切换至 "中华医学电子期刊资源库"
高级检索
中华肾病研究电子杂志

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:
全文 ( ) 下载PDF ( ) 导出引用
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/OL]. 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/OL]. 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。

关键词: 血红素加氧酶-1, 骨髓间充质干细胞, 急性肾损伤
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.

Key words: Heme oxygenase-1, Bone marrow-derived mesenchymal stem cells, Acute kidney injury
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
[1]
Li K, Han Q, Yan X, et al. Not a process of simple vicariousness, the differentiation of human adipose-derived mesenchymal stem cells to renal tubular epithelial cells plays an important role in acute kidney injury repairing [J]. Stem Cells Dev, 2010, 19(8): 1267-1275.
[2]
Morigi M, Imberti B, Zoja C, et al. Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure [J]. J Am Soc Nephrol, 2004, 15(7): 1794-1804.
[3]
Semedo P, Wang PM, Andreucci TH, et al. Mesenchymal stem cells ameliorate tissue damages triggered by renal ischemia and reperfusion injury [J]. Transplant Proc, 2007, 39(2): 421-423.
[4]
Tögel F, Weiss K, Yang Y, et al. Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury [J]. Am J Physiol Renal Physiol, 2007, 292(5): F1626-F1635.
[5]
Hoffmann J, Glassford AJ, Doyle T, et al. Angiogenic effects despite limited cell survival of bone marrow-derived mesenchymal stem cells under ischemia [J]. Thorac Cardiovasc Surg, 2010, 58(3): 136-142.
[6]
刘楠梅，程劲，黄健，等. CXCR4基因修饰对骨髓间充质干细胞向急性肾损伤微环境定向迁移的放大效应及可能机制[J]. 肾脏病与透析肾移植杂志，2013，22(2)：118-124.
[7]
刘楠梅，田军，王巍巍，等．红细胞生成素对模拟急性肾损伤微环境下培养骨髓间充质干细胞增殖的影响及机制探讨[J]．中华肾脏病杂志，2011，27(2)：112-117.
[8]
Baer PC, Bereiter-Hahn J, Schubert R, et al. Differentiation status of human renal proximal and distal tubular epithelial cells in vitro: Differential expression of characteristic markers [J]. Cells Tissues Organs, 2006, 184(1): 16-22.
[9]
Stolzing A, Scutt A. Effect of reduced culture temperature on antioxidant defences of mesenchymal stem cells [J]. Free Radic Biol Med, 2004, 41(2): 326-338.
[10]
Frippiat C, Dewelle J, Remacle J, et al. Signal transduction in H2O2-induced senescence-like phenotype in human diploid fibroblasts [J]. Free Radic Biol Med, 2002, 33(10): 1334-1346.
[11]
Zhang YG, Yang Z, Zhang H, et al. Effect of negative pressure on human bone marrow mesenchymal stem cells in vitro [J]. Connect Tissue Res, 2010, 51(1): 14-21.
[12]
Downward J. PI 3-kinase, Akt and cell survival [J]. Semin Cell Dev Biol, 2004, 15(2): 177-182.
[13]
Wei H, Li Z, Hu S, et al. Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases, p38 and JNK [J]. J Cell Biochem, 2010, 111(4): 967-978.
[14]
Mimeault M, Batra SK. Recent insights into the molecular mechanisms involved in aging and the malignant transformation of adult stem/progenitor cells and their therapeutic implications [J]. Ageing Res Rev, 2009, 8(2): 94-112.
[15]
Stocker R, Yamamoto Y, McDonagh AF, et al.Bilirubin is an antioxidant of possible physiological importance [J]．Science，1987, 235(4792)：1043-1046.
