1,25(OH)_2VD_3诱导小鼠胚胎干细胞向成骨细胞分化的研究

STUDY ON DIFFERENTIATION OF EMBRYONIC STEM CELLS INTO OSTEOBLAST IN VITRO INDUCING BY 1,25(OH)_2VD_3

目的探讨1,25(OH)2VD3在诱导小鼠胚胎干细胞(embryonic stem cells,ESCs)向成骨细胞分化过程中的重要作用。方法取2d龄昆明小白鼠颅盖骨进行成骨细胞分离培养;参照并改进zur Nieden等的方法制备拟胚体(embryoid bodies,EBs)。将EBs根据培养液不同分为4组:A组:不含白血病抑制因子EBs培养液;B组:EBs培养液中添加50μg/mL维生素C、50mmol/Lβ-磷酸甘油;C组:培养液同B组,另每孔接种5×104个第3代成骨细胞;D组与C组相同,另添加4×10-9mol/L1,25(OH)2VD3。检测ALP活性,实时定量PCR检测骨钙素mRNA的表达,茜素红S染色进行矿化骨结节计数。结果EBs贴壁后前5d,各组ALP表达未发生明显变化,5d后ALP表达逐渐增加,其中D组在第20天表达水平最高。光镜下可见轮廓清晰大小不等的红色结节。茜素红S染色测得A、B、C、D组骨结节数分别为(20±8)、(18±5)、(31±1)、(50±1)个;实时定量PCR测得A、B、C、D组骨钙素mRNA分别为10.18±1.17、20.29±1.03、18.84±4.07、32.15±5.23;C、D组与A、B组比较差异均有统计学意义(P<0.05),A、B组间比较差异无统计学意义(P>0.05),C、D组间比较差异有统计学意义(P<0.05)。结论在浓度为4×10-9mol/L1,25(OH)2VD3和维生素C、β-磷酸甘油共同作用下,有效促进了ESCs源性成骨细胞的产生。

into osteoblasts. Objective To investigate the effect of 1,25（OH）2VD3 on differentiation of embryonic stem cells （ESCs） Methods Osteoblasts were isolated and cultured from calvarium of 2-day-old Kunming white mice, embryoid bodies （EBs） were prepared with modified zur Nieden method. EBs were divided into 4 groups according to different mediums： group A, as the control group, in which EBs medium contained no leukemia inhibitory factor; group B, in which EBs medium contained supplements of Vitamin C （VC, 50 ug/mL） and β-glycerophosphate （β-GP, 50 mmol/L）; group C, in which EBs medium was the same as that of group B and 5 × 10^4 osteoblasts of 3rd passage were seeded into each well; group D, in which the medium contained supplements of VC （50 ug/mL）, β-GP （50 mmol/L） and 1,25（OH）2VD3 （4 ×10^9 mol/L）, and 5 × 10^4 osteoblasts of 3rd passage were seeded into each well. The ALP activity was determined by ALP reagent kit every 5 days, The RQ-PCR was performed to measure the mRNA expressions of osteocalcin （OCN）. Alizarin red S staining was performed to count the bone nodules. Results The expression of ALP witnessed no obvious change in each group within 5 days after adherence of EBs, but increased gradually after 5 days. The expression of ALP in group D reached the peak at 20 days. Red nodules with clear outline and different sizes were evident by microscope. Alizarin red S staining testified the number of bone noudles in groups A, B, C and D was 20 ± 8, 18 ± 5, 31 ± 1 and 50 ± 1, respectively, indicating significant differences between groups C, D and groups A, B （P 〈 0.05）, no significant difference between group A and group B （P 〉 0.05）, and a significant difference between group C and group D （P 〈 0.05）. The result of RQ-PCR showed that the mRNA expressions of OCN in groups A, B, C and D was 10.18 ± 1.17, 20.29 ± 1.03, 18.84 ± 4.07 and 32.15 ± 5.23, respectively, indicating significant differences between groups C, D and groups A, B （P 〈 0.05）, no significant difference between group A and group B （P 〉 0.05）, and a significant difference between group C and group D （P 〈 0.05）. Conclusion The combined action of 1,25 （OH）ND3 （4 × 10^-9 mol/L）, VC, and β-GP can effectively promote the differentiation of the ESCs-derived osteoblasts.