CAJ | 학술논문

背景:肝细胞生长因子是体外诱导骨髓间充质干细胞向肝干细胞方向分化的关键性细胞因子.碱性成纤维细胞生长因子不仅能提高骨髓间充质干细胞的增殖速度及其寿命,且能在增殖过程中保留骨髓间充质干细胞的多向分化潜能.目的:探讨肝细胞生长因子和碱性成纤维细胞生长因子联合诱导大鼠骨髓间充质干细胞分化为肝样细胞的可行性.方法:取大鼠股骨骨髓,用直接贴壁法分离纯化骨髓间充质干细胞并体外传代,流式细胞仪及成骨诱导对骨髓间充质干细胞进行鉴定.将细胞按以下分组进行成肝诱导: M①0组:不添加任何因子. M②1组:20μg/L肝细胞生长因子.③2组:20μg/L肝细胞生长因子+5μg/L碱性成纤维细胞生长因子. M④3组:20μg/L 肝细胞生长因子+10μg/L碱性成纤维细胞生长因子. M⑤4组:20μg/L肝细胞生长因子+20μg/L碱性成纤维细胞生长因子.倒置显微镜下观察细胞形态变化,在不同分化阶段用免疫细胞化学染色法检测不成熟肝细胞表型标志甲胎蛋白和成熟肝细胞表型标志细胞白蛋白的表达.M结果与结论:骨髓间充质干细胞诱导后呈肝样细胞形态改变.甲胎蛋白在诱导第7天细胞基本为阳性着色,在诱导第14天时表达降低,21 d表达为阴性.细胞白蛋白在诱导第14天细胞开始表达,而后持续.M0对照组未见阳性染色,同一时间点,M3、M4组细胞阳性染色率高于M2组(P <0.05).提示肝细胞生长因子和碱性成纤维细胞生长因子具有体外诱导骨髓间充质干细胞向肝样细胞分化的作用,二者有协同作用.
배경:간세포생장인자시체외유도골수간충질간세포향간간세포방향분화적관건성세포인자.감성성섬유세포생장인자불부능제고골수간충질간세포적증식속도급기수명,차능재증식과정중보류골수간충질간세포적다향분화잠능.목적:탐토간세포생장인자화감성성섬유세포생장인자연합유도대서골수간충질간세포분화위간양세포적가행성.방법:취대서고골골수,용직접첩벽법분리순화골수간충질간세포병체외전대,류식세포의급성골유도대골수간충질간세포진행감정.장세포안이하분조진행성간유도: M①0조:불첨가임하인자. M②1조:20μg/L간세포생장인자.③2조:20μg/L간세포생장인자+5μg/L감성성섬유세포생장인자. M④3조:20μg/L 간세포생장인자+10μg/L감성성섬유세포생장인자. M⑤4조:20μg/L간세포생장인자+20μg/L감성성섬유세포생장인자.도치현미경하관찰세포형태변화,재불동분화계단용면역세포화학염색법검측불성숙간세포표형표지갑태단백화성숙간세포표형표지세포백단백적표체.M결과여결론:골수간충질간세포유도후정간양세포형태개변.갑태단백재유도제7천세포기본위양성착색,재유도제14천시표체강저,21 d표체위음성.세포백단백재유도제14천세포개시표체,이후지속.M0대조조미견양성염색,동일시간점,M3、M4조세포양성염색솔고우M2조(P <0.05).제시간세포생장인자화감성성섬유세포생장인자구유체외유도골수간충질간세포향간양세포분화적작용,이자유협동작용.
BACKGROUND:Hepatocyte growth factor is a key cytokine for in vitro inducing the differentiation of bone marrow mesenchymal stem cel s into liver stem cel s. Basic fibroblast growth factor can not only increase the proliferation rate of mesenchymal stem cel s and its life, but also can maintain the multilineage differentiation potential of mesenchymal stem cel s in the proliferation process. OBJECTIVE:To investigate the possibility of rat bone marrow mesenchymal stem cel s to differentiate into hepatocyte-like cel s induced by hepatocyte growth factor and basic fibroblast growth factor in vitro. METHODS:Bone marrow mesenchymal stem cel s were col ected from femora of Sprague-Dawley rats. The harvested bone marrow mesenchymal stem cel s were separated and purified by whole bone marrow adherent culture method, and passaged in vitro. The bone marrow mesenchymal stem cel s were identified by flow cytometry and osteogenic induction. The cel s were divided into groups:(1) M0 group:as a negative control, treated without any factor;(2) M1 group:as the positive control group, treated with 20μg/L hepatocyte growth factor;(3) M2 group:treated with 20μg/L hepatocyte growth factor+5μg/L basic fibroblast growth factor;(4) M3 group:treated with 20μg/L hepatocyte growth factor+10μg/L basic fibroblast growth factor;(5) M4 group:treated with 20μg/L hepatocyte growth factor+20μg/L basic fibroblast growth factor. The morphological changes were observed under inverted microscope. At different stages of differentiation, the album expressions of of mature hepatocyte phenotype marker and alpha fetoprotein of immature hepatocyte phenotype marker were detected by immunohistochemical staining. RESULTS AND CONCLUSION:The harvested bone marrow mesenchymal stem cel s showed morphologic changes of hepatocyte after induction. The album was positively stained at 7 days after induction, its expression level was reduced at 14 days after induction, and then the expression changed into negative at 21 days after induction. The expression of alpha fetoprotein began to appear at 14 days after induction and then continued. No positive staining could be seen in the M0 group, and at the same time point, the positive staining rate in the M3 and M4 group was higher than that in the M2 group (P<0.05). It indicates that hepatocyte growth factor and basic fibroblast growth factor can induce bone marrow mesenchymal stem cel s to differentiate into hepatocyte-like cel s, and there is synergistic effect between them.