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BACKGROUND: Muscle-derived stem cells have some problems in purification, amplification and directional differentiation during in vitro culture. Basic fibroblast growth factor as a membrane of bioactive factor has been paid great attention, due to its biological activity.
OBJECTIVE: To explore effects of basic fibroblast growth factor on in vitro proliferation of rat muscle-derived stem cells.
DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Shenyang Medical College from April to July 2008.
MATERIALS: Five healthy male Wistar rats were supplied by Experimental Animal Center, Shenyang Medical College. Basic fibroblast growth factor was produced by Zhuhai Biological Product Co., Ltd.
METHODS: Left anterior limb triceps brachii was sterilely obtained from rats. Muscle-derived stem cells were harvested by isolation with enzyme digestion, purified by density gradient centrifugation and differential attachment. At the second passage, muscle-derived stem cells were treated with basic fibroblast growth factor at final concentration of 6.25, 12.50, 25.00, 50.00, 100.00 μg/L. Cells received 200 μL growth medium as blank control group, and those treated with 200 μL growth medium + muscle-derived stem cells as negative controls. MTT was added at 24, 48, 72 and 96 hours of culture.
MAIN OUTCOME MEASURES: Immunocytochemical method was used to determine rat muscle-derived stem cells. MTT assay was utilized to detect cell proliferation.
RESULTS: Muscle-derived stem cells were mostly positive for Sca-1, but few was positive for Desmin. Compared with the negative control group, various concentrations of basic fibroblast growth factor had significant proliferation effects on muscle-derived cells (P < 0.05). At 6.25-50.00 μg/L, the proliferation effect became strong with the increased concentration (P < 0.01). At 50.00 μg/L, the proliferation promotion effect nearly reached a peak. Muscle-derived stem cells significantly proliferated over time. Compared with the negative control group, significant proliferation promotion effect was detected in the basic fibroblast growth factor groups at 96 hours (P < 0.01).
CONCLUSION: Basic fibroblast growth factor can promote proliferation of rat muscle-derived stem cells in vitro in a dose-time dependent fashion. 50.00 μg/L and 96 hours are separately good concentration and time for in vitro culture. |