泡球蚴蛋白促进脂肪间充质干细胞向肝细胞分化的研究

Echinococcus multilocularis protein promotes the differentiation of adipose mesenchymal stem cells into hepatocytes

目的 探讨泡球蚴蛋白(Echinococcus multilocularis protein,EmP)促脂肪间充质干细胞(Adipose tissue Derived Mesenchymal Stem Cells,ADSCs)向肝细胞分化的作用。方法 体外分离人脂肪组织来源的ADSCs,第三代(P3)ADSCs分为对照组(HDM肝细胞诱导分化组)和Em蛋白处理组(HDM+EmP组),经无血清肝细胞诱导培养基培养7、14和21 d,检测对照组和EmP处理组肝细胞样细胞(iHep)百分比、糖原染色面积、肝细胞标志物白蛋白(Albumin,ALB)的蛋白及基因表达水平。Western-Blot法检测p-GSK-3β的表达情况。结果 P3代ADSCs具有成脂及成骨分化能力并表达干细胞标志物(CD29、CD90和CD105等)。在ADSCs向肝细胞诱导分化14 d,EmP处理组iHep细胞百分比显著高于对照组(P<0.05),EmP处理组糖原染色面积显著高于对照组(P<0.05)。免疫荧光染色结果显示两组细胞在诱导分化7 d开始表达ALB,荧光强度随诱导时间增加呈增强趋势。qPCR结果显示在诱导分化后第14 d,EmP处理组ALB表达水平显著高于对照组,两组间具有统计学差异(P<0.05)。Western-Blot结果显示,诱导分化第7 d和14 d,EmP处理组p-GSK-3β蛋白表达水平显著高于对照组(P<0.01和P<0.05)。结论 Em蛋白可能通过激活GSK-3β而具有促进ADSCs向肝细胞分化的作用,本研究为临床应用ADSCs移植治疗泡型棘球蚴病奠定了基础。

Objective To investigate the role of Echinococcus multilocularis protein(EmP) on the differentiation of adipose-derived mesenchymal stem cells(ADSCs) into hepatocytes.Methods ADSCs were isolated from human adipose tissue and expanded in vitro.P3 ADSCs were collected and induced into hepatocytes by the hepatocyte differentiation medium(HDM) and HDM plus EmP(HDM+EmP),respectively.The percentage of induced hepatocyte like cells(ihep),glycogen staining area,the gene expression and protein localization of albumin(ALB) and the p-GSK-3β protein of ADSCs of both groups were detected on Day 7,Day 14 and Day 21.Results The P3 ADSCs had adipogenic and osteogenic differentiation potential,and expressed stem cell markers(CD29,CD90 and CD105).On Day 14 of differentiation of ADSCs induced into hepatocytes,the percentage of ihep cells in the HDM+EmP group was significantly higher than that in the HDM group(39.06±9.8% vs 25.13±2.23%,P<0.05),glycogen staining area in the HDM+EmP group was significantly higher than that in the HDM group(63.39±15.92 μm~2 vs 39.99±10.61 μm~2,P<0.05).Immunofluorescence staining showed that the induced cells of both groups expressed ALB on Day 7,and the fluorescence intensity of ALB expressed in both groups was increased with induction time.On Day 14 of differentiation of ADSCs induced into hepatocytes,the gene expression of ALB in the HDM+EmP group was significantly higher than that in the HDM group(P<0.05).Western blot showed that the level of p-GSK-3β of the HDM+EmP group on Day 7 and Day 14 of differentiation of ADSCs induced into hepatocytes was significantly higher than that in the HDM group(P<0.01,P<0.05).Conclusion E.multilocularis protein could promote the differentiation of ADSCs into hepatocytes via activate GSK-3β.This study lays a foundation for the clinical application of ADSCs transplantation in the treatment of liver injury of alveolar echinococcosis.