摘要
目的探讨肝细胞生长因子(hepatocyte growth factor,HGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)诱导人骨髓间充质干细胞在体外向肝系细胞分化的可能性。方法人骨髓间充质干细胞来自成人骨髓,采用密度梯度离心法分离和反复贴壁法纯化。为有效地促进其向肝细胞分化,本实验使用了HGF和bFGF联合诱导的方法,于0、7、14、21、28d采用细胞免疫荧光法检测肝细胞表面标志甲胎蛋白(alpha_fetoprotein,AFP),细胞角蛋白(cytokeratin 18,CK_18)和细胞角蛋白(cytokeratin 19,CK_19),同时采用Periodic Acid_Schiff(PAS)染色检测诱导细胞糖原合成及贮存功能,并留取不同时段的培养上清液,用放射免疫荧光法(radioimmunoassay,RIA)法检测AFP的分泌量。结果在诱导14d后可观察到诱导细胞变成短梭形和多角形,随诱导时间延长多角形细胞增多,并可形成肝细胞样细胞集落。第14天时AFP,CK_18表达阳性,第21天诱导细胞糖原染色阳性,第28天CK_19表达阳性。培养上清液中14d时,检测到AFP的分泌,浓度为0.1ng/ml;17d时分泌量最高,为0.4ng/ml;21d时下降至0.3ng/ml。结论HGF和bFGF能够诱导人骨髓间充质干细胞在体外分化为肝细胞,这将为肝脏疾病的细胞替代治疗提供新的细胞来源。
Objective To explore whether human bone marrow mesenchymal stem cells （MSCs） are able to differentiate into functional hepatoeyte-like cells with hepatoeyte growth factor （HGF） and basic flbroblast growth factor（bFGF） in vitro. Methods MSCs from adult human bone marrow were isolated by density gradient centrifugation in combination with plastic adherence. To induce hepatic differentiation effectively, we designed aprotocol with the use of HGF and bFGF. The morphological changes of induced MSCs were observed in different culture period. The hepatic surface phenotype including AFP, CK-18 and CK-19 were identified by immunofluorescent staining at day 0,7,14,21,28. Glycogen storage was detected by Periodic Acid-Schiff（PAS） staining and the concentration of AFP in the supernatant was determined dynamically by radioimmunoassay （RIA）. Results After 14 days of induction, the fibroblast-like morphology of human MSCs was lost and cells became broadened and flattened. After prolonged culture, polygonal cells was seen and further matured hepatoeyte-like morphology was seen by day 28. lmmunofluoresee, nt staining showed the expression of AFP and CK-18 until day 14. Glycogen storage can be detected by day 21. The expression of CK-19 was detected by day 28. The concentration of AFP in the supernatant was first detected on day 14, at a concentration of 0.1 ng/ml, and increased to0.4 ng/ml by day 17, then decreased to 0.3ng/ml by day 21. Conclusions Human bone marrow MSCs are able to differentiate into functional hepatocyte-like cells and may serve as a new source of cells for cell theraov of hepatic diseases.
出处
《国际流行病学传染病学杂志》
CAS
2006年第3期150-153,156,共5页
International Journal of Epidemiology and Infectious Disease
基金
江西省科技厅重点项目
