]的抑制剂AMD3100,抑制内源性的EPCs向受损肾脏归巢,探讨抑制EPCs的归巢后对慢性肾纤维化的影响。结果表明,外源性地给予EPCs能够显著抑制周细胞-肌成细胞转分化的过程,减轻肾脏的病理变化和肾脏纤维化;而采用AMD3100抑制EPCs归巢,则肾脏纤维化明显加重,可能与EPCs旁分泌作用被抑制,导致外周血来源的T细胞的浸润增加,炎症反应上调相关。因此,EPCs输注能够显著减轻慢性肾脏纤维化的程度,抑制6EPCs向肾脏的归巢则肾纤维化的程度加重,并且EPCs或者其旁分泌作用的物质,可能会为临床肾间质纤维化的治疗提供另一种可行的干预策略。第一部分血管稀疏在慢性肾脏纤维化中的发生及机制探讨目的慢性肾脏纤维化的发生过程中常常伴随着血管稀疏,本研究的目的是探讨慢性肾脏纤维化中血管稀疏的产生及机制。方法雄性C57b1/6小鼠32只,随机分为4组：对照组,UUO-3天组,UUO-7天组和UUO-14天组,每组各8只,行假手术(对照)或单侧输尿管结扎手术,在相应的成模时间点处死小鼠。采用Western Blot及RT-PCR检测肾脏中α-SMA和Fibronectin的表达变化,采用免疫组织化学、免疫荧光和RT-PCR检测肾脏中CD31、CD34、PDGFR-β的表达情况。采用流式分析仪检测UUO过程中成熟毛细血管(CD31+)及新生血管毛细(CD34+)的变化,同时对PDGFR-β与α-SMA行免疫荧光双染。结果随着UUO时间的延长,肾间质纤维化的程度明显加重。在肾纤维化发展的过程中,肾脏的血管数目(无论是CD31,还是CD34)都显著减少,以毛细微血管网的丢失最为明显,而在单个微血管中,CD34/CD31比例增加。除此之外,周细胞的表面标记物PDGFR-β与肌成纤维细胞的表面标记物α-SMA的表达都随着纤维化的进展逐渐增加,且两者共染阳性的区域也越来越多。结论慢性肾脏纤维化的过程伴随着成熟血管和新生血管的减少,尚存的新生血管可能存在成熟障碍,这种成熟障碍是由于周细胞-肌成纤维细胞转分化增加导致血管失去周细胞的支持和营养造成的。第二部分内皮祖细胞输注通过抑制周细胞转分化抑制肾间质纤维化目的在许多血管性或缺血性疾病模型中,EPCs都被证明参与了组织缺血缺氧后组织的血管修复和血管新生。但在以血管稀疏为显著特点的慢性肾脏纤维化中,EPCs的作用尚不清楚。本研究旨在探讨EPCs输注对慢性肾间质纤维化的作用及可能机制。方法首先对骨髓来源的早期EPCs进行分离、培养和鉴定,然后通过尾静脉注射将其输注到单侧输尿管结扎的小鼠模型中。肾脏病理切片进行PAS, Masson和天狼星红染色揭示形态学的变化,采用免疫组织化学、免疫荧光和Western Blot检测肾脏a-SMA和Collagen-IV的表达情况,并检测了EPCs的输注对周细胞的表面标记物PDGFR-β与a-SMA的共表达和肾小管上皮细胞G2-M阻滞的影响。同时也评价了EPCs输注后对微血管的改善作用。结果EPCs输注显著改善了肾脏形态学的变化,减少了胶原纤维的沉积,抑制了纤维化的程度。EPCs干预组a-SMA和PDGFR-β的表达都显著减少,并且EPCs干预后a-SMA和PDGFR-β共表达和肾小管上皮细胞G2-M阻滞也显著减少。结论EPCs输注能显著改善慢性肾脏纤维化的进展,可能与其抑制了周细胞-肌成纤维细胞转分化及肾小管上皮细胞G2-M阻滞有关。第三部分AMD3100通过抑制内皮祖细胞的归巢和调节T细胞相关炎症,促进肾纤维化进展目的AMD3100是一种小分子CXCR4的抑制剂,而CXCR4存在于EPCs和多种炎症细胞的细胞膜上,报道显示AMD3100能够减轻心肌、肝脏和肺脏纤维化,鉴于肾脏纤维化正是一种伴随血管稀疏和多种炎症细胞浸润的特殊病理状态,因此本研究旨在探讨AMD3100对早期肾脏纤维化的作用及具体机制。方法雄性C57bl/6小鼠30只,随机分为3组：对照组,UUO腹腔注射PBS组,UUO腹腔注射AMD3100组,每组各10只,行假手术(对照)或UUO手术,于造模当天开始给予PBS或AMD3100腹腔注射(2mg/kg/day),直至造模第5天实验终点处死小鼠。取。肾脏行HE, PAS及Masson染色；采用Western Blot、免疫组织化学和免疫荧光法检测肾组织中α-SMA和PDGFR-p的表达变化,采用流式细胞分析仪检测EPCs (CD45+CD34+CD309+)从骨髓到外周血和肾脏中的趋化的数目多少,并检测肾脏中多种炎症细胞的变化。采用RT-PCR检测T相关趋化因子(CCL-4, CCL5, CX3CL-1, CXCL-9和CXCL-10)的变化,采用免疫荧光双染技术检测CD3+T细胞在肾脏中的增殖(PCNA+)水平。结果AMD3100腹腔注射组肾脏纤维化的程度明显加重,伴随着EPCs向肾脏的趋化减少,CD3+的T细胞的数目显著增加,并且T细胞相关趋化因子水平显著上升,但原位T细胞的增殖并无明显改变。结论AMD3100对肾脏纤维化并无保护作用,反而加重了肾纤维化的进展,可能与其抑制肾组织中EPCs向肾脏的趋化,同时促进T细胞的浸润和炎症反应有关。
[Abstract]:Renal interstitial fibrosis is a common pathological pathway of chronic renal disease progression to end-stage renal disease. Once it is difficult to reverse, there is no effective treatment for renal interstitial fibrosis. Chronic renal ischemia and hypoxia caused by the sparsity of renal vascular network is a key factor in the development and progression of renal interstitial fibrosis. Endothelial Progenitor Cells (EPCs) is a precursor cell of vascular endothelial cells, which can be returned from bone marrow and peripheral blood to ischemic anoxic organs and promote vascular repair in pathological conditions. The purpose of this study is to study the role of EPCs in renal fibrosis and its possible mechanism. A model of renal interstitial fibrosis was established by unilateral ureteral ligation (Unilateral Ureteral Occlusion, UUO) in mice. On the one hand, the bone marrow derived EPCs was cultured in vitro, and it was transfused to UUO. In the mouse model, the protection of chronic renal fibrosis was observed. On the other hand, the inhibition of endogenous EPCs to the damaged kidneys was inhibited by the use of a small molecule CXCR4 [Chemokine (C-X-C motif) Receptor 