大鼠骨髓间充质干细胞向类肝细胞体外诱导分化  

Rat bone marrow mesenchymal stem cells differentiate into hepatocyte-like cells in vitro

作　　者：何文艳[1] 刘树贤[2] 姜慧卿[1] 

机构地区：[1]河北医科大学第二医院消化科 [2]河北医科大学第一医院肝病中心实验室,河北省石家庄市050031

出　　处：《世界华人消化杂志》2008年第22期2464-2469,共6页World Chinese Journal of Digestology

摘　　要：目的:观察不同条件下骨髓间充质干细胞(MSCs)体外诱导分化为类肝细胞的差异.方法:Wistar大鼠24只随机均分为3组,分别为正常对照组、肝纤维化模型组、自拟中药干预组.采用CCl4乳剂皮下注射建立肝纤维化模型.造模成功后中药干预组采用丹金舒肝胶囊药液灌胃治疗.治疗结束后剖杀大鼠,留取大鼠肝脏标本,HE染色观察各组病理改变.采用密度梯度离心法和贴壁法分离各组大鼠的MSCs,经培养传代获得纯化的MSCs.各组纯化的MSCs采用HGF、FGF-4进行诱导培养.留取15、21、27d细胞培养液进行白蛋白(Alb)、甲胎蛋白(AFP)检测;于27d收集细胞爬片,进行糖原染色和CK-18免疫细胞化学染色.结果:3组15、21和27d各MSCs诱导组AFP水平均高于MSCs非诱导组(P<0.01),其中21dAFP水平最高(肝纤维化模型组:48.94±0.08vs9.90±0.09;中药干预组:49.86±0.29vs8.69±0.62;正常对照组:38.65±0.33vs9.04±0.11,均P<0.01);3组15d各MSCs诱导组与MSCs非诱导组白蛋白水平无统计学意义;21d、27d各MSCs诱导组白蛋白水平均高于MSCs非诱导组(1.11±0.08vs0.32±0.00,1.25±0.04vs0.32±0.00,1.06±0.03vs0.33±0.00;1.52±0.02vs0.33±0.00,1.79±0.01vs0.31±0.03,1.63±0.04vs0.32±0.01,均P<0.01),27d最高.27d各MSCs诱导组糖原染色阳性,免疫细胞化学染色CK-18均阳性,而MSCs非诱导组糖原染色、CK-18均阴性.从AFP、Alb水平综合比较,3组诱导效果发现自拟中药干预组优于其他2组.结论:HGF、FGF-4可在体外诱导实验性大鼠的MSCs分化为具有肝细胞样细胞表型和功能的类肝细胞;自体MSCs可以作为治疗临床重症肝病的一种细胞来源,而临床联合中药复方治疗可能会使MSCs体外诱导的效果更为理想.AIM： To investigate differentiation of rat bone marrow mesenchymal stem cells （MSCs） into hepatocyte-like cells. METHODS： A total of 24 Wistar rats were randomly divided into 3 groups： normal group, hepatic fibrosis model group and Chinese medicine treatment group. The model of liver fibrosis was induced by subcutaneous injection of CCl4. After the model was successfully developed, and the rats in Chinese medicine treatment group were fed with Danjin Shugan capsule. At the end of treatment, the rats were killed and the livers were obtained. Histopathological changes were evaluated by hematoxylin and eosin staining. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. The purified MSCs in each group were cultured with hepatocyte growth factor （HGF） and fibroblast growth factor4 （FGF4）. The levels of alpha-fetoprotein （AFP） and albumin （Alb） in the supernatant were determined by radioimmunoassay on days 15, 21 and 27. On day 27, the cells were collected for glycogen staining and CK-18 immunocytochemical analysis. RESULTS： Compared with those in the noninduced MSCs among the three groups, the levels of AFP in the induced-MSCs were higher on days 15, 21, and 27, and reached to the peak value on day 21 （hepatic fibrosis model group： 48.94 ± 0.08 vs 9.90 ± 0.09; Chinese medicine treatment group： 49.86 ± 0.29 vs 8.69 ± 0.62; normal group： 38.65 ± 0.33 vs 9.04 ± 0.11; all P 〈 0.01）. There were significant differences in Alb levels on days 21 and 27 between the induced and noninduced MSCs （1.11 ± 0.08 vs 0.32 ± 0.00, 1.25 ± 0.04 vs 0.32 ± 0.00, 1.06 ± 0.03 vs 0.33 ± 0.00; 1.52 ± 0.02 vs 0.33 ± 0.00,1.79 ± 0.01 vs 0.31 ± 0.03,1.63 ± 0.04 vs 0.32 ± 0.01; all P 〈 0.01）, but not on day 15; the peak level of Alb was on day 27. Both glycogen and CK-18 were positive on day 27 in the induced MSCs. According to AFP and Alb levels, the induced effects of Chinese medicine group were superior to those of the other two groups. CON

关 键 词：骨髓 间充质干细胞 类肝细胞 大鼠 放射免疫法 免疫细胞化学 肝细胞生长因子 成纤维细胞生长因子4 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]