碱性成纤维细胞生长因子对间充质干细胞增殖的影响

目的:探讨碱性成纤维细胞生长因子(bFGF)对间充质干细胞(MSCs)增殖的影响及其信号机制。方法:全骨髓贴壁法培养MSCs,流式细胞仪分析其表面MSCs标记(CD45、CD34、CD90、CD29)以及成骨、成脂肪等多向诱导分化潜能,鉴定其特征。MTT法分析不同浓度bFGF对MSCs增殖的影响,确立最佳浓度bFGF诱导MSCs增殖的时间特征,分别以磷脂酰肌醇3-激酶、促分裂素原活化蛋白激酶、磷脂酶C、蛋白激酶C、钙通道和钙泵选择性抑制剂等处理P3MSCs后,观察其对bFGF介导MSCs增殖的影响。结果:培养的MSCs表现CD90、CD29强阳性,具有成骨、成脂肪等多向分化能力;bFGF诱导MSCs增殖呈时间和剂量依赖特征,5.4×10-4mol/L bFGF作用48 h时MSCs增殖达峰值;磷脂酶C阻断剂U73122、钙通道阻断剂和选择性蛋白激酶C阻断剂等明显抑制了bFGF所介导的MSCs增殖效应;尤其是非选择性PKC抑制剂Straurosporine、钙泵选择性抑制剂thapsigargin以及蓝尼定受体抑制剂Ryandoine均抑制bFGF所诱导的MSCs增殖。结论:bFGF通过Ca2+/PKC/Erk1/2途径促进MSCs的增殖。

Effects of bFGF on Bone Mesenchymal Stem Cells Proliferation and Its Signal Mechanism

Objective To explore the effects of bFGF on mesenchymal stem cells(MSCs) proliferation and its possible signal mechanism.Methods MSCs were cultured with classical whole bone marrow adhering method,the characteristic of MSCs was identified through multi-differentiation and the surface marker(CD34,CD45,CD29,and CD90) were detected by flow cytometer.MSCs were co-cultured with different concentrations of bFGF(5.4×10-5,2.7×10-4,5.4×10-4,10.8×10-4,21.6×10-4,32.4×10-4,43.2×10-4 mol/L),in which the concentration of 5.4×10-4mol/L bFGF showed the best enhancement of proliferation,the further culture were performed under this concentration for 12 h,24 h and 48 h.Then P3MSCs were respectively treated with PI3K/Akt(5.0×10-8 mol/L Wortmannin or 1.0×10-5 mol/L LY294002),MAPK(5.0×10-5 mol/L PD98059 or 3.0×10-5 mol/L SB203580),PLC(1.0×10-5 mol/L U73122),Ca2+ signal(5.0×10-8 mol/L Verapamil,1.0×10-4 mol/L Ryandoine,7.5×10-5 mol/L 2-APB),endoplasmic reticular Ca2+ dependent ATPase(1.0×10-6 mol/L thapsigargin) or PKC(1.0×10-5 mol/L chelerythrine chloride,1.0×10-2 mol/L Straurosporine,and 6.0×10-9mol/L G06976) inhibitors for 30 min,the proliferation rate was measured by MTT assay.Results Cultured MSCs showed the ability of multi-differentiation and highly expressed CD105 and CD90.The proliferation of MSCs inducing by bFGF were in dose and time dependent manner and reached the peak at concentration of 5.4×10-4mol/L for 48 h,which were obviously inhibited by phospholipase C inhibitor U73122,calcium channel inhibitor,selective protein kinase C blocker,especially by straurosporine,thapsigargin and ryandoine.Conclusion bFGF could promote the proliferation of MSCs through Ca2+/PKC/Erk1/2 signal pathway.