摘要
目的探讨不同Wnt蛋白对小鼠胚胎肝祖细胞HP14-19分化的作用,筛选有效的肝干细胞定向分化体系,为治疗肝衰竭提供理论基础。方法携带不同Wnt蛋白基因的19种重组腺病毒,分别感染HP14-19,检测清蛋白启动子启动的荧光素酶报告基因活性;实时荧光定量PCR检测detla蛋白DLK、甲胎蛋白(AFP)、清蛋白(ALB)、上皮特异性标志物细胞角蛋白(CK18)等肝细胞分化成熟相关标志物的表达情况;糖原(PAS)染色实验和吲哚菁绿(ICG)摄取实验用于检测肝细胞合成代谢功能。结果19种Wnt腺病毒均能有效感染HP14-19细胞并表达相应的Wnt信号蛋白,随着处理时间的延长,荧光素酶值逐渐升高,其中Wnt3a的作用最明显;Ad-Wnt3a组肝干细胞标志DLK、AFP表达明显下降;肝细胞特异性成熟标志ALB、CK18明显上调;Ad-Wnt3a处理后PAS染色可见70%~80%的细胞染色阳性,细胞由多角形变为长梭形;ICG摄取实验可见大量绿色圆形或者多边形阳性细胞。结论Wnt3a蛋白是诱导HP14-19细胞成熟分化的重要蛋白,诱导分化后的细胞类似于成熟肝细胞,有较强的合成代谢功能;对进一步研究肝干细胞分化调控及肝移植应用有重要意义。
Objective To explore the effects of different Wnt proteins on the differentiation of mouse embryonic liver progenitor cells HP14-19,and to screen an effective hepatic stem cell-directed differentiation system to provide a theoretical basis for the treatment of liver failure.Methods HP14-19 were infected with 19 recombinant adenoviruses carrying different Wnt protein gene respectively,and the activity of the luciferase reporter gene initiated by the albumin promoter was detected;real-time fluorescent quantitative PCR was used to detect the expression of hepatocyte differentiation and maturation related markers such as DLK,AFP,albumin(ALB),epithelial specific marker cytokeratin(CK18);glycogen(PAS)staining test and indocyanine green(ICG)uptake test were used to detect the anabolic function of liver cells.Results All 19 Wnt adenoviruses could effectively infect HP14-19 cells and expressed the corresponding Wnt signal protein.As the treatment time increased,the luciferase value gradually increased,and Wnt3a had the most obvious effect.The expression of hepatic stem cell markers DLK and AFP in the Ad-Wnt3a group was significantly decreased;the hepatocyte-specific maturation markers ALB and CK18 were significantly increased;after Ad-Wnt3a treatment,70%-80%of the cells were stained positively in PAS staining,and the cells changed from a polygonal shape to a long spindle shape;a large number of green round or polygonal positive cells were seen in the ICG uptake experiment.Conclusion Wnt3a protein is an important protein that induces the maturation and differentiation of HP14-19 cells.The induced differentiated cells are similar to mature hepatocytes and have a strong anabolic function;it is of great significance for further research on the regulation of hepatic stem cell differentiation and liver transplantation.
作者
彭利
马文军
崔洁洁
龚梦嘉
何昀
PENG Li;MA Wenjun;CUI Jiejie;GONG Mengjia;HE Yun(Department of Clinical Laboratory,Ministry of Education Key Laboratory of Child Development and Disorders,China International Science and Technology Cooperation Base of Child Development and Critical Disorders,Chongqing Key Laboratory of Pediatrics,Children′s Hospital of Chongqing Medical University,Chongqing 400014,China;Laboratory of Stem Cell Biology and Therapy,Ministry of Education Key Laboratory of Child Development and Disorders,China International Science and Technology Cooperation Base of Child Development and Critical Disorders,Chongqing Key Laboratory of Pediatrics,Children′s Hospital of Chongqing Medical University,Chongqing 400014,China)
出处
《国际检验医学杂志》
CAS
2020年第17期2121-2124,2128,共5页
International Journal of Laboratory Medicine
关键词
Wnt信号分子
胚胎肝祖细胞
分化
Wnt signaling molecule
embryonic liver progenitor cells
differentiation
