摘要
目的:分离、鉴定小鼠骨髓基质干细胞(MSCs)并探讨在体外多种细胞因子的诱导下分化为肝细胞的可行性.方法:获取小鼠骨髓干细胞,进行体外贴壁培养、纯化,观察不同传代次数细胞形态特点.流式细胞法检测不同传代细胞的表面标志物CD45和CD90.分离后的MSCs再经含有HGF,FGF-4,EGF三种细胞因子的诱导体系继续培养21d,分别以半定量逆转录聚合酶链反应(RT-PCR)和Western blot方法检测诱导后细胞的白蛋白(ALB)、细胞角化蛋白18(CK18)、以及甲胎蛋白(AFP)在基因和蛋白水平的表达.结果:培养的骨髓干细胞随传代次数增多细胞形态趋向为长梭形.传代到第5代,基质干细胞的表面标志CD90阳性细胞从原代的25.42%增加到93.47%,造血干细胞的表面标志CD45表达阳性细胞从原代的86.49%降低到2.77%.通过RT-PCR可检测出诱导第7天细胞表达AFP mRNA,ALB mRNA及CK18 mRNA;通过Western blot可检测出诱导第21天的细胞表达ALB和CK18.结论:小鼠MSCs可以在体外被有效地分离纯化,可以被诱导为表达肝细胞表面标志的肝细胞样细胞.
AIM: To isolate and identify mouse marrow stromal stem cells (MSCs), then to explore whether MSCs cultured in the directed differentiation media including HGF, EGF and FGF-4 could differentiate into hepatocytes.
METHODS: Bone marrow cells were collected from C57BL/6 mouse by density gradient centrifugalization, and were purified by adherent culture. Cell morphology was observed, and cell surface markers were identified by flow cytometry. MSCs were cultured in the directed differentiation media including HGF, EGF and FGF-4, and the expression patterns of some genes of the hepatocytes, AFP, ALB and CK18 were validated and confirmed by RT-PCR. The ALB and CK18 expressed cells were confirmed by Western blot.
RESULTS: Cell morphology tended to be uniform fusiform shape with passage. The percentage of hematopoietic lineage marker CD45 decreased from 86.49% to 2.77% while the percentage of CD90 increased obviously from 25.42% in primary culture cells to 93.47% at the fifth generation. AFP mRNA expression appeared on culture day 7 and decreased in the later days; ALB and CK18 mRNA expression also appeared on culture day 7, but increased with the differentiation of MSCs. ALB and CK18 were confirmed to express in the differentiated MSCs by Western blot analysis.
CONCLUSION: MSCs can be isolated and purified in vitro, then induced to differentiate into hepatocytes or hepatocyte-like cells by directed differentiation media including HGF, EGF and FGF-4.
出处
《世界华人消化杂志》
CAS
北大核心
2008年第36期4051-4055,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.30371391~~
关键词
骨髓基质干细胞
肝细胞
诱导分化
半定量逆转录聚合酶链反应
Marrow stromal stem cells
Hepatocyte
Cell differentiation
Reverse transcription polymerase chain reaction