本文選題：間充質(zhì)干細胞　切入點：重癥急性胰腺炎腎損害　出處：《福建醫(yī)科大學》2013年博士論文　論文類型：學位論文

【摘要】：目的：本研究旨在通過經(jīng)鑒定的同種異體骨髓間充質(zhì)干細胞（mesenchymal stemcells, MSCs）移植到重癥急性胰腺炎(severe acute pancreatitis, SAP)腎損害的動物模型體內(nèi)，觀察MSCs對SAP合并腎損害的治療效果，并探討其對毛細血管滲漏的干預(yù)機制，為MSCs用于治療SAP合并腎損害提供理論依據(jù)。 方法： 1、建立SAP腎損害的大鼠模型，檢測腹水量、腹水淀粉酶（amylase, AMY），血清AMY、肌酐（creatinine, Cr）、尿素氮（urea nitrogen, BUN）以及腫瘤壞死因子-α（tumor necrosis factor-α, TNF-α）、白介素-1β（interleukin-1β, IL-1β）水平變化，觀察胰腺、腎臟病理形態(tài)及毛細血管內(nèi)皮超微結(jié)構(gòu)變化； 2、體外培養(yǎng)MSCs，通過細胞形態(tài)檢測、生長曲線觀察，細胞表面抗原流式鑒定以及體外定向誘導(dǎo)分化法結(jié)合檢測來所培養(yǎng)的細胞； 3、通過MSCs移植到SAP動物體內(nèi)，檢測血清AMY、Cr、BUN以及TNF-α、IL-1β水平，觀察胰腺、腎臟病理形態(tài)及毛細血管內(nèi)皮超微結(jié)構(gòu)變化，并檢測胰腺、腎臟組織中水通道蛋白1（aquaporin1, AQP1）、基質(zhì)金屬蛋白酶-9（matrixmetalloproteinase-9, MMP-9）、血管擴張刺激磷蛋白（vasodilator stimulatedphosphoprotein, VASP）mRNA及蛋白水平改變。 結(jié)果： 1、膽胰管逆行注射�；悄懰徕c配合膽胰管結(jié)扎法能成功建立SAP合并腎損害的動物模型；SAP合并腎損害發(fā)生時，腹水量增加，腹水AMY、血清AMY、Cr、BUN、TNF-α、IL-1β明顯升高，胰腺以及腎臟組織出現(xiàn)毛細血管內(nèi)皮屏障的破壞，并隨時間增加而破壞加重； 2、全骨髓貼壁法和密度梯度離心法聯(lián)合培養(yǎng)的細胞形態(tài)典型，高度表達CD29、CD90，低表達CD34、45，且能被誘導(dǎo)分化為成骨、成脂細胞，符合MSCs特性；CM-Dil對其染色，，染色熒光強，且細胞活性未見明顯下降； 3、MSCs可抑制血清TNF-α、IL-1β的過度表達，抑制SAP大鼠胰腺、腎臟中AQP1、VASPmRNA及蛋白的下降，抑制SAP大鼠胰腺、腎臟中MMP-9mRNA及蛋白的升高，減輕SAP大鼠胰腺、腎臟毛細血管內(nèi)皮屏障的破壞。 結(jié)論：在SAP腎損害發(fā)生時，移植的MSCs能夠歸巢到胰腺、腎臟受損區(qū)域，抑制炎癥因子過度表達，調(diào)節(jié)血管內(nèi)皮細胞結(jié)構(gòu)相關(guān)蛋白的表達平衡，減輕毛細血管內(nèi)皮屏障的破壞，維護內(nèi)皮屏障的完整性，從而最終達到治療SAP合并腎損害的目的。
[Abstract]:Objective: to observe the therapeutic effect of MSCs on renal damage induced by severe acute pancreatitis (SAP) by transplanting mesenchymal stem cells (MSCs) from allogeneic bone marrow mesenchymal stem cells (MSCs) into the animal model of severe acute pancretis (SAP). In order to provide theoretical basis for the treatment of SAP with renal damage, the mechanism of its intervention on capillary leakage was discussed. Methods:. 1. The rat model of SAP renal damage was established. The levels of ascitic fluid, amylase, amylase, amylase, serum amylase, creatinine, creatinine, CRU, urea urea nitrogen- (bu), tumor necrosis factor- 偽 (TNF- 偽), interleukin-1 尾 interleukin-1 尾 (IL-1 尾), tumor necrosis factor- 偽 (TNF- 偽), interleukin-1 尾 interleukin-1 尾 (IL-1 尾) were measured. The changes of renal histopathology and ultrastructure of capillary endothelium; (2) MSCs were cultured in vitro. The cells were cultured by cell morphology detection, growth curve observation, cell surface antigen flow analysis and in vitro directional induction and differentiation assay. (3) Transplantation of MSCs into SAP, the serum levels of AMYDK Cr bun and TNF- 偽 偽 -IL-1 尾 were measured, and the changes of pancreas, renal histopathology and ultrastructure of capillary endothelium were observed. The changes of aquaporin 1, aquaporin 1, matrix metalloproteinase-9, matrix metalloproteinase-9, vasodilator stimulated phosphoprotein, VASP)mRNA and protein in renal tissue were observed. Results:. 1. Retrograde injection of sodium taurocholate and ligation of biliary and pancreatic duct can successfully establish the animal model of SAP complicated with renal damage. The destruction of capillary endothelial barrier in pancreas and kidney was aggravated with the increase of time. 2. The cells cultured by whole bone marrow adherent method and density gradient centrifugation were typical in morphology, high expression of CD29 and CD90, low expression of CD34O45, and could be induced to differentiate into osteogenic and adipogenic cells. The cells were stained by CM-Dil in accordance with the characteristics of MSCs, and the fluorescence was strong. The cell activity did not decrease significantly. 3MSCs could inhibit the overexpression of serum TNF- 偽 and IL-1 尾, inhibit the decrease of AQP1 mRNA and protein in pancreas and kidney of SAP rats, inhibit the increase of MMP-9mRNA and protein in pancreas of SAP rats, and alleviate the destruction of capillary endothelial barrier in pancreas and renal capillary of SAP rats. Conclusion: at the time of renal injury of SAP, the transplanted MSCs can homing to the pancreas and the damaged area of kidney, inhibit the overexpression of inflammatory factors, regulate the balance of the expression of proteins related to the structure of vascular endothelial cells, and alleviate the destruction of capillary endothelial barrier. Maintain the integrity of endothelial barrier, so as to achieve the ultimate treatment of SAP with renal damage.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R657.51

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