本文關(guān)鍵詞： 痛風(fēng)腎 慢性腎衰竭 骨髓間充質(zhì)干細胞 上皮-間葉組織轉(zhuǎn)化 TrxR1　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討經(jīng)鼠尾靜脈移植的骨髓間充質(zhì)干細胞(BM-MSCs)對大鼠痛風(fēng)腎的影響,具體為延緩上皮到間葉組織轉(zhuǎn)化(EMT)、促進腎臟細胞分化和生長,以及抗氧化應(yīng)激等方面的作用。并進一步研究其相應(yīng)的機制。方法:1、取36只幼年雄性Wistar大鼠,質(zhì)量(90±10)g,隨機分成兩組,一組12只,為正常組;另一組24只,為造模組,造模組采用腺嘌呤200mg/kg/d灌胃4周制作痛風(fēng)腎大鼠模型。2、確保痛風(fēng)腎模型制作成功后,造模組隨機分成兩組,分別為即將接受骨髓間充質(zhì)干細胞(BM-MSCs)移植的治療組,與即將接受磷酸鹽緩沖液(PBS)注射的對照模型組�，F(xiàn)有三組大鼠,即為正常組、治療組和對照模型組。3、在造模的同時,取幼年雄性Wistar大鼠6只,質(zhì)量為(50±10)g,犧牲大鼠取其股骨和脛骨的骨髓,經(jīng)密度梯度離心法和貼壁篩選法體外培養(yǎng)骨髓間充質(zhì)干細胞,經(jīng)過流式細胞儀對細胞表型進行檢測,以及對細胞形態(tài)觀察、細胞多項分化潛能的鑒定,確定培養(yǎng)的細胞為大鼠骨髓間充質(zhì)干細胞。4、造模成功后,經(jīng)眼眶取血和留取24小時尿,測量各組的腎功能指標(biāo)尿素氮和肌酐和24小時尿蛋白。24小時后治療組于經(jīng)鼠尾靜脈移植入骨髓間充質(zhì)干細胞(BM-MSCs),同時對照模型組經(jīng)鼠尾靜脈注入相同量的磷酸鹽緩沖液(PBS)。5、移植骨髓間充質(zhì)干細胞6周后,收集大鼠的血液和尿液標(biāo)本用來測定血肌酐、尿素氮和24小時尿蛋白含量;犧牲大鼠取其腎臟做石蠟標(biāo)本,對石蠟標(biāo)本切片進行H-E染色、糖原染色和馬松染色,觀察并半定量評分評價各組腎臟病理情況。6、免疫組織化學(xué)法測定大鼠腎臟石蠟標(biāo)本切片中的轉(zhuǎn)化生長因子β1(TGF-β1)的表達水平,蛋白免疫印跡法(Western-Blot法)測定腎組織中P38、P-P38、硫氧還蛋白還原酶1(TrxR1)的表達情況。結(jié)果:1、經(jīng)腺嘌呤誘導(dǎo)制作的痛風(fēng)腎大鼠相比正常組大鼠出現(xiàn)明顯的腎功能降低,大量蛋白尿,大體標(biāo)本呈“大白腎”,形態(tài)學(xué)可見腎間質(zhì)和腎小管尿酸結(jié)晶沉積,腎間質(zhì)纖維化、炎癥細胞浸潤、腎小管上皮細胞壞死等,提示痛風(fēng)腎模型制造成功,并出現(xiàn)了慢性腎功能衰竭(CRF)。2、體外培養(yǎng)的骨髓間充質(zhì)干細胞(BM-MSCs),經(jīng)流式細胞儀檢測顯示其表面CD90高表達,CD45抗原低表達。經(jīng)成脂和成骨誘導(dǎo)成功,出現(xiàn)紅色脂滴和紅色鈣化結(jié)節(jié)。證明成功培養(yǎng)骨髓間充質(zhì)干細胞。3、移植BM-MSCs后,治療組較模型組而言,腎功能明顯改善,尿素氮和肌酐水平降低,24小時尿蛋白減少。TGF-β1和P38(尤其是P-P38)表達明顯降低,TrxR1表達明顯增加,差異具有統(tǒng)計學(xué)意義。結(jié)論:BM-MSCs改善腺嘌呤誘導(dǎo)的痛風(fēng)腎/慢性腎功能衰竭大鼠的腎功能,這可能與通過增加TrxR1的表達進而促進轉(zhuǎn)分化與腎臟細胞生長以及抗氧化應(yīng)激有關(guān);同時與降低TGF-β1水平減輕EMT有關(guān),此過程可能是通過抑制TNF-α/P-P38信號通路實現(xiàn)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 鹵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 鹵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- 偽 / P-P38 signaling pathway.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R589.7;R692

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1 韓艷;崔建軍;李秀花;賈曉靜;;CD45和CD90鑒定大鼠骨髓間充質(zhì)干細胞[J];中國藥物與臨床;2014年12期

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