摘要
目的探讨肝星状细胞是否能体外诱导分化为肝细胞样细胞以及在这过程中某些干细胞标记物的表达和变化。方法采用标准胶原酶灌注联合percoll密度梯度离心法提取并纯化原代大鼠肝星状细胞,免疫荧光鉴定细胞表型;将原代肝星状细胞分为对照组和实验组进行体外培养,对照组肝星状细胞不加任何细胞因子培养24 h,实验组肝星状细胞加入bFGF 20μg/L、FGF420μg/L、HGF 20μg/L、IL-6 1μg/L细胞因子诱导7 d;倒置相差显微镜下观察诱导细胞形态的变化,Real-time PCR和细胞免疫荧光技术检测肝星状细胞诱导前后肝细胞ALB、AFP mRNA和蛋白的表达情况,以及诱导前后干细胞标记物mRNA和蛋白水平的表达及变化。结果①通过胶原酶灌注联合percoll密度梯度离心法获得的大鼠肝星状细胞,其特异性蛋白Desmin和GFAP表达阳性率≥95%。②实验组大鼠肝星状细胞经过bFGF、FGF4、HGF、IL-6等细胞因子诱导7 d后,与对照组细胞相比细胞形态由星形变为类圆形或多角形,转录水平出现肝细胞特异性标记物ALB的表达(P<0.01)和AFP的表达(P<0.05),同时实验组ALB和AFP蛋白表达呈阳性,而对照组则呈阴性。③实验组大鼠肝星状细胞经过bFGF、FGF4、HGF、IL-6等细胞因子诱导7 d后,与对照组相比干细胞标记物P75NTR转录水平表达下降(P<0.05),CD90、c-kit、sox-2、oct-4等转录水平表达显著下降(P<0.01),并伴有干细胞标记物蛋白水平的表达下调或无表达。结论初步证明肝星状细胞可能具有潜在的干细胞特性,在体外可诱导分化形成肝细胞样细胞,在诱导过程中肝星状细胞的干细胞标记物明显下调。
Objective To investigate whether hepatic stellate cells （HSCs） can differentiate to hepatocyte-like cells in vitro, and the expression and changes of some stem cell markers during the induction. Methods Primary rat HSCs were isolated by the standard collagenase perfusion and purified by density gradient centrifugation, and then identified with fluorescent immunocytochemistry for Desmin and GFAP. The HSCs were treated with the cytokines as bFGF （20 μg/L）, FGF4 （20 μg/L）, HGF （20 μg/L） and IL-6 （1μg/L） for 7 d served as experimental group, while those cultured without any cytokine for 24 h served as control group. The morphological changes of HSCs were examined by inverted phase contrast microscopy, and the hepatocyte-like phenotypes were determined by detecting the mRNA and protein expression of AFP, ALB, and other stem cell markers expression by real time-PCR and immunocytochemical assay. Results Our primary culture method obtained a large number of primary rat HSCs, with a positive rate to Desmin and GFAP of over 95%. After a combined induction of bFGF, FGF4, HGF and IL-6 for 7 d, HSCs showed a dramatically increased expression of AFP and ALB （ P 〈 0.05） at mRNA and protein levels, with the shape changing star-like to rotund or polygonal. But no such change was found in control cells. The combined induction resulted in a significant decrease in mRNA expression of p75NTR （P 〈 0.05 ） and CD90, c-kit, sox-2 from also and oct-4 （P 〈 O. Ol ）. The expression of these markers at protein level was also significantly decreased or even undetectable. Conclusion HSCs might be of the potential as stem cells to differentiate into hepatocyte-like cells in vitro. During the induction, the expression of some stem cell rn^rlc^r~ i= =;n＇n;G^ntl.T A 1-...J
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第7期604-608,共5页
Journal of Third Military Medical University
基金
重庆市自然科学基金重点项目(CSTC2009BA5060)~~
关键词
肝星状细胞
干细胞
细胞分离
诱导
分化
hepatic stellate cells
stem cells
isolation
induction
differentiation
