人脐血间充质干细胞向肝细胞样细胞的分化

In vitro human umbilical cord blood mesenchymal stem cells differentiated into hepatocyte-like cells

目的从脐血中获得间充质干细胞(MSC),探索体外诱导MSC向肝细胞分化的实验条件。方法无菌采血,分离脐血单个核细胞。以贴壁培养法获得MSC,采用流式细胞术检测细胞表面标志。分别用联合诱导法(联合诱导组)以及阶段诱导法(阶段诱导组)诱导MSC向肝细胞分化。采用RT-PCR检测ALB、AFP及CK19mRNA的表达情况。结果MSC呈长梭形,第三代(P4)后增殖较快。流式细胞术检测表明:CD29及CD44阳性,而CD34及CD45阴性。诱导6周后,联合诱导组无变化,而阶段诱导组细胞由长梭型变为不规则型,并检测到AFP mRNA的阳性表达,而未见ALB、CK19 mRNA的表达。继续诱导2周,检测到AFP、ALB、CK19 mRNA的表达。结论脐血中的MSC对高密度、高血清的依赖性高。在体外,阶段诱导方法可将MSC诱导成肝细胞。

Objective To obtain mesenchymal stem cells（MSCs） from human umbilical cord blood （HUCB） in vitro and to study the condition in which MCSs differentiated into hepatocyte-like cells. Methods HUCB was collected after the baby was delivered, and mononuclear cells （MNCs） were separated by gradient centrifugation, and then using adherent culture, MSCs were obtained. CD29, CD44, CD34, CD45 expression levels were determined by flow cytometry. Also the albumin （ALB）, alpha-fetoprotein （AFP）, cytokeration （CK19） mRNA expression levels were determined by RT-PCR. Results The results of flow cytometry showed that CD29 （ ＋ ） and CD44 （ ＋ ） expressions were positive, however CD34 （ - ） and CD45 （ - ） expressions were negative. The adherent ceils were proved to be MSCs. The group using several cytokines step by step showed the changes of cell morphology and expressions of AFPmRNA（ ＋ ）, ALBmRNA（ ＋ ）, and CK19mRNA（ ＋ ） at week 8. But, the cell density was significantly decreased. Conclusion MSCs in HUCB are high-concentration- and high-FCS-（fetal culf serum） dependents. MSCs could be differentiated into hepatocytes by an in vitro culture with several different cytokines step by step.