近交系五指山小型猪骨髓间充质干细胞体外分泌肝细胞生长因子延缓慢性肾脏病纤维化进展

发布时间：2018-06-18 05:47

本文选题：近交系五指山小型猪 + 骨髓间充质干细胞　；参考：《中南大学学报(医学版)》2016年12期

【摘要】：目的:分离培养近交系五指山小型猪骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs),并建立其慢性肾脏病(chronic kidney disease,CKD)模型,体外研究同物种BM-MSCs对CKD肾纤维化的修复作用及其可能机制。方法:采用密度梯度法分离培养BM-MSCs,并从细胞形态、表面抗原、分化能力等方面鉴定;采用左侧输尿管部分梗阻(left partial ureteral obstruction,LPUUO)方法制作CKD模型,并通过B超、单光子发射计算机断层成像术(single-photon emission computed tomography,SPECT)、病理染色等评估;体外实验分为肾组织与BM-MSCs共培养、肾组织单独培养、BM-MSCs单独培养3组,体外培养7 d,每天收集3组上清液,酶联免疫吸附法测定肝细胞生长因子(hepatocyte growth factor,HGF)累积分泌量。肾组织行HE染色,Masson染色。结果:分离培养的BM-MSCs表达抗原CD29,CD90而不表达CD45,成骨诱导茜素红染色阳性,成软骨诱导阿利新蓝染色阳性。LPUUO术后12周,B超示左肾皮质变薄、肾盂积液;SPECT示左肾充盈延迟、梗阻性肾功能受损。上清液HGF累积含量示BM-MSCs+CKD肾组织共同培养组第1,5,6,7天HGF累积分泌量明显高于CKD肾组织单独培养组(P0.05)。HE染色显示BM-MSCs+CKD肾组织共同培养组和CKD肾组织单独培养组中肾均出现不同程度的病变,并随时间延长加重,但CKD肾组织单独培养组病变更严重;Masson染色显示BM-MSCs+CKD肾组织共同培养组术后第5,6,7天肾组织被染成蓝色的胶原纤维的累计光密度值明显低于CKD肾组织单独培养组(P0.05)。结论:近交系五指山小型猪BM-MSCs体外分泌HGF,抑制或延缓CKD纤维化进展。
[Abstract]:Objective: to isolate and culture bone marrow mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSCs) of the inbred line Wuzhishan miniature pig, and to establish its chronic kidney disease model, and to study the repair effect of BM-MSCs of the same species on renal fibrosis and its possible mechanism in vitro. Methods: BM-MSCs were isolated and cultured by density gradient method and identified from cell morphology, surface antigen and differentiation ability. Single photon emission computed tomography (SPECT) technique was used to evaluate single-photon emission computed tomphography, pathological staining, etc. In vitro, the rats were divided into three groups: renal tissue co-cultured with BM-MSCs, kidney tissue cultured alone and BM-MSCs cultured alone, cultured in vitro for 7 days, and the supernatants of three groups were collected every day. The cumulative secretion of hepatocyte growth factor was determined by enzyme linked immunosorbent assay (Elisa). The renal tissue was stained by HE staining and Masson staining. Results: BM-MSCs expressed CD29 CD90 but did not express CD45. Osteoblast-induced alizarin red staining was positive. Twelve weeks after operation, the left renal cortex was thinned by B-ultrasound, and left renal filling was delayed by SPECT. Obstructive renal function impairment. The accumulation of HGF in supernatant of BM-MSCs CKD group was significantly higher than that of CKD group and CKD tissue culture group on the 7th day of HGF accumulation in BM-MSCs CKD co-culture group and CKD renal tissue co-culture group. The results of HE staining showed that BM-MSCs CKD renal tissue co-culture group and CKD renal tissue culture group had both renal secretion. With varying degrees of disease, And increased over time, However, Masson staining showed that the cumulative optical density of the blue collagen fibers in the renal tissue of BM-MSCs CKD co-cultured group was significantly lower than that of the CKD renal tissue culture group on the 7th day after operation. Conclusion: BM-MSCs from Wuzhishan miniature pig secreted HGF in vitro and inhibited or delayed the progress of CKD fibrosis.
【作者单位】：中南大学湘雅医学院附属海口医院泌尿外科;中南大学湘雅医学院附属海口医院中心实验室;成都市第二人民医院泌尿外科;中南大学湘雅医学院附属海口医院B超诊断科;中南大学湘雅医学院附属海口医院腔镜中心;
【基金】：国家国际科技合作项目(2013DFA51290) 海南省自然科学基金(20168317) 海南省卫生厅项目(琼卫科教[2014]51-47;[2014]51-64)~~
【分类号】：R692

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