音猬因子调控BMSCs表达和分泌VEGF及bFGF的实验研究

REGULATION OF SONIC HEDGEHOG ON VASCULAR ENDOTHELIAL GROWTH FACTOR,BASIC FIBROBLAST GROWTH FACTOR EXPRESSION AND SECRETION IN BONE MARROW MESENCHYMAL STEM CELLS

目的音猬因子(Sonic hedgehog,Shh)介导的信号参与调控血管生成的重要环节。研究不同浓度的重组Shh-N(recombinant Shh N-termitant,rShh-N)对BMSCs表达和分泌VEGF和bFGF的影响。方法取健康3日龄SD大鼠骨髓分离培养BMSCs,体外扩增至第3代,分别用含0、10、100、200 ng/mL rShh-N的L-DMEM培养BMSCs,作为A、B、C、D组。培养12、24、48、72 h后行ELISA法检测各组上清液中VEGF和bFGF的浓度,实时荧光定量PCR法检测各组VEGF和bFGF mRNA的表达水平。结果在基因表达水平上,D组各时间点的VEGF和bFGF mRNA表达量均明显高于A组(P<0.05),且在12、48 h表达量高于24、72 h(P<0.01);C组在各时间点均促进bFGF mRNA表达(P<0.05),在24～72 h促进VEGF mRNA表达(P<0.05),且在72 h时表达量均最高(P<0.01);B组在12 h抑制VEGF mRNA表达(P<0.05),48 h和72 h表现出促进作用(P<0.05),在12～48 h明显促进bFGF mRNA表达(P<0.05),且在48 h时的表达量最高(P<0.01)。在蛋白水平上,D组各时间点VEGF和bFGF分泌量均高于A组(P<0.01);C组在24～72 h VEGF和bFGF分泌量明显多于A组(P<0.05);B组在12 h和48 h抑制VEGF的分泌(P<0.05),24 h增加其分泌作用(P<0.05),而在24 h和48 h促进bFGF的分泌(P<0.05)。各组在48 h和72 h时的VEGF和bFGF分泌量明显多于12 h和24 h(P<0.05)。结论 rShh-N可促进BMSCs表达和分泌VEGF和bFGF,为进一步探讨rShh-N和MSCs联合应用于治疗缺血性相关疾病以及促进骨修复重建的可行性提供了实验依据。

Objective Sonic hedgehog（Shh） signaling pathway is involved in an important part of regulating angiogenesis.To investigate the effects of recombinant Shh N-terminant（rShh-N） on the expression and secretion of angiogenesis-related factor—vascular endothelial growth factor（VEGF） and basic fibroblast growth factor（bFGF）.Methods Bone marrow mesenchymal stem scells（BMSCs） were isolated from 3-day-old healthy Sprague Dawley rats and cultured to passage 3 in vitro.rShh-N at the concentrations of 0,10,100,and 200 ng/mL were applied to culture BMSCs in groups A,B,C,and D,respectively.At 12,24,48,and 72 hours of culture,the expressions of VEGF and bFGF mRNA and the levels of VEGF and bFGF in supernatant were measured with real-time quantitative PCR and ELISA,respectively.Results At the gene level,compared with group A,the expressions of VEGF and bFGF mRNA were enhanced in group D（P 0.05） and the upregulation was more significant at 12 and 48 hours than 24 and 72 hours（P 0.01）.In group C,bFGF mRNA expression was substantially promoted at 12-72 hours（P 0.05） and VEGF mRNA level was upregulated at 24-72 hours（P 0.05）,and both reached peak at 72 hours（P 0.01）.In group B,VEGF mRNA expression was inhibited at 12 hours（P 0.05）,but the level increased at 48 and 72 hours（P 0.05）;bFGF mRNA expression was obviously promoted at 12-48 hours（P 0.05） and the maximum appeared at 48 hours（P 0.01）.At the protein level,the secretion of VEGF and bFGF in group D was significantly increased at 12-72 hours,as compared with group A（P 0.05）.In group C,VEGF and bFGF secretion was increased at 24-72 hours（P 0.05）.The secretion of VEGF in group B was inhibited at 12 and 48 hours（P 0.05） and was promoted at 24 hours（P 0.05）;bFGF secretion was up-regulated at 24 and 48 hours（P 0.05）.The secretion of VEGF and bFGF in supernatant at 48 and 72 hours were significantly more than those at 12 and 24 hours in 4 groups（P 0.05）.Conclusion rShh-N treatment can enhance the expression and secretion of VEGF and bFGF in BMSCs,which could provide the experimental evidence for the further application of Shh-MSCs in the treatment of ischemia-related diseases and bone repair.