發(fā)布時間：2018-05-31 04:14

  本文選題：內(nèi)皮祖細胞 + 腎臟纖維化　； 參考：《華中科技大學》2016年博士論文

【摘要】：腎間質(zhì)纖維化是慢性腎臟病進展至終末期腎臟病的共同病理途徑,一旦發(fā)生難以逆轉(zhuǎn)。目前臨床上尚無治療腎間質(zhì)纖維化的有效途徑。腎血管網(wǎng)的稀疏導致的慢性腎臟缺血缺氧是腎間質(zhì)纖維化發(fā)生和進展的關(guān)鍵因素,針對改善腎臟微血管網(wǎng)的修復,進而改善纖維化這一研究方向,相關(guān)認識不足,研究較少。既往報道顯示內(nèi)皮祖細胞(Endothelial Progenitor Cells, EPCs)是一種血管內(nèi)皮細胞的前體細胞,在病理情況下,能夠從骨髓和外周血歸巢至缺血缺氧的器官,促進血管修復和血管新生,改善氧供,減輕組織器官損傷。因此,本課題旨在研究EPCs在腎臟纖維化中的作用及可能機制。通過小鼠單側(cè)輸尿管結(jié)扎(Unilateral Ureteral Occlusion, UUO)建立了腎間質(zhì)纖維化的模型,一方面體外培養(yǎng)骨髓來源的EPCs,將其輸注到UUO小鼠模型中,觀察其對慢性腎纖維化的保護作用。另一方面,使用小分子的CXCR4 [Chemokine (C-X-C motif) Receptor 4]的抑制劑AMD3100,抑制內(nèi)源性的EPCs向受損腎臟歸巢,探討抑制EPCs的歸巢后對慢性腎纖維化的影響。結(jié)果表明,外源性地給予EPCs能夠顯著抑制周細胞-肌成細胞轉(zhuǎn)分化的過程,減輕腎臟的病理變化和腎臟纖維化;而采用AMD3100抑制EPCs歸巢,則腎臟纖維化明顯加重,可能與EPCs旁分泌作用被抑制,導致外周血來源的T細胞的浸潤增加,炎癥反應上調(diào)相關(guān)。因此,EPCs輸注能夠顯著減輕慢性腎臟纖維化的程度,抑制6EPCs向腎臟的歸巢則腎纖維化的程度加重,并且EPCs或者其旁分泌作用的物質(zhì),可能會為臨床腎間質(zhì)纖維化的治療提供另一種可行的干預策略。第一部分血管稀疏在慢性腎臟纖維化中的發(fā)生及機制探討目的慢性腎臟纖維化的發(fā)生過程中常常伴隨著血管稀疏,本研究的目的是探討慢性腎臟纖維化中血管稀疏的產(chǎn)生及機制。方法雄性C57b1/6小鼠32只,隨機分為4組：對照組,UUO-3天組,UUO-7天組和UUO-14天組,每組各8只,行假手術(shù)(對照)或單側(cè)輸尿管結(jié)扎手術(shù),在相應的成模時間點處死小鼠。采用Western Blot及RT-PCR檢測腎臟中α-SMA和Fibronectin的表達變化,采用免疫組織化學、免疫熒光和RT-PCR檢測腎臟中CD31、CD34、PDGFR-β的表達情況。采用流式分析儀檢測UUO過程中成熟毛細血管(CD31+)及新生血管毛細(CD34+)的變化,同時對PDGFR-β與α-SMA行免疫熒光雙染。結(jié)果隨著UUO時間的延長,腎間質(zhì)纖維化的程度明顯加重。在腎纖維化發(fā)展的過程中,腎臟的血管數(shù)目(無論是CD31,還是CD34)都顯著減少,以毛細微血管網(wǎng)的丟失最為明顯,而在單個微血管中,CD34/CD31比例增加。除此之外,周細胞的表面標記物PDGFR-β與肌成纖維細胞的表面標記物α-SMA的表達都隨著纖維化的進展逐漸增加,且兩者共染陽性的區(qū)域也越來越多。