摘要
目的:与平板培养比较,探讨悬滴三维培养对大鼠骨髓间充质干细胞(rMSCs)肝样分化的诱导效应。方法:对rMSCs进行悬滴培养,以肝细胞生长因子(HGF)作为诱导因子,同时设定平板培养诱导和非诱导对照组,在培养第7、14和21d后,通过RT-PCR法和免疫荧光技术检测白蛋白(ALB)、甲胎蛋白(APF)和角蛋白(CK-18)在mRNA和蛋白水平上的表达情况,并通过ELISA检测培养液上清中ALB的分泌。结果:HGF悬滴培养的rMSCs形成球状三维结构,内部呈现空囊样结构,大量细胞聚集在球体外侧面;在第7d就出现ALB和CK-18的强阳性表达,而AFP则表达较弱。HGF培养的单层rMSCs直到第14d才出现ALB、AFP和CK-18 mRNA的弱阳性表达,蛋白表达则在第21d才呈现阳性。ELISA检测到HGF悬滴培养后rMSCs能够胞外分泌ALB,与正常和平板培养诱导组比较差异显著(P<0.01)。结论:悬滴培养为rMSCs创造一种球形三维培养微环境,在HGF的诱导作用下,rMSCs横向分化为肝样细胞。
AIM： To establish an optimal differentiated three-dimensional microenvironment formed by hanging drops for the high efficiency of hepatic differentiation from rat mesenchymal stem cells（rMSCs）.METHODS： rMSCs were cultured in the hanging drops, which provided a three-dimensional microenvironment, for 21 days in the presence of hepatocyte growth factor（HGF, 20 μg/L）. The expression of albumin（ALB）, alpha-fetoprotein（AFP） and cytokeratin-18（CK-18） was detected by RT-PCR and immunofluorescence staining at 7th, 14th and 21st days. The secretion of albumin in the culture supernatants was measured by ELISA. RESULTS： rMSCs were aggregated in spheroid with a tubiform medium altitude in the center. In rMSCs cultured in the hanging drops with HGF, the expression of albumin, AFP and CK-18 was all detectable by RT-PCR and immunofluorescence staining at 7th day. The production of albumin in the cells cultured in the hanging drops with HGF was 50.25±5.32, 55.03±7.45 and 54.92±3.18（ng·dish-1·d-1） at 7th, 14th and 21st days, respectively, significantly higher than that in the cells in the plate cultivation with or without HGF induction at corresponding time points（P〈0.01）.CONCLUSION： In the presence of HGF, rMSCs are induced to differentiate into hepatocyte-like cells cultured in the hanging drops, where the three-dimensional spheroidal cultures are promising microenvironment for hepatic transdifferentiation of MSCs.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2010年第11期2207-2211,共5页
Chinese Journal of Pathophysiology
基金
教育部新世纪优秀人才支持计划资助项目(No.NCET-08-0611)
中国博士后科学基金资助项目(No.20090450882)
广州市科技攻关计划-科技攻关引导资助项目(No.2007Z3-E0641)
