成人脂肪间充质干细胞定向诱导为成纤维细胞的研究

Purpose:To investigate the methods of isolation and expansion of adult adipose-derived mesenchymal stem cells (ADSCs) in vitro,and their potential to differentiate into fibroblasts in vitro with the induction of cytokines. Methods: First we used the collagenase digestion method for separation of primary human ADSCs and two different culture systems were used for the continuous subculture and expansion of ADSCs in vitro.Then immunofluorescence and flow cytometric experiment were done to identify cell surface antigens CD44, CD90, CD105 and CD34 of ADSCs.In vitro adipogenic and osteogenic differentiation of ADSCs indicated that ADSCs have the same pluripotency as mesenchymal stem cells. ADSCs were induced with the cytokines EGF and TGF-β1 in vitro for a period of time, and then RNA was extracted for RT-PCR, qRT-PCR quantification; immunocytochemistry and flow cytometric analysis were also done for the comparation of expression of intracellular COL3, COL15 and FSP-1 before and after induction ;and finally qRT-PCR quantitative was done for the analysis of the expression of intracellular COL3, COL15 and FSP-1 in two different culture systems.Results: ADSCs we obtained showed positive expression of CD44, CD90, CD105, CD34-negative; oil red O staining and AKP staining showed that ADSC can differentiate into adipocytes,osteoblasts; RT-PCR,qRT-PCR,immunecytochemistry and flow analysis showed that when induced by EGF and TGF-β1, the expression of COL3, COL15 and FSP-1 in primary ADSCs was increased, and the expression of COL3, COL15 and FSP-1 was higher in high-serum culture system than in low-serum culture system. Conclusion: Adult adiose-derived mesenchymal stem cells in vitro induced by EGF and TGF-β1 could differentiate into fibroblasts, and the two different culture systems also influence the expression of intracellular COL3, COL15, and FSP-1 expression when induced.