摘要
背景:大量实验证实骨髓间充质干细胞在诱导因子及特定微环境下可诱导分化为肝细胞,并已广泛用于终末肝病的临床替代治疗,而其最佳诱导条件目前尚不清楚。目的:初步探讨中草药红景天苷联合淤胆大鼠血清体外诱导大鼠骨髓间充质干细胞向肝细胞分化的可行性和有效性。方法:采用全骨髓贴壁培养法从大鼠骨髓中获取骨髓间充质干细胞,流式法检测干细胞表型;胆总管结扎切断法制备大鼠淤胆血清。取第3代骨髓间充质干细胞分3组体外诱导培养:空白对照组,基础培养基+5%淤胆血清;红景天苷组:基础培养基+5%淤胆血清+30μmol/L红景天苷;阳性对照组:基础培养基+5%淤胆血清+20μg/L肝细胞生长因子;观察各组诱导培养过程中细胞形态变化,RT-PCR法、Western-Blot法检测各诱导组肝细胞特异性蛋白表达水平。结果与结论:骨髓间充质干细胞高表达CD90、CD105,不表达CD45、CD14、CD34、CD79a;空白对照组、红景天苷组、阳性对照组细胞在诱导培养中均出现多角及双核细胞;空白对照组、红景天苷组、阳性对照组在诱导培养7 d开始出现甲胎蛋白、白蛋白的mRNA及蛋白表达;在同一时间点空白对照组表达率最低(P<0.05),红景天苷组、阳性对照组间比较,差异无显著性意义(P>0.05)。与传统淤胆血清体外诱导相比,红景天苷联合淤胆血清能更有效诱导骨髓间充质干细胞向肝样细胞分化。
BACKGROUND：A large number of experiments have confirmed that bone marrow mesenchymal stem cells can differentiate into hepatocytes under the induction of cytokines and specific micro-environment, and have been widely used in clinical alternative treatment for terminal liver disease, but the optimal inducing conditions are unclear.
OBJECTIVE：To explore the possibility and validity of differentiation of rat bone marrow mesenchymal stem cells into hepatocytes with a culture system containing salidroside and cholestatic rat serum in vitro.
METHODS：Bone marrow mesenchymal stem cells were isolated by plastic adherence from the whole bone marrow of health rats, and cellphenotypes were identified using the flow assay;cholestatic serum was prepared by common bile duct ligation. Passage 3 bone marrow mesenchymal stem cells were randomly divided into three groups for in vitro induction by the different culture systems：blank control group：basic medium plus 5%cholestatic serum;salidroside group：basic medium plus 5%cholestatic serum plus 30 μmol/L salidroside;positive control group：basic medium plus 5%cholestatic serum plus 20 μg/L hepatocyte growth factor. Changes of cellmorphology during culture time were observed in each group, reverse transcription-PCR assay and western blot assay were used to expression of hepatocyte-specific proteins.
RESULTS AND CONCLUSION：The bone marrow mesenchymal stem cells highly expressed CD90, CD105, but did not express CD45, CD14, CD34, and CD79a. Polygonal and binucleate cells appeared in the three groups during the procedure of induction. The mRNA and protein expression of alpha-fetoprotein and albumin emerged in the three groups on the 7th day;in the same period, the lowest expression ratio was in the blank control group （P〈0.05）, while there was no significant difference between the salidroside and positive control groups （P〉0.05）. Combination of salidroside and cholestatic serum can effectively induce bone marrow mesenchymal stem cells differentiating int
出处
《中国组织工程研究》
CAS
CSCD
2013年第49期8512-8519,共8页
Chinese Journal of Tissue Engineering Research
