摘要
目的:对MMSCs来源类肝细胞进行分子生物学鉴定. 方法:从人胎儿骨髓中分离MMSCs,在10 g/L Matrigel作基质,2.5 mmoL/L AZA预处理10-12 h,HGF 10 μg/L +FGF4 10 μg/L+HGM培养基中培养和诱导.收集诱导培养4,7,14,21,28 d时的细胞爬片,SABC免疫组化法DAB显色检测肝细胞早期标志AFP,CK19及早期转录因子GATA4,成熟肝细胞标志ALB,CK18,GST-π,肝细胞转录因子HNF1α;提取诱导分化10 dN第28 d细胞RNA 及蛋白质,设计AFP,CK19,ALB,CK18,CYP1B1,CYP286 引物,进行RT-PCR,观察在mRNA水平肝细胞标志的表达,采用Western blot检测CK18,AFP,ALB的表达量. 结果:未诱导培养的MMSCs中,有较少的细胞表达AFP, 未见其他肝脏特有的转录因子或者胞质蛋白标志.免疫组化显示在诱导早期可见较多细胞呈GATA4,AFP和CK19阳性表达,至诱导后期表达下降,而ALB,CK18,GST-π和肝细胞转录因子HNF1α阳性细胞比例逐渐上升.RT-PCR显示,未诱导的MMSCs仅可表达微弱的AFP mRNA,诱导早期可见AFP mRNA和CK19 mRNA表达,诱导后期未见扩增,而ALB,CK18,CYP1B1 mRNA早期和后期均可见表达,在诱导后期可见CYP286 mRNA表达,但很弱.Western blot检测显示诱导细胞中AFP,ALB和CK18 蛋白的表达趋势同基因表达类似.在65 ku和68 ku处诱导细胞组可见AFP和ALB目的条带,在45ku处诱导细胞组可见CK18目的条带.未诱导的细胞和诱导早期细胞中AFP有表达,后期未见.诱导早期和后期CK18和ALB均有表达,且后期增强. 结论:MMSCs向类肝细胞的横向分化是先分化为肝前体细胞,再分化为成熟肝细胞,在本实验诱导条件下可获得在复制及翻译各环节肝细胞标志阳性的类肝细胞.
AIM: To identify hepatocyte-like cells derivated from the induced marrow mesenchymal stem cells (MMSCs) by molecular biological techniques. METHODS: MMSCs were isolated from fetal marrow. MMSCs were cultured and induced in vitro in 10 g/L Matrigel as matrix, 2.5 mmoL/L AZA pretreatment for 10-12 h, HGF 10 μg/L+FGF4 10μg/L+HGM medium. Creep plates of induction culture cell were collected at days 4, 7,14, 21 and 28, and early marker s for hepatocyte such as AFP, CK19, early transcription factor GATA4 and mature markers for hepatocyte such as ALB, CK18, CK8, GST-π and hepatocyte transcription factor HNF1α were detected by HAB coloration of SABC immunohistochemical method. RNA and proteins of the induced differentiation cells were extracted at days 10 and 28, hepatocyte mRNA of the AFP, CK19, ALB, CK18, CYP1B1 and CYP2B6 expression were observed by using RT-PCR, and protein expression of CK18, AFP and ALB were detected with Western blot. RESULTS: Undifferentiated MMSCs had few AFP expressed cells, and did not express any of the liver-specific transcription factors or cytoplasmic markers. Immunohistochemical detection showed that many cells in early induction were GATA4, AFP and CK19 expressed positively, and the expression reduced at the late induction, but the ratio of ALB, CK18, CK8, GST-π and hepatocyte transcription factor HNF1α positive cells increased gradually. RT-PCR detection showed that undifferentiated MMSCs expressed very weakly AFP mRNA, mRNA of AFP and CK19 expressed in early induction, but disappeared in late induction, mRNA of ALB, CK18, CYP1B1 expressed in early and late induction, CYP2B6 mRNA expressed very weakly in the late induction. Western blot detection showed the expression tendency of AFP, ALB and CK18 was similar the mRNA expression. Induction groups had AFP, ALB and CK18 objective strap at 65 ku, 68 ku and 45 ku respectively. AFP expressed in the early induction and disappeared in the late induction, CK18 and ALB expressed in early induction and enhanced in the late induction. CONCLUSION: MMSCs differentiate firstly to the precursor of hepatocyte, then differentiate to mature hepatocyte, and hepatocyte-like cells with positive hepatocyte marker in replication, transcription and translation level are obtained under this experimental condition.
出处
《世界华人消化杂志》
CAS
2004年第8期1844-1848,共5页
World Chinese Journal of Digestology
基金
国家高技术研究发展计划(863)资助项目
No.2001AA216161~~
