摘要
目的:建立人脐血间充质干细胞(umbilicalcordbloodmesenchymalstemcells,UCBMSC)的体外分离、培养方法,体外诱导UCBMSC分化为类肝细胞,观察UCBMSC细胞生物学特性,并对类肝细胞进行分子生物学及功能鉴定.方法:采用体外细胞培养技术,分离培养人脐血UCBMSC,在10g/LMatrigel作基质,2.5mmol/LAZA预处理10-12h,HGF10μg/L+FGF410μg/L+HGM培养基中诱导.用显微摄像和MTT研究细胞增殖及生长特征,用流式细胞仪、免疫组织化学、RT-PCR鉴定细胞表型.采用ELISA法检测培养上清中人白蛋白水平.结果:每份脐血可获得150±20个贴壁细胞:细胞种植后6d达到对数生长期,连续传10代后,每份脐带血UCBMSC可扩增达109-1010个细胞.UCBMSC表型为CD44及CD166阳性,CD34及CD45阴性.在添加FGF4和HGF的Matrigel上诱导培养的UCBMSC在21-28d时,形态由长梭形变为三角形,多角形或类圆形.细胞转圆率为40-50%,双核细胞比率5-7%.免疫组化,RT-PCR检测显示未诱导培养的UCBMSC中,有较少的细胞表达AFP及其mRNA,未见其他肝脏特有的转录因子或者胞质蛋白标志.诱导早期可见较多细胞表达GATA4,AFP和CK19及其mRNA,至诱导后期表达下降,而ALB,CK18,GST-π和肝细胞转录因子HNF1α表达逐渐上升.ALB,CK18阳性细胞比例达61-65%.未诱导分化的UCBMSC没有分泌ALB和产生尿素,诱导分化的UCBMSC以时间依赖方式产生白蛋白.结论:人脐血UCBMSC先分化为肝前体细胞,再分化为成熟肝细胞,获得了在复制及翻译各环节肝细胞标志阳性的类肝细胞,已具备肝细胞特有的分泌白蛋白功能.
AIM： To differentiate human umbilical cord blood mesenchymal stem cells （UCBMSCs） into functional hepatocytelike cells in vitro, and to identify the molecular biology and function of hepatocyte-like cells. METHODS： Human UCBMSCs were isolated and cultured in vitro. Matrigel （10 g/L） was used as matrix, and after pretreated with 2.5 mmol/L AZA for 10-12 h, UCBMSCs were induced in HGF （10 μg/L）＋FGF4 （10 μg/L）＋HGM medium in vitro. The proliferation of UCBMSCs was detected by MTT. The phenotypes of UCBMSCs were identified by flow cytometry, immunohistochemistry, and reverse transcription polymerase chain reaction （RT-PCR）. The level of albumin （ALB） in the supernatant was determined with enzyme linked immunosorbent assay （ELISA）. RESULTS： About 150±20 adherent cells were obtained in each cord blood unit, and the growth of the cells entered log phase 6 days after plantation. The number of UCBMSCs reached 10^9-10^10 after 10 generations. The CD44 and CD166 phenotypes were positive in UCBMSCs, whereas CD34 and CD45 were negative. On day 21-28, UCBMSCs changed from long fusiform to polygonal or round-like shape （40-50%）. The rate of double-nucleus cells was 5-7%. A few parts of the undifferentiated UCBMSCs expressed alpha-fetoprotein （AFP） and AFP mRNA, but did not express any of the liver-specific transcription factors or cytoplasmic markers. In the early period of induction, many cells expressed GATA4, AFP and CK19 proteins and their mRNA, and their expression reduced in the late period of induction. However, the expression of ALB, CK18, GST-π and hepatic nuclear factor-1α（HNF-1 α） increased gradually in the process of induction. The rates of ALB- and CK18-positive cells were 61-65%. The undifferentiated UCBMSCs did not secret ALB and urea while the differentiated UCBMSCs produced ALB in a time-dependent manner. CONCLUSION： Human UCBMSCs can be successfully induced to differentiate into hepatocyte-like cells, which are able to secret ALB.
出处
《世界华人消化杂志》
CAS
北大核心
2005年第15期1814-1818,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~~
