摘要
目的探索半乳糖凝集素-3(Gal-3)联合肝细胞生长因子(HGF)诱导大鼠骨髓间充质干细胞(BMSCs)向肝样细胞分化的可行性及最佳诱导条件,并对诱导后的细胞进行鉴定。方法离心、贴壁培养分离大鼠BMSCs,流式细胞术鉴定表面标志。取第3代细胞进行分组诱导培养,A组:0μg/mL Gal-3+20ng/mL肝细胞生长因子(HGF);B组:0.1μg/mL Gal-3+20ng/mL HGF;C组:0.5μg/mL Gal-3+20ng/mL HGF;D组:1.0μg/mL Gal-3+20ng/mL HGF;E组:2.0μg/mL Gal-3+20ng/mL HGF;阳性对照组:SD大鼠肝脏细胞IAR20。分别于诱导后7、14、21、28d时段进行细胞形态学观察、免疫荧光染色检测、实时荧光定量核酸扩增检测(Q-PCR)鉴定其诱导分化情况。结果分组诱导培养后,各组BMSCs形态逐渐向肝样细胞转化,与大鼠原代肝脏细胞IAR20相似,其中28d时间段,C组转化成IAR20相似细胞比例最高;各组甲胎蛋白(AFP)、清蛋白(ALB)荧光染色阳性率明显增高,并且在28d时间点C组AFP、ALB的荧光染色阳性率最高;C组AFP、ALB mRNA表达明显增加,同时以28d时间点表达量达到峰值。结论 Gal-3联合HGF能有效诱导大鼠BMSCs分化为肝样细胞,分化后的肝样细胞能分泌肝细胞特异性产物AFP、ALB,且其诱导效率与诱导培养时间和Gal-3浓度有关。
Objective To explore the feasibility and optimum concentration of direct differentiation of rats BMSCs into hepatocyte-like cells induced by Gal-3joint HGF and identify the induced cells.Methods Rats BMSCs were separated used gradient centrifugation and adherence culture,and then identified by flow cytometry.The third generation cells were divided into 5groups.Group A：0μg/mL Gal-3＋20ng/mL HGF;group B：0.1μg/mL Gal-3＋20ng/mL HGF;group C：0.5μg/mL Gal-3＋20ng/mL HGF;group D：1.0μg/mL Gal-3＋20ng/mL HGF;group E：2.0μg/mL Gal-3＋20ng/mL HGF;positive control group：primary rat hepatic cells IAR20.The morphology observation,Q-PCR and immunofluorescent staining were applied respectively after induced 7,14,21,28 dto identify the cells differentiation.Results After induction,the morphological characteristics gradually transformed into hepatocyte-like cells,similar to primary rat hepatic cells IAR20,the translation rate of group C in the 28-days time period was the highest;Immunohistochemical examination demonstrated that fluorescence staining positive rate of ALB and AFP in each group increased over induced time,and group C was the highest in the 28-days time period.Q-PCR demonstrated that the expression of ALB and AFP mRNA in group C increased over induced time,and peaked in the 28-days time period.Conclusion Gal-3joint HGF could induce the differentiation of rats BMSCs into hepatocyte-like cells which secrete hepatocyte specific product AFP and ALB,and the induction efficiency is related to the induction time and the concentration of Gal-3.
出处
《重庆医学》
CAS
北大核心
2016年第15期2043-2047,共5页
Chongqing medicine
基金
国家高技术发展研究计划"863"项目(2012AA020505)
广东省自然科学基金项目(2014A030313279)
广东省省级科技计划项目(2014B020227002)
关键词
间质干细胞
甲胎蛋白类
肝细胞生长因子
骨髓间充质干细胞
半乳糖凝集素-3
诱导
肝样分化
mesenchymal stem cells
alpha-fetoproteins
hepatocyte growth factor
bone marrow-derived mesenchymal stem cells
Gal-3
induce
hepatocyte-like differentiation
