本文選題：糖尿病腎病　切入點：骨髓間充質(zhì)干細(xì)胞　出處：《中國免疫學(xué)雜志》2017年05期

【摘要】：目的:觀察骨髓間充質(zhì)干細(xì)胞分離、鑒定方法及在糖尿病大鼠腎臟保護(hù)中的機制。方法:取大鼠31只,1只大鼠采用貼壁培養(yǎng)法分離大鼠骨髓間充質(zhì)干細(xì)胞,利用相差顯微鏡觀察細(xì)胞形態(tài),Real-time PCR檢測培養(yǎng)細(xì)胞表面分子量。30只大鼠采用鏈脲佐菌素腹腔注射構(gòu)建糖尿病大鼠模型。根據(jù)處理方法不同分為對照組(n=15)和干細(xì)胞治療組(n=15)。對照組大鼠采用胰島素聯(lián)合普羅布考治療,干細(xì)胞治療組植入干細(xì)胞治療,治療前、后檢測2組大鼠空腹血糖、24 h尿蛋白排泄、肌酐清除率、腎重比體重;采用PAS染色觀察2組腎組織情況;采用Western blot檢測腎臟組織相關(guān)基因和蛋白水平。結(jié)果:原代骨髓間充質(zhì)干細(xì)胞培養(yǎng)24 h后換液時可見細(xì)胞分布不均勻,在培養(yǎng)瓶呈分片聚集,形態(tài)不規(guī)則。培養(yǎng)7 d后細(xì)胞集落多,細(xì)胞規(guī)則,呈長梭形、橢圓形,折光性強,細(xì)胞間相互融合鋪滿瓶底;骨髓間充質(zhì)干細(xì)胞傳代培養(yǎng)后細(xì)胞形態(tài)均一,呈長梭形,輪廓清除,折光性強,生長迅速,部分細(xì)胞呈漩渦狀;Real-time PCR檢測結(jié)果顯示:SH2、CD44、CD31呈陽性表達(dá),CD34呈陰性表達(dá);觀察組治療后空腹血糖、24 h尿蛋白排泄、肌酐清除率、腎重比體重,顯著低于對照組(P0.05);干細(xì)胞治療組大鼠腎臟組織PAS染色結(jié)果顯示:腎小球結(jié)構(gòu)清晰,形態(tài)規(guī)則,腎小球內(nèi)細(xì)胞排列規(guī)則,基底膜和包曼氏囊清晰。對照組大鼠腎小球體積增大,系膜區(qū)增寬,基質(zhì)明顯增多;干細(xì)胞治療組治療后Bcl-2蛋白水平得到顯著提高,Bax和Caspase-3蛋白水平得到顯著下降。對照組Bcl-2蛋白水平得到顯著降低,Bax和Caspase-3蛋白水平得到提高(P0.05)。2組治療前氧化應(yīng)激ROS、MDA、SOD指標(biāo)差異無統(tǒng)計學(xué)意義(P0.05);干細(xì)胞治療組治療后氧化應(yīng)激ROS、MDA、SOD指標(biāo),低于對照組(P0.05)。結(jié)論:利用貼壁培養(yǎng)法能分離大鼠骨髓間充質(zhì)干細(xì)胞并利用綠色熒光蛋白進(jìn)行體外標(biāo)記,植入骨髓間充質(zhì)干細(xì)胞能保護(hù)機體腎臟,抑制糖尿病腎臟的氧化應(yīng)激反應(yīng)。
[Abstract]:Objective: to observe the isolation, identification and mechanism of bone marrow mesenchymal stem cells (BMSCs) in diabetic rats. Methods: bone marrow mesenchymal stem cells (BMSCs) were isolated from 31 rats by adherent culture. A diabetic rat model was established by intraperitoneal injection of streptozotocin (streptozotocin) and stem cell therapy. Rats in the control group were treated with insulin combined with probucol. Stem cell group was implanted with stem cell therapy. Before and after treatment, 24 h urinary protein excretion, creatinine clearance rate and weight ratio of kidney were detected in rats of two groups. The renal tissue was observed by PAS staining. Western blot was used to detect the level of genes and proteins related to renal tissue. Results: after 24 h culture of primary bone marrow mesenchymal stem cells, the distribution of cells was not uniform and the cells were clustered in culture flask. After 7 days of culture, the cells were in the shape of long spindle shape, elliptical shape, strong refraction, and the intercellular fusion was covered with bottle bottom, the morphology of bone marrow mesenchymal stem cells was uniform and fusiform after subculture. Outline clearance, strong refraction and rapid growth. The results of real-time PCR analysis of some cells showed that the positive expression of CD31 was negative and the urine protein excretion, creatinine clearance rate and weight of kidney were 24 h after treatment in the observation group. The results of PAS staining showed that glomerular structure was clear, morphology was regular, glomerular cell arrangement was regular, basement membrane and Bowman's sac were clear, and glomerular volume in control group was increased. The Mesangial area was widened and the matrix was increased obviously. The levels of Bcl-2 protein and Caspase-3 protein were significantly increased after treatment in stem cell treatment group, and Bcl-2 protein and Caspase-3 protein level in control group were significantly decreased after treatment. In the control group, the levels of Bcl-2 protein and Caspase-3 protein were increased significantly after treatment, and the oxidative stress rosier MDA-SOD finger was increased before treatment in the control group. There was no significant difference in the standard value (P 0.05). The oxidative stress rosier MDA-SOD index was found in the stem cell treatment group after treatment. Conclusion: rat bone marrow mesenchymal stem cells can be isolated by adherent culture, labeled with green fluorescent protein in vitro, and transplanted into bone marrow mesenchymal stem cells to protect kidney. Inhibition of oxidative stress in diabetic kidneys.
【作者單位】： 河北醫(yī)科大學(xué)第一醫(yī)院;
【基金】：河北省衛(wèi)生廳重點科技研究計劃(160211K102)
【分類號】：R587.2;R692.9

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本文編號：1669578