肌成纤维细胞促进小鼠胚胎肝干细胞的增殖和分化

移植细胞的增殖和分化需要微环境支持。作为最重要的微环境成分,肌成纤维细胞在肿瘤的生长过程中发挥着重要作用。该实验Hepa1-6肿瘤细胞上清液在体外激活成纤维细胞分化为肌成纤维细胞,探讨肌成纤维细胞上清对小鼠胚胎肝干细胞（embryonic hepatic stem cells,EHSCs）HP14.5增殖和分化的影响。实验将EHSCs HP14.5分为三组：DMEM培养液处理组（DMEM组）、成纤维细胞上清液处理组（CMFb组）及肌成纤维细胞上清液处理组（CMAFb组）。MTT法绘制三组HP14.5细胞生长曲线图,免疫荧光法及Real-time PCR法检分别测白蛋白（albumin,ALB）、甲胎蛋白（alpha fetoprotein,AFP）、细胞角蛋白18（cytokeratin 18,CK18）的蛋白及mRNA表达情况,PAS染色法检测糖原合成状况。MTT法检测显示,CMAFb组胚胎肝干细胞增殖明显速度较其他两组快。免疫荧光染色及Real-time PCR结果显示,HP14.5培养5 d后,CMAFb组ALB和CK18的蛋白及mRNA表达水平以及糖原合成水平显著高于CMFb组及DMEM组,而AFP蛋白和mRNA表达水平明显降低。该实验表明,Hepa1-6激活的成纤维细胞能促进胚胎干肝细胞的增殖以及分化为有功能的成熟肝细胞。

Myofriboblast Promotes the Proliferation and Differentiation of Mouse Embryonic Hepatic Stem Cells

Microenvironment is very important to proliferation and differentiation of transplanted cells. As a crucial composition of the microenvironment in tumor, myofibroblast influence the development of tumor cells deeply. This study focuses on the effect of fibroblast and myofibroblast on the proliferation of embryonic hepatic stem cells （EHSCs） HP14.5. In this research, two sorts of conditioned medium were harvested from the supemate of fibroblast （CMFb） and the supemate of myofibroblast （CMAFb） which came from the Hepal-6 induced differentiation of fibroblast in vitro. The EHSCs HP14.5 were divided in 3 groups： DMEM group, CMFb group and CMAFb group. MTT was used to draw the curves of cell growth. Immunofluorescence staining and real-time quantitative polymerase chain reaction （Real-time PCR） were performed for detecting the albumin （ALB）, alpha- fetoprotein （AFP） and cytokeratin 18 （CK18） in protein and mRNA expression level. And the PAS staining was used to detect Glycogen synthesis. In the results, MTT showed faster proliferation of liver stem cells in CMAFb group compared with other groups; The Real-time PCR showed that the mRNA expression of ALB, AFP and CK18 inCMAFb group were much higher than in CMFb group and DMEM group after 5 days culture, while the number alpha-fetoprotein and mRNA expression level declined remarkably. Also, the similar results were observed in immunofluorescence. And the result of PAS staining suggested that the Glycogen synthesis in CMAFb group was much higher than the other two groups. Therefore, this research highly suggests that the fibroblasts activated by Hepa 1-6 are able to increase the proliferation of EHSCs and differentiate the functional mature liver cells.