不同浓度地塞米松在体外定向诱导分化兔骨髓间充质干细胞为成骨细胞的实验研究

Empirical study on the influence of different concentration of DEX on induction and differentiation of rabbit's BMSCs into osteoblasts

目的探讨兔骨髓间充质干细胞(MSCs)体外定向诱导分化为成骨细胞所需的地塞米松的最佳浓度。方法抽取兔骨髓,采用全骨髓贴壁培养法体外培养,贴壁细胞进行传代。取第3代细胞,随机分为5组,在培养基中添加成骨分化诱导剂β-甘油磷酸钠、L-抗坏血酸,5组添加的地塞米松的浓度分别为1×10-6mol/L、1×10-7mol/L、1×10-8mol/L、1×10-9mol/L、1×10-10mol/L。培养10 d后,在倒置显微镜观察细胞形态,计算细胞浓度,采用5-溴-4-氯-3-吲哚磷酸盐(BCIP)/氯化硝基四氮唑蓝(NBT)底物显色试剂检测碱性磷酸酶(ALP),茜素红染色检测钙结节。结果较高浓度(1×10-8mol/L、1×10-7mol/L、1×10-6mol/L)的地塞米松相对于较低浓度(1×10-9mol/L、1×10-10mol/L)的地塞米松可显著增加ALP表达(P均<0.05),并可增加钙结节的形成;1×10-9mol/L及其以下浓度的地塞米松对细胞增殖无明显影响(P均>0.05),1×10-7mol/L以上浓度的地塞米松可明显抑制细胞的增殖(P<0.01)。结论地塞米松在体外定向诱导分化兔骨髓间充质干细胞为成骨细胞,最佳的浓度为1×10-8mol/L。

Abstract： Objective It is to approach the optimal concentration of dexamethasone （DEX） in directional induction and differentiation of rabbit＇ s BMSCs into osteoblasts in vitro. Methods The bone marrow was aspirated from the iliac crest of New Zealand white rabbit and cultured with adherent culture technique. BMSCs of third passage were divided into 5 groups random- ly and subcultured in osteogenic induction medium. The osteogenic induction medium contained 10 mmol/L β- glycerol phos-phate and 50 mg/L L - ascorbic acid. The concentration of DEX in the medium was 1 × 10^-6 mol/L, 1 × 10^-7 mol/L, 1 ×10^-8 mol/L, 1 × 10^-9 mol/L and 1 × 10-10 mol/L respectively. After cultured for 10 days, the appearance of cells was ob-served with inverted microscope and the concentration of cells was calculated. In addition, ALP was detected by BCIP/NBTand calcium nodus was detected by alizarin bordeaux. Results The DEX with high concentration （ 1 × 10 -8 mol/L, 1× 10-7mol/L, 1 × 10-6 mol/L） had more ability in promotion of ALP expression （ P 〈 0. 05 ） and calcium nodus formation. DEX with concentration under 1 × 10-9 mol/L had no influence on proliferation of BMSCs （P 〉 0.05 ） , while it with concentration beyond 1 × 10-7 mol/L could inhibit proliferation （P 〈 0. 01 ）. Conclusion The optimal DEX concentration on directional induction and differentiation of rabbit＇ s BMSCs into osteoblasts in vitro is 1 ×10 -8 mol/L.