同种异体骨髓间充质干细胞治疗重症急性胰腺炎肾损害效果评价及对毛细血管渗漏干预机制的实验研究

发布时间：2018-03-13 05:04

本文选题：间充质干细胞　切入点：重症急性胰腺炎肾损害　出处：《福建医科大学》2013年博士论文　论文类型：学位论文

【摘要】：目的：本研究旨在通过经鉴定的同种异体骨髓间充质干细胞（mesenchymal stemcells, MSCs）移植到重症急性胰腺炎(severe acute pancreatitis, SAP)肾损害的动物模型体内，观察MSCs对SAP合并肾损害的治疗效果，并探讨其对毛细血管渗漏的干预机制，为MSCs用于治疗SAP合并肾损害提供理论依据。方法： 1、建立SAP肾损害的大鼠模型，检测腹水量、腹水淀粉酶（amylase, AMY），血清AMY、肌酐（creatinine, Cr）、尿素氮（urea nitrogen, BUN）以及肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、白介素-1β（interleukin-1β, IL-1β）水平变化，观察胰腺、肾脏病理形态及毛细血管内皮超微结构变化； 2、体外培养MSCs，通过细胞形态检测、生长曲线观察，细胞表面抗原流式鉴定以及体外定向诱导分化法结合检测来所培养的细胞； 3、通过MSCs移植到SAP动物体内，检测血清AMY、Cr、BUN以及TNF-α、IL-1β水平，观察胰腺、肾脏病理形态及毛细血管内皮超微结构变化，并检测胰腺、肾脏组织中水通道蛋白1（aquaporin1, AQP1）、基质金属蛋白酶-9（matrixmetalloproteinase-9, MMP-9）、血管扩张刺激磷蛋白（vasodilator stimulatedphosphoprotein, VASP）mRNA及蛋白水平改变。结果： 1、胆胰管逆行注射牛磺胆酸钠配合胆胰管结扎法能成功建立SAP合并肾损害的动物模型；SAP合并肾损害发生时，腹水量增加，腹水AMY、血清AMY、Cr、BUN、TNF-α、IL-1β明显升高，胰腺以及肾脏组织出现毛细血管内皮屏障的破坏，并随时间增加而破坏加重； 2、全骨髓贴壁法和密度梯度离心法联合培养的细胞形态典型，高度表达CD29、CD90，低表达CD34、45，且能被诱导分化为成骨、成脂细胞，符合MSCs特性；CM-Dil对其染色，，染色荧光强，且细胞活性未见明显下降； 3、MSCs可抑制血清TNF-α、IL-1β的过度表达，抑制SAP大鼠胰腺、肾脏中AQP1、VASPmRNA及蛋白的下降，抑制SAP大鼠胰腺、肾脏中MMP-9mRNA及蛋白的升高，减轻SAP大鼠胰腺、肾脏毛细血管内皮屏障的破坏。结论：在SAP肾损害发生时，移植的MSCs能够归巢到胰腺、肾脏受损区域，抑制炎症因子过度表达，调节血管内皮细胞结构相关蛋白的表达平衡，减轻毛细血管内皮屏障的破坏，维护内皮屏障的完整性，从而最终达到治疗SAP合并肾损害的目的。
[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- 伪 (TNF- 伪), interleukin-1 尾 interleukin-1 尾 (IL-1 尾), tumor necrosis factor- 伪 (TNF- 伪), 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- 伪伪 -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- 伪 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.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R657.51

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