碱性成纤维细胞生长因子和表皮生长因子对骨骼肌源性干细胞的促增殖效应

目的观察碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）和表皮生长因子（epidermal growth factor，EGF）单独及联合应用对骨骼肌源性干细胞（muscle derived stem cells，MDSCs）生长的影响。方法取出生24h内的昆明小鼠15只，采用连续预贴壁法从小鼠后肢肌分离培养MDSCs，用含2％胎牛血清的DMEM培养基促进其向骨骼肌细胞分化。取原代MDSCs及MDSCs分化细胞，采用免疫细胞化学染色检测干细胞标志Sca-1和骨骼肌细胞标志α-Sarcomeric肌动蛋白的表达。HE染色观察细胞肌管形成。MTT比色法检测不同浓度（6．25、12．50、25．00、50．00、100．00ng／ml）的bFGF和EGF单独应用96h对MDSCs增殖的影响以及二者（100．00ng／ml）联合作用24、48、72和96h对MDSCs增殖的影响。结果从新生小鼠后肢肌成功分离培养MDSCs，免疫细胞化学染色90％以上的MDSCs呈Sca-1阳性，分化形成的肌管呈α—Sarcomeric肌动蛋白阳性。HE染色可见肌管形成。bFGF、EGF对MDSCs的促增殖效应随浓度的增加而增加。与阴性对照组比较，bFGF于12．50ng／ml出现促增殖效应（P〈0．05）；25．00ng／ml组与12．50ng／ml组比较，作用提高（P〈0．01）；50．00、100．00ng／ml组较25．00ng／ml组无明显提升（P〉0．05）；EGF的作用与bFGF类似，但于50．00ng／ml时趋于饱和。与阴性对照组比较，EGF于72h、bFGF于96h表现促增殖效应（P〈0．01），而二者联合应用于24h即表现促增殖效应（P〈0．01），并于48、72和96h增殖效应均较单独应用显著提高（P〈0．05）。结论bFGF和EGF都能促进MDSCs的增殖，联合作用更快、更强。

PROLIFERATIVE EFFECT OF BASIC FIBROBLAST GROWTH FACTOR AND EPIDERMAL GROWTH FACTOR ON MUSCLE DERIVED STEM CELLS

OBJECTIVE: To explore the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on the growth of muscle derived stem cells (MDSCs). METHODS: MDSCs were isolated from hindlimb muscle of 15 new born Kunming mice through serial preplates. 2% fetal bovine serum-containing DMEM was used to induce MDSCs to differentiate into skeletal muscle lineage. The expressions of stem cell marker Sca-1 and skeletal muscle cell marker alpha-Sarcomeric actin were examined by immunocytochemistry. The effect of bFGF and EGF on the proliferation of MDSCs was determined by MTT colorimetric microassay. The solo effect of bFGF or EGF at different concentrations (6.25, 12.50, 25.00, 50.00, and 100.00 ng/ml) was examined at 96 h and the combined effect (100.00 ng/ml) was examined at 24, 48, 72 and 96 h. RESULTS: MDSCs were successfully isolated from the hindlimb of neonatal mice. Over 90% of MDSCs showed Sca-1 positive immunoreactivity. MDSCs could give rise to alpha-Sarcomeric actin positive myotubes in differentiation cultures. The proliferative effect of bFGF and EGF on MDSCs increased with the elevated concentration. bFGF began to show significant proliferative effect at 12.50 ng/ml (P<0.05). The effect increased significantly when the concentration reached 25.00 ng/ml from 12.50 ng/ml (P < 0.01) and reached a saturation point. The effect at 50.00 ng/ml or 100.00 ng/ml showed no significant increase when compared with that at 25.00 ng/ml. EGF had a similar effect to bFGF except that the saturation concentration was 50.00 ng/ml. EGF showed significant effect at 72 h and bFGF at 96 h (P<0.01). When they were applied together, significant effect was shown at 24 h (P<0.01) and much higher effect was observed at 48, 72 and 96 h (P<0.05). CONCLUSION: Both bFGF and EGF can promote the proliferation of MDSCs. The combined application reacts faster and stronger.