4] inhibitor AMD3100, and the effect of inhibiting the homing of EPCs on the chronic renal fibrosis was investigated. The results showed that EPCs could be given exogenous. The process of transdifferentiation of pericyte and myoblast can be inhibited significantly, and the pathological changes of kidney and renal fibrosis are alleviated, and the renal fibrosis is obviously aggravated by AMD3100 inhibition of EPCs homing, which may be inhibited by EPCs paracrine, resulting in the infiltration of T cells from the peripheral blood and up up of the inflammatory response. Therefore, EPCs infusion can be shown to be significant. To reduce the degree of chronic renal fibrosis and to inhibit the degree of 6EPCs's homing to the kidneys, the degree of renal fibrosis is aggravated. And EPCs or its paracrine substance may provide another feasible intervention strategy for the treatment of clinical renal interstitial fibrosis. The purpose of this study is to explore the generation and mechanism of vascular sparsity in chronic renal fibrosis. Methods 32 male C57b1/6 mice were randomly divided into 4 groups: control group, UUO-3 day group, UUO-7 day group and UUO-14 day group, 8 rats in each group, and a sham operation (control) or a single group. The expression of the expression of alpha -SMA and Fibronectin in the kidneys was detected by Western Blot and RT-PCR. The expression of CD31, CD34 and PDGFR- beta in the kidneys was detected by immunohistochemistry, immunofluorescence and RT-PCR. The flow analyzer was used to detect the mature capillaries in the UUO process (CD3). (CD3 (CD3) 1+) and the changes of neonatal vascular capillary (CD34+) and both PDGFR- beta and alpha -SMA double staining with immunofluorescence. With the prolongation of UUO time, the degree of renal interstitial fibrosis is obviously aggravated. In the course of the development of renal fibrosis, the number of blood vessels in the kidneys (whether CD31, or CD34) is significantly reduced, with the most obvious loss of capillary net of hair. In addition, the expression of the surface marker of the surface marker of the pericytes, PDGFR- beta and the surface marker of the myofibroblast, increased with the progression of fibrosis, and more and more regions were co dyed positive. Conclusion the process of slow renal fibrosis is accompanied by mature blood vessels and new CD34/CD31. The decrease of the blood vessels and the survival of the neovascularization may have mature obstacles, which are caused by the increase of the pericytes - myofibroblast transdifferentiation that causes the vascular loss of peripheral blood cells to support and nutrition. The second part of endothelial progenitor cells transfused in many vessels through inhibition of pericytes conversion inhibition of renal interstitial fibrosis. In the model of sexual or ischemic disease, EPCs has been proved to be involved in vascular repair and angiogenesis after tissue ischemia and hypoxia. But the role of EPCs in chronic renal fibrosis with significant vascular sparsity is not clear. The purpose of this study is to explore the effect and possible mechanism of EPCs infusion on chronic renal fibrosis. The early EPCs of the bone marrow source was isolated, cultured and identified, and then injected into the mouse model of unilateral ureteral ligature by injection of the tail