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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(P<0.05)。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(P<0.05)。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。
OBJECTIVE: To isolate and culture bone marrow mesenchymal stem cells (BM-MSCs) of Wuzhishan mini-inbred line and to establish a model of chronic kidney disease (CKD) in vitro. Repair effect of CKD on renal fibrosis and its possible mechanism. Methods: BM-MSCs were isolated and cultured by density gradient method and identified from cell morphology, surface antigen and differentiation ability. CKD model was made by left partial ureteral obstruction (LPUUO) Single-photon emission computed tomography (SPECT), pathological staining and other assessment; in vitro experiments were divided into renal tissue and BM-MSCs co-culture, kidney tissue cultured alone, BM-MSCs cultured alone in 3 groups, in vitro Three groups of supernatants were collected daily for 7 days. The cumulative secretion of hepatocyte growth factor (HGF) was measured by enzyme-linked immunosorbent assay. Kidney tissue HE staining, Masson staining. Results: The isolated BM-MSCs expressed antigens CD29, CD90 but not CD45, osteogenic induction alizarin red staining positive cartilage-induced Aliskiren staining. After 12 weeks of LPUUO, B-ultrasound showed left renal cortex thinning and hydronephrosis; SPECT showed delayed filling of the left kidney and impaired renal function. The accumulation of HGF in supernatant showed that the cumulative amount of HGF accumulated on the 1st, 5th, 6th and 7th day in BM-MSCs + CKD renal tissue co-culture group was significantly higher than that in CKD alone group (P <0.05). Hematoxylin-eosin staining showed that the kidney of BM-MSCs + CKD renal tissue co-culture group and CKD renal tissue culture group were different degrees of lesions, and with the prolongation of aggravation, but CKD renal tissue alone group more serious lesions; Masson staining showed BM -MSCs + CKD renal tissue co-culture group was significantly lower than CKD renal tissue group (P <0.05) on the 5th, 6th and 7th day after operation. Conclusion: Inbred Wuzhishan mini-pig BM-MSCs can secrete HGF in vitro to inhibit or delay the progression of CKD fibrosis.