[16]
Zeng B, Ren X, Lin G, et al. Paracrine action of HO-1-modified mesenchymal stem cells mediates cardiac protection and functional improvement [J]. Cell Biol Int, 2008, 32(10): 1256-1264.
[17]
Hill-Kapturczak N, Chang SH, Agarwal A. Heme oxygenase and the kidney [J]. DNA Cell Biol, 2002, 21(4): 307-321.
[18]
Gonzalez-Zulueta M, Feldman AB, Klesse LJ, et al. Requirement for nitric oxide activation of p21(ras)/extracellular regulated kinase in neuronal ischemic preconditioning [J]. Proc Natl Acad Sci, 2000, 97(1): 436-441.
[19]
Brunet A, Datta SR, Greenberg ME. Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway [J]. Curr Opin Neurobiol, 2001, 11(3): 297-305.
[20]
Dai Z, Li Y, Quarles LD, et al. Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation [J]. Phytomedicine, 2007, 14(12): 806-814.
[21]
Isele NB, Lee H-S, Landshamer S, et al. Bone marrow stromal cells mediate protection through stimulation of PI3-K/Akt and MAPK signaling in neurons [J]. Neurochem Int, 2007, 50(1): 243-250.
[22]
Platt MO, Wilder CL, Wells A, et al. Multipathway kinase signatures of multipotent stromal cells are predictive for osteogenic differentiation: tissue-specific stem cells [J]. Stem Cells, 2009, 27(11): 2804-2814.
[1] 张婧琦, 江洋, 孙佳璐, 唐兴喆, 赵宇飞, 崔颖, 李信响, 戴景月, 傅琳, 彭新桂. 基于肾周CT特征结合血清肌酐水平探讨脓毒症伴急性肾损伤的早期识别[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(04): 285-292.
[2] 樊恒, 孙敏, 朱建华. 红景天苷通过抑制PI3K/AKT/mTOR信号通路对大鼠脓毒症急性肾损伤的保护作用[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(03): 188-195.
[3] 张锦丽, 席毛毛, 褚志刚, 栾夏刚, 陈诺, 王德运, 谢卫国. 大面积烧伤患者发生早期急性肾损伤的危险因素分析[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(04): 282-287.
[4] 彭瑞, 杨瑞文, 魏澹宁, 夏永良. 琥珀酸受体1加重肾脏缺血再灌注损伤的作用研究[J/OL]. 中华移植杂志(电子版), 2024, 18(03): 159-164.
[5] 杨阳, 王琤, 周文土, 周冰. Caveolae/Caveolin-1与膜胆固醇共同调控小鼠BMSCs成骨分化[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(03): 137-142.
[6] 中华医学会器官移植学分会, 中国医师协会器官移植医师分会. 中国肝移植受者肾损伤管理临床实践指南（2023版）[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(03): 276-288.
[7] 杜霞, 马梦青, 曹长春. 造影剂诱导的急性肾损伤的发病机制及干预靶点研究进展[J/OL]. 中华肾病研究电子杂志, 2024, 13(05): 279-282.
[8] 郭俊楠, 林惠, 任艺林, 乔晞. 氨基酸代谢异常在急性肾损伤向慢性肾脏病转变中的作用研究进展[J/OL]. 中华肾病研究电子杂志, 2024, 13(05): 283-287.
[9] 袁楠, 黄梦杰, 白云凤, 李晓帆, 罗从娟, 陈健文. 急性肾损伤-慢性肾脏病转化小鼠模型制备的教学要点及学习效果分析[J/OL]. 中华肾病研究电子杂志, 2024, 13(04): 226-230.
[10] 林玲, 李京儒, 沈瑞华, 林惠, 乔晞. 基于生物信息学分析小鼠急性肾损伤和急性肺损伤的枢纽基因[J/OL]. 中华肾病研究电子杂志, 2024, 13(03): 134-144.
[11] 司楠, 孙洪涛. 创伤性脑损伤后肾功能障碍危险因素的研究进展[J/OL]. 中华脑科疾病与康复杂志(电子版), 2024, 14(05): 300-305.
[12] 汪鹏飞, 程莹莹, 赵海康. 骨髓间充质干细胞改善神经病理性疼痛的机制探讨[J/OL]. 中华脑科疾病与康复杂志(电子版), 2024, 14(04): 230-234.
[13] 沈炎, 张俊峰, 唐春芳. 预后营养指数结合血清降钙素原、胱抑素C及视黄醇结合蛋白对急性胰腺炎并发急性肾损伤的预测价值[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(06): 536-540.
[14] 崔秋子, 姚红曼, 艾迎春. 监测NLR、PLR、CAR、白蛋白、血钙及血糖指标水平对急性胰腺炎患者急性肾损伤的预测价值分析[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(03): 244-248.
[15] 颜世锐, 熊辉. 感染性心内膜炎合并急性肾损伤患者的危险因素探索及死亡风险预测[J/OL]. 中华临床医师杂志(电子版), 2024, 18(07): 618-624.
阅读次数
全文


摘要

版权所有 中华医学会 中华医学电子音像出版社有限责任公司  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号