結(jié)論慢性腎臟纖維化的過程伴隨著成熟血管和新生血管的減少,尚存的新生血管可能存在成熟障礙,這種成熟障礙是由于周細胞-肌成纖維細胞轉(zhuǎn)分化增加導致血管失去周細胞的支持和營養(yǎng)造成的。第二部分內(nèi)皮祖細胞輸注通過抑制周細胞轉(zhuǎn)分化抑制腎間質(zhì)纖維化目的在許多血管性或缺血性疾病模型中,EPCs都被證明參與了組織缺血缺氧后組織的血管修復和血管新生。但在以血管稀疏為顯著特點的慢性腎臟纖維化中,EPCs的作用尚不清楚。本研究旨在探討EPCs輸注對慢性腎間質(zhì)纖維化的作用及可能機制。方法首先對骨髓來源的早期EPCs進行分離、培養(yǎng)和鑒定,然后通過尾靜脈注射將其輸注到單側(cè)輸尿管結(jié)扎的小鼠模型中。腎臟病理切片進行PAS, Masson和天狼星紅染色揭示形態(tài)學的變化,采用免疫組織化學、免疫熒光和Western Blot檢測腎臟a-SMA和Collagen-IV的表達情況,并檢測了EPCs的輸注對周細胞的表面標記物PDGFR-β與a-SMA的共表達和腎小管上皮細胞G2-M阻滯的影響。同時也評價了EPCs輸注后對微血管的改善作用。結(jié)果EPCs輸注顯著改善了腎臟形態(tài)學的變化,減少了膠原纖維的沉積,抑制了纖維化的程度。EPCs干預組a-SMA和PDGFR-β的表達都顯著減少,并且EPCs干預后a-SMA和PDGFR-β共表達和腎小管上皮細胞G2-M阻滯也顯著減少。結(jié)論EPCs輸注能顯著改善慢性腎臟纖維化的進展,可能與其抑制了周細胞-肌成纖維細胞轉(zhuǎn)分化及腎小管上皮細胞G2-M阻滯有關(guān)。第三部分AMD3100通過抑制內(nèi)皮祖細胞的歸巢和調(diào)節(jié)T細胞相關(guān)炎癥,促進腎纖維化進展目的AMD3100是一種小分子CXCR4的抑制劑,而CXCR4存在于EPCs和多種炎癥細胞的細胞膜上,報道顯示AMD3100能夠減輕心肌、肝臟和肺臟纖維化,鑒于腎臟纖維化正是一種伴隨血管稀疏和多種炎癥細胞浸潤的特殊病理狀態(tài),因此本研究旨在探討AMD3100對早期腎臟纖維化的作用及具體機制。方法雄性C57bl/6小鼠30只,隨機分為3組：對照組,UUO腹腔注射PBS組,UUO腹腔注射AMD3100組,每組各10只,行假手術(shù)(對照)或UUO手術(shù),于造模當天開始給予PBS或AMD3100腹腔注射(2mg/kg/day),直至造模第5天實驗終點處死小鼠。取。腎臟行HE, PAS及Masson染色；采用Western Blot、免疫組織化學和免疫熒光法檢測腎組織中α-SMA和PDGFR-p的表達變化,采用流式細胞分析儀檢測EPCs (CD45+CD34+CD309+)從骨髓到外周血和腎臟中的趨化的數(shù)目多少,并檢測腎臟中多種炎癥細胞的變化。采用RT-PCR檢測T相關(guān)趨化因子(CCL-4, CCL5, CX3CL-1, CXCL-9和CXCL-10)的變化,采用免疫熒光雙染技術(shù)檢測CD3+T細胞在腎臟中的增殖(PCNA+)水平。結(jié)果AMD3100腹腔注射組腎臟纖維化的程度明顯加重,伴隨著EPCs向腎臟的趨化減少,CD3+的T細胞的數(shù)目顯著增加,并且T細胞相關(guān)趨化因子水平顯著上升,但原位T細胞的增殖并無明顯改變。結(jié)論AMD3100對腎臟纖維化并無保護作用,反而加重了腎纖維化的進展,可能與其抑制腎組織中EPCs向腎臟的趨化,同時促進T細胞的浸潤和炎癥反應有關(guān)。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R692