vein. The pathological sections of the kidney were examined by PAS, Masson and Sirius red staining to reveal the morphological changes. Immunohistochemistry, immunofluorescence and Western Blot were used to detect the renal a-SMA and Collage. The expression of n-IV and the effect of EPCs infusion on the co expression of PDGFR- beta and a-SMA and G2-M block in renal tubular epithelial cells were detected. Meanwhile, the effect of EPCs infusion on the microvascular improvement was also evaluated. Results EPCs infusion significantly improved the changes in the morphology of the kidneys, reduced the deposition of collagen fibers and suppressed the deposition of collagen fibers. The expression of a-SMA and PDGFR- beta in.EPCs intervention group decreased significantly, and a-SMA and PDGFR- beta co expression and renal tubular epithelial cell G2-M block decreased significantly after EPCs intervention. Conclusion EPCs infusion can significantly improve the progress of chronic renal fibrosis, which may inhibit the transdifferentiation of pericytes myofibroblast and renal minor. G2-M block of tubular epithelial cells. Third part AMD3100 is a small molecule CXCR4 inhibitor by inhibiting the homing of endothelial progenitor cells and regulating inflammation of T cells and promoting the progression of renal fibrosis, and CXCR4 exists on the membrane of EPCs and many inflammatory cells. It is reported that AMD3100 can reduce the myocardium, liver and lung. In this study, the purpose of this study was to explore the role and specific mechanism of AMD3100 on early renal fibrosis. Methods 30 male C57bl/6 mice were randomly divided into 3 groups: group A, UUO intraperitoneal injection of PBS, UUO intraperitoneal injection of AMD310 The 0 groups, 10 in each group, were treated with PBS or AMD3100 intraperitoneal injection (2mg/kg/day) on the day of making the model, and the mice were sacrificed at the end of the model for fifth days. The kidneys were stained with HE, PAS and Masson; Western Blot, immunohistochemistry and immunofluorescence were used to detect the expression of alpha -SMA and PDGFR-p in the kidney tissues. Change, the number of chemotactic numbers of EPCs (CD45+CD34+CD309+) from bone marrow to peripheral blood and kidney were detected by flow cytometry, and changes of various inflammatory cells in the kidney were detected. The changes of T related chemokines (CCL-4, CCL5, CX3CL-1, CXCL-9 and CXCL-10) were detected by RT-PCR, and CD3+T fines were detected by immunofluorescence double staining technique. The level of cell proliferation (PCNA+) in the kidney results in a significant increase in the degree of renal fibrosis in the AMD3100 intraperitoneal injection group, with the decrease in the chemotaxis of EPCs to the kidneys, the number of T cells in CD3+ increased significantly, and the level of T cell related chemokines increased significantly, but the proliferation of in situ T cells was not significantly changed. Conclusion AMD3100 is a kind of renal fiber. Instead of protective effect, it aggravates the progress of renal fibrosis, which may inhibit the chemotaxis of EPCs to the kidneys in the kidney tissue and promote the infiltration of T cells and the inflammatory reaction.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692

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