Xiang Yang, Long Jiale, Xing Jiansheng, Gao Yuanhui, Cheng Qing, Cai Yong, Liu Zhenxiang, Zhang Shufang, Chen Lie, Yang Chao, Bai Zhiming
Department of Urology, Affiliated Haikou Hospital, Xiangya School of Medicine, Central South University, Haikou 570208, China.
Department of Central Laboratory, Affiliated Haikou Hospital, Xiangya School of Medicine, Central South University, Haikou 570208, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2016 Dec 28;41(12):1260-1269. doi: 10.11817/j.issn.1672-7347.2016.12.003.
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.
分离五指山（WZS）小型猪骨髓间充质干细胞（BM-MSCs）并建立慢性肾脏病（CKD）模型，研究BM-MSCs对体外CKD诱导的肾纤维化的修复作用。方法：采用密度梯度法分离培养BM-MSCs。通过形态学、表型、分化等对细胞进行鉴定。采用左侧输尿管部分梗阻（LPUUO）建立CKD模型，通过B超、单光子发射计算机断层扫描（SPECT）、HE和Masson染色进行评估。将细胞分为3组，分别为组织加BM-MSCs组、组织组和BM-MSCs组。7天后，收集上清液观察肝细胞生长因子（HGF）累积释放变化。采用HE和Masson染色观察肾组织变化。结果：分离的BM-MSCs具有以下特点：呈成纤维细胞样贴壁生长；CD29和CD90表达阳性，CD45表达阴性；成骨诱导及茜素红染色阳性；软骨诱导后阿利新蓝染色阳性。LPUUO术后12周，B超显示肾皮质变薄伴肾盂积液；SPETCT显示左肾灌注延迟及肾功能损害。组织加BM-MSCs组在第1、5、6、7天HGF的累积量分别显著高于单纯组织组（P<0.05）。HE染色显示组织加BM-MSCs+CKD组与单纯组织组之间存在不同程度的肾损伤，且随时间加重。Masson染色显示组织加BM-MSCs组在第5至7天蓝色染色胶原纤维的累积光密度显著低于组织组（P<0.05）。结论：WZS小型猪的BM-MSCs在体外可通过自分泌HGF抑制或延缓CKD诱导的肾纤维化进程。
