插头框旋转录因子C2转染BMSCs后成骨作用的研究

A study on osteogenesis of BMSCs transfected with Foxc2

[目的]观察过表达Foxc2基因对兔骨髓间充质干细胞(BMSCs)增殖及成骨分化的影响。[方法]构建携带Foxc2和绿色荧光蛋白(GFP)基因的重组慢病毒表达载体,分离、培养兔BMSCs,分别以Lenti-GFP(GFP组)、Lenti-Foxc2(Foxc2组)转染BMSCs,另设未转染BMSCs对照组。MTT法检测过表达Foxc2对细胞增殖能力;Western blot、Real time PCR检测转染后7d各组细胞Foxc2蛋白、m RNA表达;以及转染后1、2、4周各组细胞骨钙蛋白(OCN)、骨桥蛋白(OPN)、一型胶原(COLⅠ)蛋白及m RNA表达;转染后2周,各组行碱性磷酸酶(ALP)染色和ALP活性检测。[结果]Foxc2组Foxc2 m RNA和蛋白高效表达;对照组、GFP组无Foxc2 m RNA及蛋白表达;MTT法检测示过表达Foxc2对BMSCs的增殖未产生显著影响;Foxc2组OCN、OPN、COLⅠm RNA和蛋白表达显著高于其他组(P<0.01),余组比较差异无统计学意义(P>0.05);Foxc2组ALP染色阳性区域大于其余两组;ALP活性显著高于其他各组(P<0.01)。[结论]过表达Foxc2不影响细胞增殖,可持续表达基因产物,并可促进BMSCs向成骨细胞分化。

[ Objective ] To investigate the influence of forkhead box protein C2 （Foxc2） overexpression on the prolifera- tion and osteogenic differentiation of bone mesenchymal stem cells （BMSCs） in rabbits. [Methods] The recombinant lentivi- rus vectors carrying Foxc2 and green fluorescent protein （GFP） were constructed, and the rabbit bone marrow-derived BM- SCs were isolated and cultured in vitro. BMSCs were transfected with Lenti-GFP （GFP group） or Lenti-Foxc2 （Foxc2 group）, while the BMSCs without transfection were used as control group. The influence of Foxc2 on BMSCs proliferation was evaluated using MTY assay, additionally the expression of Foxc2 mRNA and protein was determined by real-time PCR and Western blot at 7 days after transfection, furthermore the mRNA and protein expression of osteocalcin （OCN）, osteopontin （OPN） and type Ⅰ collagen （COL Ⅰ） were measured at 1, 2, and 4 weeks after transfection, moreover the alkaline phosphatase （ALP） staining and ALP activity measurement in each group were performed at 2 weeks after transfection. [Results] The MTT assay showed that Foxc2 overexpression had no significant impact on the proliferation of BMSCs. The Foxc2 mRNA and protein had high-level expression in the Foxc2 group, whereas those were not expressed in the control group and GFP group. The Foxc2 group had significantly higher mRNA and protein expression of OCN, OPN, and COL Ⅰ than the other two groups （P 〈0.01）, however, these parameters were not statistically significant between the other two groups （P 〉0.05） . In addition, the Foxc2 group had a significantly larger area of positive staining of ALP and significantly higher ALP ＇activity compared with the other groups （P〈0.01） . [Conclusions] Although Foxc2 overexpression does not affect the cell proliferation, continuously expression of this gene product does improve osteogenic differentiation of BMSCs.