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目的通过基因水平和蛋白水平研究不同诱导浓度以及不同诱导时间重组人BMP-2(recombinanthuman BMP-2,rhBMP-2)调节人脂肪来源干细胞(adipose-derived stem cells,ADSCs)表达VEGF的生物学特点。方法从成人脂肪组织中分离培养ADSCs。取第3代细胞,分别用终浓度为0、50、100、200 ng/mL rhBMP-2诱导,24 h后收集样本;另采用浓度为100 ng/mL rhBMP-2诱导(rhBMP-2诱导组),3、6、12、18、24、36、48 h后收集样本,并设未诱导的相应时间点细胞作为对照组;采用RT-PCR和ELISA检测VEGF mRNA和蛋白表达。结果 RT-PCR及ELISA检测示,不同浓度rhBMP-2诱导下VEGF mRNA相对表达量和蛋白表达均呈浓度依赖性,其中100 ng/mL组表达最高。50、100、200 ng/mL组VEGF mRNA相对表达量均高于0 ng/mL组(P<0.05);100 ng/mL组VEGF mRNA相对表达量最高,与其余两组比较差异有统计学意义(P<0.05)。100 ng/mL组VEGF蛋白表达量显著高于其余各组,差异均有统计学意义(P<0.05)。100 ng/mL rhBMP-2诱导ADSCs后VEGF mRNA及蛋白表达呈时间依赖性。rhBMP-2诱导组诱导3、6 h时VEGF mRNA相对表达量及蛋白表达均低于对照组(P<0.05);12 h时VEGF表达较低,两组比较差异无统计学意义(P>0.05);18、24 h达高峰,rhBMP-2诱导组显著高于对照组(P<0.05);36、48 h两组VEGF表达均明显减少,比较差异无统计学意义(P>0.05)。结论 rhBMP-2调节人ADSCs表达VEGF具有浓度依赖性和时间依赖性。rhBMP-2最适诱导浓度为100 ng/mL,诱导至18～24 h是利用rhBMP-2促进组织工程骨血管化的关键时间段。
Objective To study the biological characteristics of recombinant human BMP-2 (rhBMP-2) in regulating the expression of VEGF in human adipose-derived stem cells (ADSCs) at different inducing concentrations and different inducing time by gene level and protein level . Methods ADSCs were isolated and cultured from adult adipose tissue. The third generation cells were induced with rhBMP-2 at the final concentrations of 0, 50, 100 and 200 ng / mL, respectively. The samples were collected after 24 h. The rhBMP-2 induced group ), 3, 6, 12, 18, 24, 36, and 48 h after transplantation. Samples were collected at 3, 6, 12, 18, 24, 36, and 48 h after treatment. Cells at the corresponding time points were used as the control group. VEGF mRNA and protein were detected by RT- Results RT-PCR and ELISA showed that the relative expression and protein expression of VEGF mRNA in a concentration-dependent manner induced by different concentrations of rhBMP-2, of which 100 ng / mL group had the highest expression. The relative expression of VEGF mRNA in 50,100,200 ng / mL group was higher than that in 0 ng / mL group (P <0.05), and the relative expression level of VEGF mRNA in 100 ng / mL group was the highest, with significant difference compared with the other two groups (P <0.05). The expression of VEGF protein in 100 ng / mL group was significantly higher than the other groups (P <0.05). The expression of VEGF mRNA and protein in ADSCs induced by 100 ng / mL rhBMP-2 was time-dependent. The mRNA and protein expressions of VEGF mRNA in rhBMP-2 induced group were lower than those in control group at 3 and 6 h (P <0.05), and the expression of VEGF was lower at 12 h, but there was no significant difference between the two groups (P> 0.05 ), Peaked at 18 and 24 h, and the induced group of rhBMP-2 was significantly higher than that of the control group (P <0.05). The expression of VEGF in both groups decreased significantly at 36 and 48 h, with no significant difference (P> 0.05). Conclusion rhBMP-2 regulates human ADSCs expression of VEGF in a concentration-dependent and time-dependent manner. The optimal inducing concentration of rhBMP-2 is 100 ng / mL, and the induction of rhBMP-2 for 18-24 h is the key period for rhBMP-2 to promote tissue engineering bone vascularization.