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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) -mediated signaling is involved in the regulation of angiogenesis. To investigate the effect of different concentrations of recombinant Shh-N-termitant (rShh-N) on the expression of VEGF and bFGF in BMSCs. Methods BMSCs were isolated from bone marrow of healthy 3-day-old SD rats and expanded to the third passage in vitro. BMSCs were cultured with L-DMEM containing 0, 10, 100, and 200 ng / mL rShh-N as A, C, D group. After cultured for 12, 24, 48 and 72 h, the concentrations of VEGF and bFGF in supernatants of each group were detected by ELISA. The expression of VEGF and bFGF mRNA in each group were detected by real-time fluorescence quantitative PCR. Results At the gene expression level, the expression of VEGF and bFGF mRNA in group D at each time point were significantly higher than those in group A (P <0.05), and were higher at 12 and 48 h (P <0.01) (P <0.05). The expression of bFGF mRNA was increased at 24 h and 72 h in group C (P <0.05), and the expression of bFGF mRNA was highest at 72 h (P <0.01). In group B (P <0.05) at 12 h, and promoted the expression of VEGF at 48 h and 72 h (P <0.05), and promoted the expression of bFGF mRNA at 12 h to 48 h (P <0.05) The highest expression level (P <0.01). At the protein level, the secretion of VEGF and bFGF in group D at each time point was higher than that in group A (P <0.01); the secretion of VEGF and bFGF in group C at 24-72 h was significantly higher than that of group A (P <0.05); B Group inhibited VEGF secretion at 12 h and 48 h (P <0.05), increased secretion at 24 h (P <0.05), and promoted bFGF secretion at 24 h and 48 h (P <0.05). The secretion of VEGF and bFGF at 48 h and 72 h in each group was significantly higher than that at 12 h and 24 h (P <0.05). Conclusion rShh-N can promote the expression of VEGF and bFGF in BMSCs, which provides experimental evidence for further exploring the feasibility of combined application of rShh-N and MSCs in the treatment of ischemic related diseases and the promotion of bone repair and reconstruction.