中国医学科学院学报

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中国医学科学院学报

中国医学科学院学报 ›› 2019, Vol. 41 ›› Issue (3): 291-299.doi: 10.3881/j.issn.1000-503X.11157

• 论著 • 上一篇    下一篇

间介中胚层样细胞在肾脏再生中的应用

张建烨1,管勇1,孔峰2,赵升田3()   

  1. 1 山东大学第二医院 泌尿外科,济南 250033
    2 山东大学第二医院 中心实验室,济南 250033
    3 山东大学附属山东省立医院泌尿外科,济南 250012
  • 收稿日期:2019-01-08 出版日期:2019-06-30 发布日期:2019-07-04
  • 通讯作者: 赵升田 E-mail:zhaoshengtian@sdu.edu.cn
  • 基金资助:
    国家自然科学基金面上项目(81670625)

Application of Intermediate Mesoderm-like Cells in Kidney Regeneration

ZHANG Jianye1,GUAN Yong1,KONG Feng2,ZHAO Shengtian3()   

  1. 1 Department of Urology, the Second Hospital of Shandong University,Jinan 250033,China
    2 Department of Central Research Lab,the Second Hospital of Shandong University,Jinan 250033,China
    3 Department of Urology,Shandong Provincial Hospital of Shandong University,Jinan 250012,China
  • Received:2019-01-08 Online:2019-06-30 Published:2019-07-04
  • Contact: Shengtian ZHAO E-mail:zhaoshengtian@sdu.edu.cn
  • Supported by:
    Supported by the National Nature Sciences Foundation of China(81670625)

摘要:

目的 体外诱导脂肪干细胞分化为间介中胚层样细胞,对肾脏脱细胞支架进行再细胞化,经共培养构建有部分肾脏结构的组织工程肾脏。方法 手术获取Wistar大鼠腹股沟脂肪垫,从中分离、培养原代脂肪干细胞后对其进行干细胞特性鉴定。经过在不同阶段添加糖原合成酶激酶3抑制剂(CHIR99021)和成纤维生长因子9(FGF9),诱导脂肪干细胞分化为间介中胚层样细胞,并于诱导7 d后进行间介中胚层相关蛋白鉴定。将诱导成功的间介中胚层样细胞结合制备好的肾脏脱细胞支架进行共培养,10 d后获取再细胞化的组织工程肾脏,并对其进行肾脏分化的鉴定。结果 获取的脂肪干细胞具有成骨成脂及成软骨分化的能力,能表达脂肪干细胞表面标志物。体外诱导7 d后得到的间介中胚层样细胞经过免疫荧光鉴定可以观察到锌指蛋白转录因子和配对盒基因-2的阳性表达,Western blot验证可得到同样的阳性结果。结合肾脏脱细胞支架后培养10 d,通过免疫荧光鉴定可以观察到Wilms肿瘤基因1、GATA连接蛋白-3和E-钙黏素的阳性表达。结论 脂肪干细胞可被诱导分化为间介中胚层样细胞,诱导后的间介中胚层样细胞结合肾脏脱细胞支架可以观察到肾系分化的现象。

关键词: 脂肪干细胞, 间介中胚层样细胞, 脱细胞支架, 诱导分化

Abstract:

Objective To induce adipose-derived stem cells (ADSCs) to differentiate into intermediate mesoderm (IM)-like cells in vitro,with IM-like cells for recellularizing kidney scaffolds,and then to obtain a tissue-engineering kidney with renal structures and functions through co-culture.Methods After inguinal fat pads of Wistar rats were surgically harvested,the primary ADSCs were isolated,induced,and cultured for stem cell identification. ADSCs were inducted to differentiate into IM-like cells by adding glycogen synthase kinase-3 inhibitor (CHIR99021) and fibroblast growth factor 9 (FGF9) at different stages. Seven days later,the IM-like cells were identified. The induced IM-like cells and well-prepared kidney decellularized scaffolds were co-cultured for 10 days to obtain recellularized tissue-engineered kidneys and their differentiation was identified.Results The ADSCs harvested had osteogenic and adipogenic abilities and could express the stem cell surface markers. After 7 days of in vitro induction,the positive expressions of odd-skipped related 1 and paired-box 2 were observed in IM-like cells by immunofluorescence technique. After 10 days of co-culture with kidney decellularized scaffolds,the positive expressions of Wilms’tumor 1,GATA-binding protein-3,and E-cadherin were observed by immunofluorescence technique.Conclusion ADSCs can be induced into IM-like cells,and renal cell differentiation can be observed through combining the induced IM-like cells with kidney decellularized scaffolds.

Key words: adipose-drived stem cells, intermidate mesoderm-like cells, decellularized scaffold, differentiation

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