胚胎肝细胞提取液体外诱导骨髓干细胞向肝系细胞分化的实验研究  

Study of differentiation of bone marrow stem cells into hepatocyte lineage induced by embryonic hepatocyte extract in vitro

作　　者：谢杏榕[1] 李云静[1] 李儒贵[1] 雷飞飞[1] 李金科[1] 姚柏春[1] 谭华炳[1] 

机构地区：[1]湖北医药学院附属人民医院,湖北十堰442000

出　　处：《现代预防医学》2013年第1期124-126,133,共4页Modern Preventive Medicine

基　　金：2009年十堰市科学技术研究与开发计划项目(SYKJ20090001)

摘　　要：目的探讨胚胎肝细胞提取液体外诱导骨髓干细胞向肝系细胞分化的可行性。方法通过密度梯度分离法分离出骨髓中单个核细胞，进行体外培养和鉴定。在BMSCs传代培养7d后，把培养细胞分为诱导组和非诱导组；诱导组加入胚胎肝细胞提取液的培养体系，非诱导组加入不舍胚胎肝细胞提取液的基础培养体系。在诱导培养d0，d10，d20，d30．d40对诱导分化的细胞进行形态学观察、肝系细胞特异性标志物的鉴定。结果①形态学：诱导培养2周后，长梭形细胞大多数转变成圆形、卵圆形及多角形，细胞呈肝干细胞样圆形。②免疫细胞化学检测：诱导培养d10—20细胞表达AFP、CK19逐渐增强，d30～40逐渐减弱；诱导培养d10～40细胞表达ALB呈上升趋势；阳性染色细胞胞浆内出现棕黄色颗粒。非诱导组细胞不表达AFP、CK19、ALB3种蛋白，为阴性染色。⑧糖原染色：诱导培养的细胞d10—40BMSCs均有糖原表达．非诱导细胞未见糖原表达（P〈0．05）。结论胚胎肝细胞提取液在体外可以诱导骨髓干细胞向肝系细胞分化，并分化为具有合成、代谢、分泌及储存功能的肝系细胞。OBJECTIVE To investigate whether embryonic hepatocyte extract induces bone marrow stem cells to differentiate into hepatocyte lineage in vitro. METHODS The bone marrow mononuelear cell was separated by density gradient centrifugal method, and was cultured and identified in vitro. After 7 days subculture, the cuhured cells were divided into induced group and control group. The induced group was treated with embryonic hepatocyte extract, and control group without embryonic hepa- tocyte extract. On days 0, 10, 20, 40, the morphologie characteristics of the cells were observed, and the specific markers of hepatocyte lineage were identified. RESULTS （~） Morphology： After 2 weeks culture, most elongated spindle cells turned into round, oval and polygonal, they were round hepatic stem cells. （~） Immunocytochemical detection： In the induced group, the AFP, CKI9 tended to increase during 10-20 d, decreased during 30-40 d; the expression of ALB increased during 10-40 d, and the brown granules were detected in the cytosolic of positive staining cells.But in the group without embryonic hepatocyte extract, AFP, CK19, ALB could not be detected in each phases. ~） Staining for glycogen： Bone marrow stem cells in induced group expressed glycogen during 10-40 d, and in control group didn＇t express glycogen （P 〈 0.05）. CONCLUSION The bone marrow stem cells can be induced into hepatocyte lineage by embryonic hepatoeyte extract in vitro, and differentiate into hepato- cyte-like cells with metabolism, secretion, and storage functions.

关 键 词：终末期肝病 胚胎肝细胞提取液 骨髓干细胞 肝干细胞 肝细胞 分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]