To isolate bone marrow mesenchymal stem cells (BM-MSCs) and establish the model of chronic kidney disease (CKD) of Wuzhishan (WZS) mini-pig, and to study the repairment effect of BM-MSCs on CKD-induced renal fibrosis in vitro. Methods: Density gradient method was used to isolate and culture BM-MSCs. The cells were verified by morphology, phenotype, differentiation and so on. The left partial ureteral obstruction (LPUUO) was used to establish the CKD model, which was evaluated by B-ultrasound, single-photon emission computed tomography (SPECT), HE and Masson staining. The cells were divided into 3 groups, the tissue plus BM-MSCs group, the tissue group, and the BM-MSCs group, respectively. Seven days later, the supernatants were collected to observe the changes of hepatocyte growth factor (HGF) cumulative release. HE and Masson staining was used to observe the changes of renal tissue. Results: The isolated BM-MSCs possessed the features as follow: fibroblast-like adherent growth; positive in CD29 and CD90 expression while negative in CD45 expression; osteogenic induction and alizarin red staining were positive; alcian blue staining were positive after chondrogenic induction. Twelve weeks after the operation of LPUUO, B-ultrasound showed the thin renal cortical with pelvis effusion; SPETCT showed the left kidney delayed filling and renal impairment. The accumulation of HGF in the tissue plus BM-MSCs group was significantly higher than that in the tissue alone group at the 1st, 5th, 6th, 7th day, respectively (P<0.05). HE staining showed the different degree of renal lesions between the tissue plus BM-MSCs+CKD group and the tissue alone group, which was aggravated with the time going. Masson staining showed that the cumulative optical density of blue-stained collagen fibers in tissue plus BM-MSCs group was significantly lower than that in the tissue group at the 5th to 7th day (P<0.05). Conclusion: BM-MSCs from WZS mini-pig can inhibit or delay the progress of CKD-induced renal fibrosis through autocrine HGF in vitro.
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。.