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 real-time fluorescence quantitative PCR (RT-qPCR); 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 no dule 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.
Conclusions: BMSCs conditioned medium can alleviate the inhibitory effect of dexamethasone on osteoblasts function.
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1 μmol/L地塞米松；C组于细胞中按1:1比例添加1 μmol/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-qPCR）测定细胞内α1-Ⅰ型胶原（collagen typeⅠ-α 1，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-qPCR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。.
Keywords: Bone marrow mesenchymal stem cells; Conditioned medium; Dexamethasone; Mouse; Osteoblasts; Osteogenesis.