Bone marrow mesenchymal stem cells stably expressing NICD which is controlled by Tet-On system in the treatment of renal ischemia-reperfusion injury

doi:10.3969/j.issn.2095-4344.0911

Abstract

Abstract:

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is the main cause of acute kidney injury (AKI). The treatment of renal IRI by bone marrow mesenchymal stem cells (MSC) has always been an issue of concern.
OBJECTIVE: To construct BMSCs stably expressing NICD controlled by Tet-On system in vitro (NICD-Tet-BMSCs), and to explore its therapeutic effect on renal IRI.
METHODS: We separated and cultured rat BMSCs by density gradient centrifugation, and constructed NICD-Tet-BMSCs in vitro. The cells were then transplanted to renal IRI rats. Serum creatinine level, the distribution of BMSCs in renal tissue and the expression of vascular endothelial growth factor and tumor necrosis factor α in the kidney were detected thereafter.
RESULTS AND CONCLUSION: NICD protein stably expressed in the NICD-Tet-BMSCs which was regulated by the doxycycline. Compared with the control group, the renal IRI rats undergoing NICD-Tet-BMSCs transplantation had significantly lower serum creatinine levels (P < 0.05), sustained cell proliferation, increased vascular endothelial growth factor protein expression and decreased tumor necrosis factor α protein expression in the kidney (P < 0.05). Therefore, NICD-Tet-BMSCs can be successfully established in vitro, and promote the reparation of renal IRI in rats.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Kidney, Reperfusion Injury, Tissue Engineering

CLC Number:

R394.2

Xi Xiao-qing, Hu Hong-lin, Zou Cong, Fang Zhou-qing, Kuang Ren-rui, Ye Zhen-feng, Huang Ya-wei. Bone marrow mesenchymal stem cells stably expressing NICD which is controlled by Tet-On system in the treatment of renal ischemia-reperfusion injury[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 4035-4040.

Figures/Tables 2

2.1 实验动物数量分析 40只SD大鼠，随机分3组：NICD-Tet-BMSCs组14只、Tet-BMSCs组14只和肾缺血再灌注损伤组12只，术后1 d NICD-Tet-BMSCs组和Tet-BMSCs组各死亡1只，其余动物均存活。 2.2 NICD稳定诱导表达的NICD-Tet-BMSCs建立 pTRE-NICD质粒不含抗性筛选标记，将100 ng Linear Puromycin Marker与2 μg pTRE-NICD质粒共同转染Tet-on 3G-BMSCs，用1 mg/L 嘌呤霉素筛选抗性克隆。挑取单细胞克隆传代于24孔细胞培养板，分别用1 mg/L强力霉素或等量ddH2O处理，24 h后用Western blot检测不同细胞克隆的NICD蛋白表达情况。结果显示，在挑取的不同嘌呤霉素抗性克隆中，1#和7#克隆见NICD表达受强力霉素调控，其他克隆强力霉素诱导后未发现NICD蛋白表达，但7#表达水平比较低，只有1#克隆NICD蛋白高效表达且受强力霉素的诱导调控(图1A)，将1#克隆视为NICD-Tet-BMSCs，并传代培养。而对照组Tet-BMSCs的NICD蛋白表达较低，并不受强力霉素调控(图1B)。 2.3 NICD-Tet-BMSCs移植对肾缺血再灌注损伤大鼠肾功能的影响肾缺血再灌注后1，3，7，10，14 d，分别抽血检测大鼠血清肌酐水平。结果显示：术后1 d，各组大鼠血清肌酐均升高，组间差异无显著性意义(P > 0.05)；而术后3-14 d，与肾缺血再灌注损伤组相比，NICD-Tet-BMSCs组和Tet-BMSCs组血清肌酐水平均明显降低，NICD-Tet-BMSCs组降低尤为明显，组间差异有显著性意义(P < 0.05)，见图2。 2.4 移植的NICD-Tet-BMSCs在大鼠肾组织中的分布肾脏缺血再灌注后2，5，10 d，处死大鼠，取出肾脏，做快速冰冻切片，在荧光显微镜下观察DAPI标记的BMSCs在肾组织中的分布。结果显示：NICD-Tet-BMSCs组和Tet-BMSCs组细胞移植后第2天在肾组织中均未见DAPI阳性细胞；第5天则有大量的DAPI阳性细胞。而NICD-Tet-BMSCs组第10天在肾组织中仍可见一些DAPI阳性细胞，但Tet-BMSCs组肾组织中未见明显DAPI阳性细胞，DAPI阳性细胞的胞核呈蓝色荧光，主要分布于肾脏外髓质肾小管中，见图3。 2.5 移植NICD-Tet-BMSCs对肾缺血再灌注损伤后肾组织中血管内皮生长因子和肿瘤坏死因子α的影响血管内皮生长因子是促进血管形成的强有力的生长因子，肾脏缺血再灌注后2，5 d，采用Western blot法检测各组肾组织中血管内皮生长因子蛋白表达，结果显示NICD-Tet-BMSCs组和Tet-BMSCs组血管内皮生长因子蛋白表达量均升高，NICD-Tet-BMSCs组升高更为明显(图4)。肿瘤坏死因子α是器官损伤后最早产生的多功能炎症细胞因子之一，肾脏缺血再灌注后2，5 d，采用Western blot法检测各组肾组织中肿瘤坏死因子α蛋白表达，结果显示：NICD-Tet-BMSCs组和Tet-BMSCs组肿瘤坏死因子α蛋白表达量均降低，而且NICD-Tet-BMSCs组降低更为明显(图5)。"

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