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目的探讨培养液中不同浓度FBS对成骨生长肽(osteogenic growth peptide,OGP)促进BMSCs增殖和分化的影响。方法取8只5周龄SD大鼠四肢骨,贴壁法分离纯化BMSCs并传代,倒置相差显微镜下观察细胞形态。取第3代BMSCs分组培养:分别采用浓度为1×10-10、1×10-9和1×10-8 mol/L的OGP进行培养,以正常培养细胞作为对照组;同时每组设FBS浓度为0、2%、5%、8%和10%5个梯度。培养后1、3、5、7、9、12 d采用MTT法检测细胞增殖,9 d时采用对硝基苯磷酸二钠法测定早期分化指标细胞内ALP活性。结果倒置相差显微镜下观察BMSCs贴壁生长,增殖迅速,呈纤维状涡旋生长,形态典型。MTT检测示,FBS低于5%时各组细胞不能持续增殖,当FBS浓度为8%以上时细胞可持续增殖;OGP 1×10-8 mol/L和1×10-9 mol/L组在FBS各浓度中促增殖效果均大于对照组(P<0.05);FBS浓度低于10%时,OGP 1×10-8 mol/L组促增殖效果显著优于其余OGP浓度组(P<0.05),但FBS浓度为10%时OGP 1×10-8 mol/L组无促增殖优势。ALP检测示,各组组内随FBS浓度增加,ALP活性增加(P<0.05);当FBS浓度为5%、8%时,各OGP浓度组ALP活性均大于对照组(P<0.05),且OGP 1×10-8 mol/L组最高(P<0.05);当FBS浓度为10%时,各OGP组ALP活性仍大于对照组(P<0.05),但OGP1×10-8 mol/L组与OGP 1×10-9 mol/L组差异无统计学意义(P>0.05)。结论 8%FBS浓度为OGP促进BMSCs增殖分化的最佳血清浓度,且OGP促进BMSCs增殖分化的最适浓度为1×10-8 mol/L。
Objective To investigate the effects of different concentrations of FBS in culture medium on the proliferation and differentiation of BMSCs induced by osteogenic growth peptide (OGP). Methods BMSCs were isolated and purified from 8 limbs of 5-week-old SD rats by adherent method. The morphological changes of the cells were observed under inverted phase contrast microscope. The 3rd generation of BMSCs were cultured in groups: OGP cultured at concentrations of 1 × 10-10, 1 × 10-9, and 1 × 10-8 mol / L, respectively. Normal cultured cells were used as the control group. At the same time, FBS Concentrations of 0, 2%, 5%, 8% and 10% of 5 gradients. Cell proliferation was detected by MTT assay at 1, 3, 5, 7, 9 and 12 days after culture. On the 9th day, ALP activity in early differentiated cells was determined by p-nitrophenyl disodium phosphate assay. Results The inverted microscope was used to observe the adherent growth of BMSCs with rapid proliferation and fibrous swirling growth. MTT assay showed that the cells could not proliferate continuously when the FBS was below 5%. When the FBS concentration was above 8%, the cells could proliferate continuously. OGP 1 × 10-8 mol / L and 1 × 10-9 mol / L (P <0.05). When the concentration of FBS was less than 10%, the proliferation of OGP 1 × 10-8 mol / L group was significantly better than that of the other OGP groups (P <0.05) , But there was no advantage of proliferation in OGP 1 × 10-8 mol / L group when FBS concentration was 10%. ALP assay showed that ALP activity was increased with the increase of FBS concentration in each group (P <0.05); ALP activity of each OGP concentration group was significantly higher than that of control group (P <0.05) when FBS concentrations were 5% and 8% OGP 1 × 10-8 mol / L group was the highest (P <0.05). When the concentration of FBS was 10%, the ALP activity of each OGP group was still higher than that of the control group (P <0.05) And OGP 1 × 10-9 mol / L group, the difference was not statistically significant (P> 0.05). Conclusion 8% FBS concentration is the optimal serum concentration for OGP to promote the proliferation and differentiation of BMSCs, and the optimal concentration of OGP to promote the proliferation and differentiation of BMSCs is 1 × 10-8 mol / L.