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中华肾病研究电子杂志 ›› 2015, Vol. 04 ›› Issue (04) : 200 -207. doi: 10.3877/cma.j.issn.2095-3216.2015.04.008

所属专题: 文献

论著

HO-1基因修饰对急性肾损伤微环境下骨髓间充质干细胞增生分化的影响
刘楠梅1, 王会玲1, 韩国锋1, 于秀峙1, 田军1, 胡伟锋1, 张金元1,()   
  1. 1. 200052 上海,中国人民解放军第四五五医院肾脏科 南京军区肾脏病研究所
  • 出版日期:2015-08-28
  • 通信作者: 张金元
  • 基金资助:
    上海市基础研究重大项目(12DJ1400203); 国家自然科学基金青年项目(81300568); 全军医学科技青年培育基金(13QNP050); 上海市优秀青年医学人才培养计划基金(XYQ2013088); 上海市青年科技启明星计划项目(12QA1405000)

Effect of HO-1 gene modification on proliferation and differentiation of bone marrow-derived mesenchymal stem cells under the acute kidney injury microenvironment

Nanmei Liu1, Huiling Wang1, Guofeng Han1, Xiuzhi Yu1, Jun Tian1, Weifeng Hu1, Jinyuan Zhang1,()   

  1. 1. Department of Nephrology, The 455th Hospital of PLA, Kidney Research Institute of Nanjing Military Area, Shanghai 200052, China
  • Published:2015-08-28
  • Corresponding author: Jinyuan Zhang
  • About author:
    Corresponding author: Zhang Jinyuan, Email:
引用本文:

刘楠梅, 王会玲, 韩国锋, 于秀峙, 田军, 胡伟锋, 张金元. HO-1基因修饰对急性肾损伤微环境下骨髓间充质干细胞增生分化的影响[J]. 中华肾病研究电子杂志, 2015, 04(04): 200-207.

Nanmei Liu, Huiling Wang, Guofeng Han, Xiuzhi Yu, Jun Tian, Weifeng Hu, Jinyuan Zhang. Effect of HO-1 gene modification on proliferation and differentiation of bone marrow-derived mesenchymal stem cells under the acute kidney injury microenvironment[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2015, 04(04): 200-207.

目的

观察经血红素加氧酶-1(HO-1)基因修饰的骨髓间充质干细胞(BMSCs)在急性肾损伤(AKI)微环境下的增生分化,并探讨其机制。

方法

Gateway技术构建含HO-1目的基因的质粒,同时构建仅含示踪基因eGFP的对照载体;脂质体法转染293FT细胞获得慢病毒原液lenti-HO-1-eGFP和lenti-eGFP;稀释后分别感染BMSCs获得HO-1-BMSCs和eGFP-BMSCs(空载体对照),检测转染细胞的活力、分化潜能。制作缺血再灌注诱导AKI大鼠的肾脏匀浆上清(AKI-KHS),以正常大鼠肾脏匀浆上清(N-KHS)作为对照,分别干预BMSCs、eGFP-BMSCs和HO-1-BMSCs,构成空白组(BMSCs组)、对照组(BMSCs/N-KHS组)、BMSCs/AKI-KHS组、eGFP-BMSCs/AKI-KHS组和HO-1-BMSCs/AKI-KHS组,于37℃、5% CO2培养箱中培养3 d。MTT法检测培养BMSCs的生长抑制率,流式细胞仪检测细胞凋亡,透射电镜观察细胞超微结构变化,免疫组化法检测细胞内角蛋白18(CK18)表达,Western印迹对各组细胞内HO-1、磷酸化丝氨酸/苏氨酸蛋白激酶(pAKT)、磷酸化细胞外信号调节激酶(pERK)水平进行检测。用SPSS19.0统计软件进行统计学分析。

