上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

体外诱导人iPS细胞向肾脏细胞定向分化的实验研究

黄佳卉1,2，牛 鑫1，胡 斌1，周树敏1，李 青1，郭尚春1，汪 泱1   

  1. 1.上海交通大学附属第六人民医院四肢显微外科研究所， 上海 200233； 2.中山大学附属第六医院检验科， 广州 510655
  • 出版日期:2014-07-28 发布日期:2014-08-11
  • 通讯作者: 汪 泱, 电子信箱: wangy63cn@126.com。
  • 作者简介:黄佳卉（1984—）， 女， 硕士生; 电子信箱: jiahuihuang33006@163.com。
  • 基金资助:

    国家自然科学基金（81170640, 30960385）

Experimental study on differentiating human induced pluripotent stem cells towards renal cells in vitro

HUANG Jia-hui1,2, NIU Xin1, HU Bin1, ZHOU Shu-min1, LI Qing1, GUO Shang-chun1, WANG Yang1   

  1. 1.Shanghai Institute for Microsurgery of Extremities, the Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, China; 2.Department of Clinical Laboratory, the Sixth Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510655, China
  • Online:2014-07-28 Published:2014-08-11
  • Supported by:

    National Nature Science Foundation of China, 81170640, 30960385

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摘要：

目的 探讨人诱导性多能干细胞（iPS）体外定向分化为肾脏细胞的方法，为应用干细胞进行肾脏再生治疗奠定实验基础。方法 以正常人iPS细胞为实验对象，运用生肾因子活化素A （Activin-A）、骨形成蛋白7（BMP7）、人血管内皮生长因子(hVEGF)、碱性成纤维细胞生长因子（b-FGF）、锂盐、维甲酸（RA）等细胞因子联合肾脏上皮细胞培养液对iPS进行定向诱导肾脏细胞分化，分别于诱导后第14、21和28日，细胞免疫荧光染色及Real-Time PCR检测肾脏发育相关蛋白Brachyury （Bry）、Paired box gene 2 （Pax2）、水通道蛋白1 （AQP1）和E钙粘素（E-cad）蛋白及mRNA的表达水平，RT-PCR检测分化各阶段多能性基因Nanog和OCT4的表达水平。结果 诱导培养液分别诱导14、21和28 d后，细胞免疫荧光染色观察发现；分化后的细胞分别表达Bry、Pax2、AQP1和E-cad蛋白，而在未分化的iPS细胞中未见表达。Real-Time PCR结果显示：诱导后细胞中Bry、Pax2、AQP1、E-cad mRNA的表达分别较未分化的iPS细胞上调了4、30、37和25倍（P<0.05），分化各阶段多能性基因OCT4和Nanog的表达水平逐渐下调（P<0.05）。结论 细胞因子联合肾脏上皮细胞培养液可有效诱导人iPS细胞定向分化为肾脏细胞。

关键词: 细胞因子, 分化, 诱导性多能干细胞, 肾脏细胞

Abstract:

Objective To investigate the method of differentiating human induced pluripotent stem (iPS) cells towards renal cells in vitro and to provide the experimental basis for the application of stem cells in kidney regeneration. Methods The normal human iPS cells were treated by cytokines such as Activin-A, BMP7, hVEGF, b-FGF, lithium, and retinoic acid (RA) in renal epithelial cell growth medium (REGM) for inducing the differentiation towards renal cells. After 14, 21, and 28 d of differentiation, the expressions of proteins relevant to the renal development, i.e. Bry, Pax2, AQP1, and E-cad, were detected by the immunofluorescence staining and Real-Time PCR. The expressions of pluripotent genes of Nanog and OCT4 of each differentiation stage were measured by the RT-PCR. Results After being induced by the medium for 14, 21, and 28 d, the results of immunofluorescence staining showed that the differentiated cells expressed the proteins of Bry, Pax2, AQP1, and E-cad, while undifferentiated iPS cells did not express. The results of Real-Time PCR indicated that the expressions of Bry, Pax2, AQP1, and E-cad of differentiated cells were about 4, 30, 37, and 25 times higher than those of undifferentiated iPS cells (P<0.05). The expressions of pluripotent genes of Nanog and OCT4 of differentiation stages decreased gradually (P<0.05). Conclusion Human iPS cells can be efficiently induced and differentiate towards renal cells by cytokines in renal epithelial cell growth medium.

Key words: cytokines, differentiation, induced pluripotent stem cells, renal cells