BMSCs条件培养基改善地塞米松对成骨细胞成骨功能抑制效应的研究

CONDITIONED MEDIUM OF BONE MARROW MESENCHYMAL STEM CELLS ALLEVIATE INHIBITING EFFECT OF DEXAMETHASONE ON OSTEOGENETIC CAPABILITY OF OSTEOBLAST

目的探讨BMSCs的旁分泌作用对地塞米松所致成骨细胞成骨功能抑制作用的影响。方法首先通过培养小鼠BMSCs 24 h制备无血清条件培养基备用。将小鼠MC3T3-E1细胞株复苏并传至第3代用于实验,分为4组:A组为对照组;B组于细胞中添加1μmol/L地塞米松;C组于细胞中按1∶1比例添加1μmol/L地塞米松和BMSCs条件培养基;D组仅添加BMSCs条件培养基。培养24 h后收集细胞测定ALP含量,Western blot测定细胞内RUNX2、骨钙素(Osteocalcin)蛋白表达,实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-q PCR)测定细胞内α1-Ⅰ型胶原(collagen typeⅠ-α1,COL1A1)、RUNX2、ALP、Osteocalcin基因表达;21 d后行茜素红染色观察钙结节形成情况。结果培养24 h后,B、C、D组ALP含量均显著低于A组,B组低于C、D组(P<0.05),C、D组间差异无统计学意义(P>0.05)。Western blot检测示,B组RUNX2蛋白相对表达量显著低于A、C、D组(P<0.05),A、C、D组间比较差异无统计学意义(P>0.05);B组Osteocalcin蛋白相对表达量均显著低于A、C、D组,A、C组低于D组(P<0.05),A、C组间差异无统计学意义(P>0.05)。RT-q PCR检测示,B、C、D组RUNX2、Osteocalcin、COL1A1、ALP基因相对表达量均显著低于A组(P<0.05);D组RUNX2、Osteocalcin、ALP基因相对表达量均显著高于B、C组,C组显著高于B组(P<0.05);D组COL1A1基因相对表达量显著高于B组(P<0.05),B、C组间及C、D组间比较差异均无统计学意义(P>0.05)。培养6 d时A组细胞死亡;21 d时B、C、D组均可见钙结节阳性染色,且逐渐增强。结论小鼠BMSCs条件培养基能改善地塞米松对成骨细胞成骨功能的抑制效应。

Objective To explore the paracrine effect of bone marrow mesenchymal stem cells（BMSCs） on dexamethasone-induced inhibition of osteoblast function in vitro. Methods The serum free conditioned medium of mouse BMSCs cultured for 24 hours was prepared for spare use. The 3rd passage of MC3T3-E1 cells were divided into 4 groups： the control group（group A）, dexamethasone group（group B）, dexamethasone＋BMSCs conditioned medium（1 ∶ 1） group（group C）, and BMSCs conditioned medium group（group D）. After 24 hours of culture, the alkaline phosphatase（ALP） content was determined; the protein expressions of RUNX2 and Osteocalcin were detected by Western blot; and the gene expressions of collagen type I-α 1（COL1A1）, RUNX2, ALP, and Osteocalcin were detected by realtime fluorescence quantitative PCR（RT-q PCR）; alizarin red staining was used to observe calcium nodules formation at 21 days. Results After cultured for 24 hours, ALP content was significantly lower in groups B, C, and D than group A, and in group B than groups C and D（P〈0.05）, but no significant difference was found between groups C and D（P〈0.05）. The relative protein expression of RUNX2 of group B was significantly lower than that of groups A, C, and D（P〈0.05）, but difference was not significant between groups A, C, and D（P〉0.05）. The relative protein expression of Osteocalcin was significantly lower in group B than groups A, C, and D, in groups A and C than group D（P〈0.05）, but difference had no significance between groups A and C（P〉0.05）. The relative gene expressions of RUNX2, Osteocalcin, COL1A1, and ALP of groups B, C, and D were significantly lower than those of group A（P〈0.05）; the relative gene expressions of RUNX2, Osteocalcin, and ALP were significantly higher in group D than groups B and C, in group C than group B（P〈0.05）. The gene expression of COL1A1 was significantly higher in group D than group B（P〈0.05）, but difference was not significant between groups B and C, and between groups C and D（P〉0.05）. The cells of group A all died at 6 days after culture; at 21 days, the calcium nodule staining was positive by alizarin red in groups B, C and D, and the degree of the staining gradually increased from groups B to D. Conclusion BMSCs conditioned medium can alleviate the inhibitory effect of dexamethasone on osteoblasts function.