结果

基因修饰并未改变BMSCs活力及多向分化潜能。与对照组比,BMSCs/AKI-KHS组的细胞生长抑制率和凋亡阳性细胞比例显著增加(t=12.581,t=16.283;P<0.05);经HO-1基因修饰后,HO-1-BMSCs/AKI-KHS组的细胞生长抑制率和凋亡阳性细胞比例均有显著下降(t=5.958,t=7.957;P<0.05)。AKI-KHS可诱导BMSCs出现肾小管上皮细胞样分化的超微结构改变,胞浆中可观察到CK18表达,以HO-1-BMSCs/AKI-KHS组的CK18细胞比例最高(t=4.057,P<0.05)。与BMSCs/AKI-KHS组相比,HO-1-BMSCs/AKI-KHS组的细胞内HO-1蛋白水平显著增高(t=4.163,P<0.05),并伴随着细胞内pAKT、pERK蛋白水平显著增高(tpAKT=14.305,tpERK=7.148;P<0.05)。

结论

HO-1基因修饰可改善AKI微环境下培养BMSCs的增殖,细胞凋亡减轻,向肾小管上皮样细胞转分化能力增加,HO-1的过表达及其下游的AKT、ERK为发挥作用的可能信号通路。

Objective

To investigate the effect of heme oxygenase-1 (HO-1) gene modification on the proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) under the acute kidney injury (AKI) microenvironment in vitro and the possible mechanism.

Methods

Plasmids were constructed by the gateway technology that contained HO-1 target gene (eGFP as the tracing-gene) or only eGFP as the control, and were transfected into the 293FT cells through the liposome method to get the lenti-HO-1-eGFP/puro and the lenti-eGFP/puro, which then infected BMSCs to obtain HO-1-BMSCs and eGFP-BMSCs, testing the activity and differentiation potential of transfected cells. The ischemia/reperfusion-AKI kidney homogenate supernatant (KHS) and the control N-KHS were harvested and used to cultivate with BMSCs, eGFP-BMSCs, or HO-1-BMSCs, respectively, consisting of five groups: the blank group (BMSCs group), control group (BMSCs/N-KHS group), BMSCs/AKI-KHS group, eGFP-BMSCs/AKI-KHS group, and HO-1-BMSCs/AKI-KHS group, at 37 ℃ in 5% CO2 for 3 days. The MTT method was used to detect the growth inhibitory rate of BMSCs, flow cytometry to detect the cell apoptosis, the transmission electron microscope (TEM) to observe the cell ultrastructure changes, immunohistochemistry to detect the expression of cytokeratin 18 (CK18), and Western blot to detect the protein expressions of HO-1, phospho-AKT (pAKT), and phospho-ERK (pERK).

Results

The cell viability and differentiation potential of BMSCs were not changed by the gene modification. Compared with the BMSCs/N-KHS group, the growth inhibitory rate of the BMSCs/AKI-KHS group as well as the proportion of apoptotic cells increased significantly (t=12.581, t=16.283, P<0.05), which, after HO-1 gene modification, however, significantly decreased in the HO-1-BMSCs/AKI-KHS group (t=5.958, t=7.957, P<0.05). AKI-KHS induced ultrastructural change of the renal tubular epithelial differentiation in the cultured BMSCs with CK18 expression in the cytoplasm. The proportion of the CK18+ cells was the highest in the HO-1-BMSCs/AKI-KHS group (t=4.057, P<0.05). Compared with the BMSCs/AKI-KHS group, the HO-1-BMSCs/AKI-KHS had significantly increased cellular expression of HO-1 (t=4.163, P<0.05), pAKT (tpAKT=14.305, P<0.05), and pERK (tpERK=7.148; P<0.05).

Conclusions

In the AKI microenvironment, HO-1 gene modification could improve the proliferation of BMSCs, enhance the ability of BMSCs to differentiate into renal tubular epithelial cells, and decrease the apoptosis of BMSCs. HO-1 overexpression with the downstream AKT and ERK signaling pathway might be the possible mechanism.

图1 慢病毒感染并经药物筛选获得的纯化HO-1-BMSCs和eGFP-BMSCs
图2 转染BMSCs的生长情况及生物学特性检测结果
图3 各组细胞的生长抑制率比较
图4 各组细胞的凋亡率比较
图5 透射电镜下BMSCs的超微结构
图6 各组细胞内CK18表达率
图7 各组细胞内HO-1表达强度
图8 各组细胞内pAKT和pERK表达
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