小鼠骨髓干细胞诱导分化为肝细胞的实验研究

Study of mouse marrow cells differentiation into a hepatocyte lineage in vitro

目的 探讨成年小鼠骨髓干细胞在肝细胞生长因子(HGF)作用下分化为肝细胞的可能性及其分化特性,为肝细胞移植提供实验基础。 方法 HGF 100 ng/ml体外诱导小鼠骨髓干细胞向肝细胞分化,于诱导的第0、7、14、21、28天,观察细胞形态特征;半定量逆转录聚合酶链反应(RT-PCR)法检测细胞白蛋白(ALB)、甲胎蛋白(AFP)mRNA水平的表达;免疫细胞化学法检测ALB、AFP和细胞角蛋白19(CK 19)蛋白水平的表达。 结果 新鲜分离的骨髓干细胞ALB及AFP mRNA呈弱阳性。在非诱导组,培养7 d时ALB mRNA已检测不到,AFP mRNA明显减弱,14 d时消失。在诱导组,7 d时ALB mRNA检测不到,14 d时再次出现阳性条带,21 d时表达量最大,而AFP mRNA在诱导组始终呈阳性,14 d时表达量最大,以后逐渐减少。免疫细胞化学结果与RT-PCR结果基本一致,但CK 19蛋白始终阴性。 结论 小鼠骨髓干细胞在HGF单独作用下能诱导分化为肝细胞样细胞,诱导分化的最佳时期是2-3周。

Objective To explore whether bone marrow stem cells (MSCs) from adult mice can be induced to differentiate into hepatocytes by hepatocyte growth factor (HGF) alone and the time phase characteristics in the differentiation progress. Methods Adult mouse MSCs were treated with or without 100 ng/ml HGF, on days 0, 7, 14, 21, and 28. The morphologic characteristics of the cells were examined; the albumin (ALB), AFP mRNA were analyzed sub-quantively using reverse transcription polymerase chain reaction (RT-PCR) and immumohistochemistry techniques. The expression of ALB, AFP and CK19 were detected by using anti-ALB, AFP and CK19 antibodies. Result Freshly isolated adult mouse MSCs expressed ALB and AFP mRNA weakly; in the group without HGF, no ALB mRNA was detected on day 7. The expression of AFP mRNA was reduced significantly on day 7, and could not be detected anymore after day 14. In the HGF treated group, ALB mRNA was not detected on day 7, but the positive lane appeared again on day 14, and the expression of ALB mRNA was increased on day 21 but reduced in the following days. The AFP mRNA was positive at all times, however it tended to decrease after day 14 in the HGF treated groups. The result of immumohistochemistry was consistent with that of RT-PCR, and CK19 was always negative. Conclusion Adult mouse MSCs can be induced into hepatocyte differentiation in vitro. The optimal time for the induction was 2 to 3 weeks.