兔肾间质纤维化模型的建立及自体骨髓间充质干细胞移植治疗的实验研究

发布时间：2018-04-02 22:03

本文选题：肾间质纤维化　切入点：骨髓间充质干细胞　出处：《昆明医学院》2009年硕士论文

【摘要】： 目的建立兔肾单侧输尿管结扎制作肾间质纤维化动物模型,将体外分离标记的自体骨髓间充质干细胞经股动脉插管,肾动脉灌注回输细胞,观察移植自体骨髓间充质干细胞能否归巢至损伤肾脏组织,及其在肾组织中的分布,能否促进其再生修复,从而改善肾功能。方法 ①兔肾间质纤维化模型的建立:用单侧输尿管结扎建立兔肾间质纤维化模型,动态观察4周,于造模的第0、1、2、3周末检测血尿素氮(Bun)、肌酐(Scr)含量及第3周采用单光子发射电子计算机断层成像仪(SPECT),扫描监测肾功能变化;4周末采用HE染色、PAS染色,观察肾组织变化。②细胞分离纯化:氯化铵破碎红细胞联合贴壁培养法分离培养兔骨髓间充质干细胞。③Brdu标记移植细胞:Brdu标记移植骨髓间充质干细胞,观察标记率。④移植细胞:于造模成功后的第4周经右股动脉插管行自体骨髓间充质干细胞肾动脉灌注回输细胞,分别在移植前、移植后的第1、3、5、7周行血生化Bun、Scr检测,肾组织病理学分析、Brdu免疫组化分析。结果 ①动物模型的血生化指标、病理组织学变化及SPECT监测:造模前两组血Bun、Scr值没有显著性差异(P＞0.05)。于造模后的第1周起两组血Bun、Scr逐渐升高,第3周达到最高水平,有显著差异性,与对照组比较有统计学意义(P＜0.05)。SPECT显示患侧肾明显变形,肾显影变淡,呈新月形,肾小球滤过率明显低于对照组,模型组兔肾间质纤维增生,呈纤维化改变,炎症细胞浸润,肾小管上皮部分脱落,浊肿,部分肾小管萎缩,肾小球有出血,其余未见明显异常。②移植细胞的标记:以30umol/L终浓度的Brdu孵育骨髓间充质干细胞72小时的阳性标记率均达95%以上,而标记组细胞形态、生长和增殖与对照组未见明显异常。③移植后肾功能检测:移植后第3周起血Bun、Scr逐渐降低,与造模后及对照组比较有统计学意义(P＜0.05)。④移植后肾病理组织学变化:移植后第1周所见肾小管上皮细胞变性、坏死脱落,腔内可见透明管型,在移植的第3周、第5周可见肾小管上皮细胞混浊肿胀,随着时间延长,坏死肾小管逐渐修复。⑤肾组织Brdu免疫组化分析:移植组于移植后的第1周逐渐显示肾小管和部分肾小球,Brdu阳性染色持续至实验结束,阳性染色主要分布于肾组织外的髓质和皮髓交界处,部分肾小球也呈阳性染色,而对照组Brdu染色则呈阴性。结论 ①通过病理学及SPECT监测,单侧输尿管结扎制作该模型具有明显的肾间质纤维化特征,为临床实验研究提供了实验模型。②经肾动脉灌注途径移植的骨髓间充质干细胞能迁移并定居于肾脏,参与了损伤肾脏结构的修复。③兔肾间质纤维化后,移植的自体骨髓间充质干细胞对血Bun和Scr的升高有一定的抑制作用,促进了肾脏功能的恢复。④骨髓间充质干细胞移植后第1周后即可进入肾脏,主要分布于肾脏的外髓区。
[Abstract]:PurposeAn animal model of renal interstitial fibrosis was established by unilateral ureteral ligation in rabbits. Autologous bone marrow-derived mesenchymal stem cells (MSCs) isolated and labeled in vitro were intubated through femoral artery and injected back into renal artery.To observe whether autologous bone marrow mesenchymal stem cells (BMSCs) can homing to the injured kidney tissue and its distribution in the renal tissue, and whether it can promote its regeneration and repair, thereby improving the renal function.Method1 Establishment of rabbit renal interstitial fibrosis model: rabbit renal interstitial fibrosis model was established by unilateral ureteral ligation and observed dynamically for 4 weeks.Blood urea nitrogen (bun) and creatinine (SCR) contents were detected at the end of the 3rd week of the model, and SPECTX was used to detect the changes of renal function by single photon emission computed tomography (SPECTX) at the 3rd week. The changes of renal function were detected by HE staining and pas staining at the end of 4 weeks.Observe the renal tissue changes .2 cell isolation and purification: ammonium chloride broken red blood cells combined with adherent culture method to isolate and culture rabbit bone marrow mesenchymal stem cells. 3 Brdu labeled transplantation cells labeled bone marrow mesenchymal stem cells labeled with Brdu labeled bone marrow mesenchymal stem cells.To observe the labeling rate of transplanted cells: the renal artery infusion of autologous bone marrow mesenchymal stem cells was performed through right femoral artery catheterization at the 4th week after the model was successfully established, and the blood biochemistry Bunn SCR was detected before transplantation and 57 weeks after transplantation, respectively.Renal histopathology and Brdu immunohistochemical analysis.Result1Blood biochemical indexes, histopathological changes and SPECT monitoring of the animal model: there was no significant difference between the two groups before the establishment of the model (P > 0.05).There was no obvious abnormal renal function in the growth and proliferation group. 3. The blood BunScr level decreased gradually from the third week after transplantation.At the 5th week, the tubule epithelial cells were turbid and swollen, and the tubuloepithelial cells showed turbid swelling over time.The positive staining was mainly located at the junction of medulla and dermis outside renal tissue, and some glomeruli were also positive staining, but Brdu staining was negative in the control group.Conclusion1 by pathological and SPECT monitoring, the model of unilateral ureteral ligation had obvious characteristics of renal interstitial fibrosis.The experimental model was provided for the clinical study. 2 the bone marrow mesenchymal stem cells transplanted by renal artery perfusion were able to migrate and settle in the kidney and participated in the repair of renal interstitial fibrosis in rabbits.Autologous bone marrow mesenchymal stem cells (BMSCs) could inhibit the increase of Bun and Scr and promote the recovery of renal function. 4. BMSCs could enter the kidney 1 week after transplantation, mainly distributed in the extramedullary area of the kidney.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R692.9;R-332

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