妊娠早期人脐带血管周围细胞的分离培养及定向分化胰岛样细胞的研究

DIFFERENTIATING INTO ISLET-LIKE CELL CLUSTERS FROM HUMAN FIRST TRIMESTER UMBILICAL CORD PERIVASCULAR CELLS IN VITRO

目的研究妊娠早期人脐带血管周围细胞（humanumbilical cord perivascular cells，HUCPVC）分离培养条件及定向分化胰岛样细胞能力。方法取患者自愿捐赠的妊娠一期（8～12周）脐带组织，体外分离培养妊娠早期HUCPVC，记录传代代数并观察细胞分化情况；采用免疫组织化学染色法检测细胞表面标志物阶段特异性胚胎抗原1（stage．specificembryonicantigen1，SSEA-1）、SSEA-3、SSEA-4、OCT-4、TRA-1．60和TRA-1-81表达。利用分段培养法（5个阶段）对细胞进行拟胚体诱导和胰岛样细胞定向分化，采用免疫组织化学染色方法检测拟胚体特异标志物甲胎蛋白（α-fetoprotein，AFP）、巢蛋白（Nestin）、平滑肌肌动蛋白（smoothmuscleactin，SMA）表达，采用葡萄糖刺激胰岛素分泌实验检测胰岛素分泌能力。结果成功分离纯化出妊娠早期HUCPVC并可稳定连续传代10代以上，细胞均显著表达干细胞标志物SSEA-3、SSEA-4、OCT-4、TRA-1．60和TRA-1-81，具有分化潜能。经拟胚体体外诱导可形成表面有膜包被的一中空、分层并具有规则球状形态的拟胚体，标志物AFP、Nestin和SMA均呈阳性。可定向分化为独立的胰岛样细胞结构，胰岛素表达阳性；葡萄糖刺激胰岛素分泌实验结果显示，分化的细胞在高糖培养基中的胰岛素含量为（23．2±5．3）mU／L，明显高于低糖培养基中的含量（7．0±0．5）mU／L（t=-7．444，P=0．002）。结论通过分离、传代和诱导，妊娠早期HUCPVC可定向分化为胰岛样细胞结构并分泌胰岛素。

isolated from human in vitro. Methods Objective To study the characteristics of the human umbilical cord perivascular cells （HUCPVC） first trimester umbilical cord perivascular layer tissues and the differentiation into islet-like cell clusters The HUCPVC derived from human first trimester umbilical cord which was donated by the volunteers were isolated and subcultured. The surface markers such as stage-specific embryonic antigen 1 （SSEA-1）, SSEA-3, SSEA-4, OCT-4, TRA-1-60, and TRA-1-81 were detected by immunohistochemical method. The first trimester HUCPVC were induced to embryoid bodies （EB）-like cell aggregations and isletqike cell dusters in vitro through a simple stepwise culture protocol （5 steps）. The expressions of specific markers [ct-fetoprotein （AFP）, Nestin, and smooth muscle actin （SMA）] were measured by immunohistochemical method; and the ability of glucose-stimulated insulin secretion was analyzed. Results The first trimester HUCPVC were successfully isolated and could be passaged steadily more than 10 generations, which expressed SSEA-3, SSEA-4, OCT-4, TRA-1-61, and TRA-1-81. The first trimester HUCPVC were successfully induced into EB-like cell aggregations and islet-like cell clusters. The EB-like cell aggregations could express markers of three germ lineages： AFP, Nestin, and SMA. The islet-like cell clusters could release insulin significantly in response to elevated concentrations of glucose in vitro （t=7.444, P=0.002）. The insulin contents were （23.2 ± 5.3） mU/L and （7.0± 0.5） mU/L in high and low glucose media, respectively. Conclusion The first trimester HUCPVC has the ability to differentiate into islet-like cell clusters which can secret insulin in vitro.