摘要
目的探索肝细胞生长因子(HGF)+成纤维生长因子-4(FGF-4)诱导人骨髓来源多能成体祖细胞(hMAPCs)分化为肝细胞的可行性,为肝组织工程提供新的种子细胞来源。方法(1)取志愿者适量骨髓后采用梯度密度离心+贴壁培养获取骨髓间充质干细胞(MSCs),将MSCs通过CD45、GlyA免疫微磁珠负分选得到hMAPCs。(2)将hMAPCs用HGF+FGF-4进行诱导分化。实验分组:A组:HGF (20 ng/ml)+(FGF-4)10ng/ml诱导hMAPCs;B组(阳性对照组):L-02人肝细胞株; C组(阴性对照组):未加任何诱导因素的hMAPCs。(3)免疫细胞化学鉴定不同诱导分化阶段细胞的白蛋白(Alb)、甲胎蛋白(AFP)、细胞角蛋白-18(CK-18)等肝细胞特征的表型变化并计数阳性细胞比率。(4)逆转录-聚合酶链反应检测不同诱导分化阶段细胞的Alb、AFP、CK-18的mRNA转录。(5)Western blot检测诱导分化第21、35天后细胞的Alb表达。结果(1)免疫细胞化学结果:Alb、CK-18在诱导组中不同时间段基本为阳性着色;AFP在诱导分化第7天为阳性着色,在诱导第14、21天为阴性着色。(2)逆转录-聚合酶链反应结果:作为不成熟肝细胞表型的AFP,在诱导分化的第7天有mRNA阳性表达;作为成熟肝细胞表型的Alb及CK-18,在不同时间段mRNA均为阳性表达。(3)Western blot检测诱导分化第21、35天后细胞的Alb表达。结论hMAPCs在一定诱导条件下具有向肝样细胞分化的潜能。
Objective To investigate the possibility of the human bone marrow multipotent adult progenitor cells （hMAPCs） to differentiate into hepatocytes with hepatocyte growth factor （HGF）/fibroblast growth factor-4（FGF - 4） in vitro. Methods （1） Obtaining the hMAPCs. Bone marrow was obtained from volunteers and then centrifuged through density gradient centrifugation methods. The collected mononuclear cells were cultured through adheret culture to get mesenchymal stem cells （MSCs）. The hMAPCs were obtained through collecting and isolating the MSCs by magnetic activated cell sorting （MACS） through depletion selection by use of CD45 and GlyA microbeads. （2） Differentiation of the hMAPCs with HGF＋FGF-4. GroupA： HGF （20 ng/ml） ＋ FGF-4 （10 ng/ml） induced hMAPCs; group B （positive control group）： L-02 human hepatocytes（cell lines）; and group C （negative control group）： the undifferentiated hMAPCs. （3） The expressions of albumin （Alb）, alpha fetoprotein （AFP）, cytokeratin-18 （CK-18）, and cytokeratin-19 （CK-19） were detected with immunocytochemistry to identify the characteristics of the differentiated cells at different times and the ratio of the positive cells was determined. （4） ALB, AFP, CK-18, and CK-19 expressions of the differentiated cells were detected by RT-PCR assayto investigate the mRNA transcriptions of characteristic hepatic proteins. （5） Alb expressions of the differentiated cells at different times were detected by Western blot on the 21stand 35th days. Results （1） The results of immunocytochemistry. The staining of Alb, CK18 were essentially positive in group A. As an early marker of immature hepatocytes, AFP staining was positive on the 7th day but negative in later differentiating periods in group A. （2） The results of RT-PCR. On the 7th day, the differentiated hMAPCs expressed AFP mRNA but were negative in later differentiating periods. On the contrary, the mRNA of Alb and CK-18 were positive at all times. （3） T
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2005年第9期652-655,共4页
Chinese Journal of Hepatology
基金
广东省科技计划项目(2002A3020206)