【相似文獻】

相關(guān)期刊論文 前10條

1 王先梅;楊麗霞;宋武戰(zhàn);齊峰;郭傳明;石燕昆;趙穎;魏玲;王燕;;鹽酸貝那普利對自發(fā)性高血壓大鼠腎臟纖維化的影響[J];第四軍醫(yī)大學學報;2006年20期

2 王偉銘;陳永熙;陳楠;;慢性腎臟病與腎臟纖維化和炎癥[J];內(nèi)科理論與實踐;2007年06期

3 袁偉杰;張郁苒;;干細胞在腎臟纖維化治療中的研究進展[J];中國中西醫(yī)結(jié)合腎病雜志;2009年05期

4 楊云華;艾維;;慢性腎臟病與腎臟纖維化研究進展[J];實用醫(yī)學雜志;2009年20期

5 肖紅波;劉瑞洪;凌光輝;;熱休克蛋白47在腎臟纖維化中的作用及其機制[J];中國中西醫(yī)結(jié)合腎病雜志;2009年12期

6 陳麗平;王保興;;腎臟纖維化治療的研究進展[J];河北醫(yī)藥;2009年23期

7 盧穎;王偉銘;;過氧化物酶體增殖物激活受體-γ與腎臟纖維化的研究進展[J];上海交通大學學報(醫(yī)學版);2010年01期

8 王興華;徐再春;;腎臟纖維化臨床研究進展[J];山東中醫(yī)雜志;2010年03期

9 李曉忠;;腎臟纖維化能逆轉(zhuǎn)嗎?[J];實用兒科臨床雜志;2011年17期

10 黃松明;朱春華;;過氧化物酶體增殖物激活受體γ在腎臟纖維化中的作用[J];實用兒科臨床雜志;2011年17期

相關(guān)會議論文 前8條

1 劉必成;;腎臟纖維化發(fā)生機制研究進展[A];中華醫(yī)學會腎臟病學分會2010學術(shù)年會專題講座匯編[C];2010年

2 袁偉杰;邊琪;;維生素D在抗腎臟纖維化中的作用[A];中華醫(yī)學會腎臟病學分會2006年學術(shù)年會專題講座[C];2006年

3 于永綱;羅后宙;;轉(zhuǎn)錄因子GATA-3在腎臟纖維化中的作用研究[A];第十五屆全國泌尿外科學術(shù)會議論文集[C];2008年

4 劉必成;;腎臟纖維化研究新進展[A];中國生理學會腎臟生理專業(yè)委員會第二屆學術(shù)年會論文匯編[C];2013年

5 孫偉;魏明剛;;腎臟纖維化的逆轉(zhuǎn)機制及其中藥的干預[A];中華中醫(yī)藥學會第二十一屆全國中醫(yī)腎病學術(shù)會議論文匯編（下）[C];2008年

6 張學凱;趙宗江;;巨噬細胞在腎臟纖維化過程中的作用(摘要)[A];第十九次全國中醫(yī)腎病學術(shù)交流會論文匯編[C];2006年

7 喬玉峰;蔣云生;;鉛對大鼠腎臟纖維化相關(guān)因子表達的影響[A];中華醫(yī)學會腎臟病學分會2006年學術(shù)年會論文集[C];2006年

8 李素敏;楊林;王建榮;楊萬霞;曾文;傅淑霞;王彥;;CTGF、P-P38MAPK在TGF-β_1在IgAN腎臟纖維化中的作用[A];中華醫(yī)學會腎臟病學分會2006年學術(shù)年會論文集[C];2006年

相關(guān)重要報紙文章 前1條

1 程守勤;抗腎臟纖維化：控制腎衰的關(guān)鍵[N];健康報;2007年

相關(guān)博士學位論文 前10條

1 劉丁;二次諧波與雙光子激發(fā)熒光（SHG/TPEF）顯微成像技術(shù)診斷腎臟纖維化的系統(tǒng)研究[D];南方醫(yī)科大學;2015年

2 孫玉靜;Klotho蛋白干預腎臟纖維化的機制研究[D];山東大學;2016年

3 汪妍;促紅細胞生成素對骨髓間充質(zhì)干細胞來源微泡修復腎臟纖維化影響的研究[D];南京醫(yī)科大學;2016年

4 曹玉涵;基于尿液mRNA芯片檢測的腎臟纖維化生物標志物研究[D];東南大學;2016年

5 楊娟;內(nèi)皮祖細胞對腎臟纖維化的保護作用及其機制研究[D];華中科技大學;2016年

6 宋鍇;硫化氫在腎臟纖維化中的作用及其機制研究[D];蘇州大學;2013年

7 肖紅波;熱休克蛋白47在腎臟纖維化中的作用及機制研究[D];中南大學;2010年

8 辛欣;五甲基槲皮素抗糖尿病大鼠腎臟纖維化的作用及機制研究[D];華中科技大學;2013年

9 王暢;靶向基因敲除cPLA_2α延緩細胞周期進程以減輕單側(cè)輸尿管梗阻所致腎臟纖維化[D];中南大學;2011年

10 蓋志博;Trps1表達單倍不足通過上調(diào)Arkadia水平促進腎臟纖維化[D];山東大學;2013年

相關(guān)碩士學位論文 前10條

1 張園園;活性維生素D_3對糖尿病腎病大鼠維生素D受體和腎臟纖維化因子的影響[D];新疆醫(yī)科大學;2015年

2 王晨;11β-HSD1抑制劑治療高脂飲食所致腎臟纖維化的機制研究[D];南京醫(yī)科大學;2015年

3 俞瑞;1，25雙羥維生素D_3減少糖尿病大鼠腎臟纖維化的機制研究：抑制TGF-β1、FN及VEGF的表達[D];新疆醫(yī)科大學;2015年

4 孫欣欣;NAC在大鼠心腎綜合征腎臟纖維化中的作用及其機制的研究[D];山西醫(yī)科大學;2016年

5 李敏;基于腎臟纖維化靶向和修復的水溶性殼聚糖研究[D];華南理工大學;2016年

6 劉倩;RDN抑制IPiCMP心臟和腎臟纖維化的機制[D];南京醫(yī)科大學;2016年

7 龍英杰;羅蓋全對5/6腎衰大鼠腎臟纖維化的影響及機理的實驗研究[D];南華大學;2016年

8 李娜;鉤藤堿對自發(fā)性高血壓大鼠腎臟纖維化的影響[D];皖南醫(yī)學院;2016年

9 郭薇;孕期炎癥刺激致子代腎臟纖維化的機制及干預初探[D];第三軍醫(yī)大學;2016年

10 肖靜;依那普利聯(lián)合纈沙坦對實驗性大鼠腎臟纖維化的影響[D];鄭州大學;2002年

，

本文編號：1958244