重组pcDNA3.1-hBMP-2转染兔脂肪干细胞体外诱导的成骨分化

背景：骨形态发生蛋白2具有很强的诱导干细胞成骨活性。 目的：构建人骨形态发生蛋白2基因真核表达载体，探讨其转染脂肪干细胞后的成骨效果。 方法：通过噬菌斑原位杂交筛选人混合细胞cDNA文库获得人骨形态发生蛋白2基因，与真核表达载体pcDNA3.1-连接，构建重组质粒pcDNA3.1-hBMP-2。利用脂质体Lipofectamine? 2000分别介导人骨形态发生蛋白2 基因、EGFP基因转染第4代脂肪干细胞，并经G418进行筛选。 结果与结论：酶切鉴定及DNA测序结果证实重组质粒pcDNA3.1-hBMP-2构建成功。经计算脂质体介导的脂肪干细胞瞬时转染率为(18.0±0.42)%，并经过G418筛选后获得了稳定转染的细胞。细胞生长曲线表明转染后对脂肪干细胞生长、增殖无明显影响。ELISA检测发现人骨形态发生蛋白2组的人骨形态发生蛋白2因子表达量均高于EGFP组及未转染组，且能够稳定表达。经人骨形态发生蛋白2基因转染的脂肪干细胞的Ⅰ型胶原含量、碱性磷酸酶活性以及钙结节数目均比EGFP组及未转染组有明显升高。

Osteogenic differentiation of rabbit adipose derived stem cells transfected with recombinant pcDNA3.1-hBMP-2 in vitro

BACKGROUND:Bone morphogenetic protein 2 (BMP-2) shows strong potential to induce osteogenesis of stem cells. OBJECTIVE:To construct an eukaryotic expression vector of human BMP-2 (hBMP-2) gene and then to investigate its effect in osteogenic differentiation of human adipose-derived stem cells (ADSCs). METHODS:hBMP-2 gene was obtained by screening human mixed cell cDNA library by plaque in situ hybridization and connected with the eukaryotic expression vector pcDNA3.1. The recombinant plasmid pcDNA3.1-hBMP-2 was identified by BamHⅠ/ EcoRⅠenzyme digestion and DNA sequencing. ADSCs were isolated from fat tissues at the back of a 3-month-old New Zealand white rabbit neck, then cultured and proliferated for further use. The hBMP-2 group (experimental group), EGFP group (negative control group) and non-transfected group (blank control group) were used. Mediated by Lipofectamine? 2000, hBMP-2 gene and EGFP gene were transfected with the 4th generation of ADSCs respectively. At 2 days after transfection,    400 μg/mL G418 was used for screening the transfected cells. At 48 hours after transfection, green fluorescent cells were counted for determining transient transfection efficiency under fluorescence microscope. Cell growth curves of each group were drawn by MTT colorimetry. The hBMP-2 content in cell supernatants of each group was quantitated by ELISA. CollagenⅠcontent was determined by immunocytochemical staining, alkaline phosphatase (ALP) activity in the cell supernatant was measured through the use of ALP activity detection kit, and the number of calcium nodules was calculated by alizarin red staining. RESULTS AND CONCLUSION:After BamHⅠ/ EcoRⅠdigestion and DNA sequencing, the recombinant plasmid was constructed successfully. The transfection of pcDNA3.1-hBMP-2 and pcDNA3.1-EGFP can be mediated by Lipofectamine? 2000. The transient transfection efficiency was (18.0 ± 0.42) %. With the screening of 400 μg/mL G418, cells stably transfected were harvested. The cell growth curve of each group was drawn by MTT colorimetry, indicating that there was no significant effect on the growth and proliferation of ADSCs with gene transfection mediated by Lipofectamine? 2000. The hBMP-2 levels in the cell supernatant quantified by ELISA showed that hBMP-2 expression was higher in the hBMP-2 group than in the EGFP group and non-transfected group at the corresponding period (between groups P < 0.05). The cells of hBMP-2 group provided a steady hBMP-2 expression (intra-group P > 0.05). ADSCs transfected by hBMP-2 gene showed greater collagenⅠ content and higher activity of ALP, more calcium nodules compared with the EGFP and non-transfected groups (P < 0.05). The recombinant plasmid pcDNA3.1-hBMP-2 was constructed successfully; the transfection of hBMP-2 gene into ADSCs can be mediated by Lipofectamine? 2000 successfully, and there is no significant effect on the growth and proliferation of the transfected cells. With the screening of G418, the stably transfected cells can be harvested. The hBMP-2 expression of ADSCs transfected by hBMP-2 gene is stable and efficient, and with the induction of hBMP-2, they self-expressed. ADSCs can carry on the differentiation into osteoblast cells.