微重力对骨髓间充质干细胞向成骨细胞分化影响的研究

Effect of microgravity on bone mesenchymal stem cells differentiation to osteogenesis

目的研究微重力对骨髓间充质干细胞（BMSCs）向成骨细胞（OB）分化的影响。方法小鼠股骨离断法提取BMSCs，传代扩增。取第4代BMSCs分为4组：①正常重力组（NG组），正常重力条件下无成骨诱导。②微重力组（MG组），微重力条件下无成骨诱导。③正常重力诱导组（NG＋I组），正常重力条件下行成骨诱导。④微重力诱导组（MG＋I组），微重力条件下行成骨诱导，培养10d。噻唑蓝（MTT）法测定各组BMSCs的增殖；茜素红染色检测细胞基质钙沉积；细胞碱性磷酸酶（ALP）活性测定以计算其在细胞基质中的表达；实时定量PCR（qPCR）及WesternBlot检测相关基因及蛋白表达。结果MG组细胞培养4d后数量开始多于NG组（P〈0．05）；茜素红染色示BMSCs诱导10d，NG＋I组出现较多的矿化结节，而MG组基本无矿化结节，NG组和MG＋I组出现的结节均较少；qPCR结果示NG组和MG组中COLI、ALP、RUNX-2基因的相对值分别为2．41±0．30VS0．56±0．19、2．15±0．12vs0．16±0．15、0．98±0．12VS0．16±0．05，差异均有统计学意义（P〈0．05）；MG＋I组上述3种基因相对值也均低于NG＋I组：1．79±0．10vs0．59±0．04、1．57±0．19vs0．57±0．08、1．30±0．14VS0．74±0．05，差异均有统计学意义（P〈0．05）；WesternBlot检测结果与qPCR基本一致，即微重力条件能明显抑制成骨相关蛋白的表达。结论微重力条件可促进BMSCs增殖，抑制BMSCs向成骨细胞的分化。

Objective To study the effect of microgravity on bone mesenchymal stem cells （BMSCs） differentiating to osteobtast （OB）. Methods BMSCs were harvested from femur of mouse and then subcuhured in vitro to the 4th generation. The cells were then divided into four groups： （1） normal gravity group（NG group）, normal gravity without osteogenic induction. （2） microgravity group （MG group）, microgravity without osteogenic induction. （3） normal gravity induction group （NG＋I group）, normal gravity with osteogenic induction. （4） microgravity induction group （MG＋I group）, mierogravity with osteogenic induction, and all ceils were cultured for 10 d. Cell proliferation was tested by thiazolyl blue tetrazolium bromide （MTT） assay, alizarin red staining was used to determine extracellular matrix mineralization, and alkaline phosphatase activity （ALP） was measured to calculate its expression in the extracellular matrix. Real-time quantitative PCR （qPCR） and Western Blot were used to evaluate the expression of osteoblast-related gene and protein. Results The numbers of BMSCs in microgravity condition were more than that in normatgravity condition after 4 days＂ culture （P〈0.05）. Alizarin red staining results showed that after 10 days＂ induction, lots of calcium nodules were found in NG＋I group, while barely nodules could be found in microgravity group at the same time. Some nodules also could be found in NG group and MG＋I group. The qPCR demonstrated that microgravity condition inhibits the expression of osteogenic gene （COL I, ALP, RUNX-2）. The relative values of the three genes mentioned above in NG group and MG group were 2.41±0.30 vs 0.56±0.19, 2.15±0.12 vs 0.16±0.15, 0.98±0.12 vs 0.16±0.05 （P〈0.05）. The relative values of the three genes in MG＋I group were also less than that in NG＋I group： 1.79±0.10 vs 0.59±0.04, 1.57±0.19 vs 0.57±0.08, 1.30±0.14 vs 0.74±0.05 （P〈0.05）. The Western Blot resuhs were nearly consistence with qPCR, and the osteogenesis-related protein was inhibited in microgravity condition. Conclusions Microgravitv can oromote the oroliferation of BMSCs and inhibit the differentiation to osteoeenesis.