Ping Jian, Chen Hong-Yun, Yang Zhou, Yang Cheng, Xu Lie-Ming
Zhongguo Zhong Xi Yi Jie He Za Zhi. 2014 Mar;34(3):348-54.
To observe the effect of Yiguan Decoction (YGD) on differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro.
Rat BMSCs were isolated using whole bone marrow adherent method. The properties of BMSCs were identified by analyzing the expression of surface cytokines by flow cytometry. The third passage cells were differentiated into fat cells to identify their features. BMSCs were incubated with hepatocyte growth factor (HGF) plus fibroblast growth factor 4 (FGF4) or YGD containing serum YGD for 21 days. The mRNA expression of alpha-fetoprotein (alphaAFP), albumin (Alb), and hepatocyte nuclear factor 4alpha (HNF4alpha) were detected by real time PCR. Expression of AFP and cytokeratin 18 (CK18) protein was detected by cell immunofluorescence. Glycogen synthesis was observed using periodic acid-Schiff stain (PAS). CK18, Wnt 3alpha, and alphacatenin protein expressions were detected by Western blot.
High expression of CD90, CD29, and CD44, and low expression of CD34 and CD11b were observed in BMSCs isolated by whole bone mar- row adherent method, and numerous lipid droplets were observed in BMSCs using oil red O staining. Both YGD containing serum and growth factor stimulated the expression levels of Alb, AFP, HNF4alpha mRNA and CK18 protein. The down-regulated expression of Wnt 3alpha and beta-catenin could be detected at 21 days after induction. The synthesized glycogen granule could be seen. Down-regulated Wnt 3alpha and beta-catenin expression could also be observed.
YGD could induce the differentiation of rat BMSCs into hepatocyte-like cells, which was related to down-regulating Wnt/beta-catenin signal pathway.
观察一贯煎（YGD）对体外培养的骨髓间充质干细胞（BMSCs）向肝细胞样细胞分化的影响。
采用全骨髓贴壁法分离大鼠BMSCs。通过流式细胞术分析表面细胞因子的表达来鉴定BMSCs的特性。将第三代细胞诱导分化为脂肪细胞以鉴定其特征。将BMSCs与肝细胞生长因子（HGF）加碱性成纤维细胞生长因子4（FGF4）或含血清的一贯煎（YGD含药血清）孵育21天。通过实时荧光定量PCR检测甲胎蛋白（αAFP）、白蛋白（Alb）和肝细胞核因子4α（HNF4α）的mRNA表达。通过细胞免疫荧光检测AFP和细胞角蛋白18（CK18）蛋白的表达。采用过碘酸希夫染色（PAS）观察糖原合成情况。通过蛋白质免疫印迹法检测CK18、Wnt 3α和α连环蛋白的蛋白表达。
采用全骨髓贴壁法分离的BMSCs中观察到CD90、CD29和CD44高表达，CD34和CD11b低表达，用油红O染色在BMSCs中观察到大量脂滴。含血清的一贯煎和生长因子均能刺激Alb、AFP、HNF4α mRNA和CK18蛋白的表达水平。诱导21天后可检测到Wnt 3α和β连环蛋白的表达下调。可见合成的糖原颗粒。也可观察到Wnt 3α和β连环蛋白表达下调。
一贯煎可诱导大鼠BMSCs向肝细胞样细胞分化，这与下调Wnt/β连环蛋白信号通路有关。
