论文部分内容阅读
目的研究妊娠早期人脐带血管周围细胞(human umbilical 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-specific embryonic antigen 1,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(smooth muscle actin,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。
Objective To study the isolation and culture conditions of human umbilical cord perivascular cells (HUCPVC) in early pregnancy and the ability of differentiating islet-like cells. Methods The umbilical cord tissues of one (8-12 weeks) pregnant women who were voluntarily donated by the patients were collected. HUCPVC was isolated and cultured in early pregnancy. The passage algebra was recorded and the cell differentiation was observed. The cell surface marker-specific embryonic antigens were detected by immunohistochemical staining 1 (SSEA-1), SSEA-3, SSEA-4, OCT-4, TRA-1-60 and TRA-1-81. The embryoid body-induced and islet-like cell differentiation were induced by cell culture (5 stages). The expression of α-fetoprotein (AFP), nestin (Nestin) and smooth muscle actin (SMA). Glucose-stimulated insulin secretion assay was used to detect insulin secretion. Results HUCPVC was successfully isolated and purified in early pregnancy and could be stably passaged for more than 10 passages. The stem cells markers SSEA-3, SSEA-4, OCT-4, TRA-1-60 and TRA-1-81 were significantly expressed in cells . After being induced in vitro by simulated embryoid body, a hollow, layered and regularly globular embryoid body with membrane-coated surface could be formed. The markers AFP, Nestin and SMA were all positive. The results showed that the insulin content of the differentiated cells in high glucose medium was (23.2 ± 5.3) mU / L, which was significantly higher than that in low glucose medium The basal content (7.0 ± 0.5) mU / L (t = 7.444, P = 0.002). Conclusions HUCPVC can be differentiated into islet-like cell structure and insulin secretion through the separation, passage and induction of early pregnant HUCPVC.