骨髓间充质干细胞移植治疗大鼠痛风肾

发布时间：2018-02-26 06:31

本文关键词： 痛风肾慢性肾衰竭骨髓间充质干细胞上皮-间叶组织转化 TrxR1　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨经鼠尾静脉移植的骨髓间充质干细胞(BM-MSCs)对大鼠痛风肾的影响,具体为延缓上皮到间叶组织转化(EMT)、促进肾脏细胞分化和生长,以及抗氧化应激等方面的作用。并进一步研究其相应的机制。方法:1、取36只幼年雄性Wistar大鼠,质量(90±10)g,随机分成两组,一组12只,为正常组;另一组24只,为造模组,造模组采用腺嘌呤200mg/kg/d灌胃4周制作痛风肾大鼠模型。2、确保痛风肾模型制作成功后,造模组随机分成两组,分别为即将接受骨髓间充质干细胞(BM-MSCs)移植的治疗组,与即将接受磷酸盐缓冲液(PBS)注射的对照模型组。现有三组大鼠,即为正常组、治疗组和对照模型组。3、在造模的同时,取幼年雄性Wistar大鼠6只,质量为(50±10)g,牺牲大鼠取其股骨和胫骨的骨髓,经密度梯度离心法和贴壁筛选法体外培养骨髓间充质干细胞,经过流式细胞仪对细胞表型进行检测,以及对细胞形态观察、细胞多项分化潜能的鉴定,确定培养的细胞为大鼠骨髓间充质干细胞。4、造模成功后,经眼眶取血和留取24小时尿,测量各组的肾功能指标尿素氮和肌酐和24小时尿蛋白。24小时后治疗组于经鼠尾静脉移植入骨髓间充质干细胞(BM-MSCs),同时对照模型组经鼠尾静脉注入相同量的磷酸盐缓冲液(PBS)。5、移植骨髓间充质干细胞6周后,收集大鼠的血液和尿液标本用来测定血肌酐、尿素氮和24小时尿蛋白含量;牺牲大鼠取其肾脏做石蜡标本,对石蜡标本切片进行H-E染色、糖原染色和马松染色,观察并半定量评分评价各组肾脏病理情况。6、免疫组织化学法测定大鼠肾脏石蜡标本切片中的转化生长因子β1(TGF-β1)的表达水平,蛋白免疫印迹法(Western-Blot法)测定肾组织中P38、P-P38、硫氧还蛋白还原酶1(TrxR1)的表达情况。结果:1、经腺嘌呤诱导制作的痛风肾大鼠相比正常组大鼠出现明显的肾功能降低,大量蛋白尿,大体标本呈“大白肾”,形态学可见肾间质和肾小管尿酸结晶沉积,肾间质纤维化、炎症细胞浸润、肾小管上皮细胞坏死等,提示痛风肾模型制造成功,并出现了慢性肾功能衰竭(CRF)。2、体外培养的骨髓间充质干细胞(BM-MSCs),经流式细胞仪检测显示其表面CD90高表达,CD45抗原低表达。经成脂和成骨诱导成功,出现红色脂滴和红色钙化结节。证明成功培养骨髓间充质干细胞。3、移植BM-MSCs后,治疗组较模型组而言,肾功能明显改善,尿素氮和肌酐水平降低,24小时尿蛋白减少。TGF-β1和P38(尤其是P-P38)表达明显降低,TrxR1表达明显增加,差异具有统计学意义。结论:BM-MSCs改善腺嘌呤诱导的痛风肾/慢性肾功能衰竭大鼠的肾功能,这可能与通过增加TrxR1的表达进而促进转分化与肾脏细胞生长以及抗氧化应激有关;同时与降低TGF-β1水平减轻EMT有关,此过程可能是通过抑制TNF-α/P-P38信号通路实现的。
[Abstract]:Objective: to investigate the effects of bone marrow mesenchymal stem cells (BM-MSCs) transplanted through tail vein on gout kidney in rats, in order to delay the transformation of epithelium to mesenchymal tissue and promote the differentiation and growth of renal cells. Methods 36 young male Wistar rats, 90 卤10 g in weight, were randomly divided into two groups, one group (12 rats) as normal group, the other group (24 rats) as model group. The rat model of gout kidney was made by administration of adenine 200 mg / kg / d for 4 weeks. After the model was made successfully, the model group was randomly divided into two groups: the treatment group, which was about to receive BM-MSCstransplantation of bone marrow mesenchymal stem cells. There were three groups of rats, namely normal group, treatment group and control group. 6 young male Wistar rats were taken at the same time. The bone marrow of femur and tibia was harvested from rats with a mass of 50 卤10g. Bone marrow mesenchymal stem cells (BMSCs) were cultured by density gradient centrifugation and adherent screening in vitro. Cell phenotypes were detected by flow cytometry, and cell morphology was observed. The differentiation potential of the cells was identified as rat bone marrow mesenchymal stem cells (BMSCs) .4. after the successful establishment of the model, blood was taken from the orbit and urine was retained for 24 hours. Renal function parameters of each group were measured: urea nitrogen and creatinine and 24 hour urine protein. 24 hours later, the treatment group was transplanted into bone marrow mesenchymal stem cells (BM-MSCsN) through the tail vein of mice, while the control group was injected with the same amount of phosphate bradycardia through the tail vein of mice. After 6 weeks of transplantation of bone marrow mesenchymal stem cells, The blood and urine samples of rats were collected to determine the contents of creatinine, urea nitrogen and 24 hour urine protein. The kidneys of the rats were taken as paraffin samples, and the paraffin sections were stained with H-E, glycogen and Ma Song. Renal pathology was evaluated by observing and semi-quantitative scoring. The expression of TGF- 尾 _ 1 in paraffin section of rat kidney was determined by immunohistochemical method, and the expression of TGF- 尾 _ 1 in paraffin section of kidney was determined by immunohistochemistry, and the expression of TGF- 尾 _ 1 was determined by immunohistochemistry. The expression of P38 P-P38, thioredoxin reductase (1) TrxR1) in renal tissue was determined by Western blot. Results: 1. Compared with normal rats, the renal function of gout kidney induced by adenine was significantly decreased, and a large amount of proteinuria was found in the rats with gout induced by adenine. The gross specimens showed "large white kidney", and the renal interstitial and renal tubulointerstitial uric acid crystal deposition, renal interstitial fibrosis, inflammatory cell infiltration, renal tubular epithelial cell necrosis and so on were observed in morphology, which indicated that the model of gout kidney was made successfully. The bone marrow mesenchymal stem cells (BM-MSCs1) were cultured in vitro. Flow cytometry showed that the high expression of CD45 antigen and the high expression of CD45 antigen on the surface of the bone marrow mesenchymal stem cells were successfully induced by lipogenesis and osteogenesis. It was proved that bone marrow mesenchymal stem cells. 3 was cultured successfully. After transplantation of BM-MSCs, the renal function of the treatment group was significantly improved than that of the model group. The levels of urea nitrogen and creatinine decreased the expression of urinary protein. TGF- 尾 1 and P38 (especially P-P38) decreased significantly, and the expression of TrxR1 increased significantly. Conclusion: BM-MSCs can improve the renal function of rats with adenine-induced gout / chronic renal failure, which may be related to the growth of renal cells and antioxidant stress by increasing the expression of TrxR1. At the same time, it is related to the reduction of TGF- 尾 1 level to reduce EMT, which may be achieved by inhibiting the TNF- 伪 / P-P38 signaling pathway.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.7;R692

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