An Experimental Study On Osteogenic Differentiation Of Rabbit's ADSCs Induced By Transfecting With Recombinant PcDNA3.1-hBMP-2 In Vitro

Objective:To construct an eukaryotic expressing vector of human bone morphogenetic protein-2 gene-recombinant plasmid pcDNA3.1-hBMP-2, the rabbit adipose-derived stem cells are transfected with pcDNA3.1-hBMP-2 which mediated by LipofectamineTM 2000. Then explore the effect of the osteogenic differentiation of ADSCs induced by hBMP-2 expressed all by themselves. And prepare for the construction of tissue engineering bone further. Methods:1, The primers was designed and synthesized, and hBMP-2 gene was amplificated by PCR.2, hBMP-2 gene and pcDNA3.1 were digested by the BamHI/EcoRI enzyme respectively, and then connected with each other; after transformation and amplification, the recombinant plasmid was extracted for enzyme digestion and DNA sequencing.3, Adipose-drived stem cells were isolated from fat tissues at the back of a three month-old New Zealand white rabbit neck, then cultured and proliferated for further use.4, Mediated by LipofectamineTM 2000, hBMP-2 gene and EGFP gene were transfected with the 4th generation of adipose drived stem cells respectively. Two days Afer transfection, G418 was used at a concentration of 400μg/ml for screening the transfected cells.5,48h after transfection, the number of the green fluorescent cells was counted for determining transient transfection efficiency under fluorescence microscope.6, Cell growth curves of hBMP-2 group (the Experimental group), EGFP group (the negative control group) and non-transfected group (the blank control group) were drawn by MTT colorimetry.7, ELISA was used for quantitating the hBMP-2 content in the three consecutive days cell supernatants of each group which were collected at the 3rd day, the 7th day, the 14th day, the 21th day.8, Detection of osteogenic differentiation indicators:Collagen I immunocytochemical staining was carried out on the cells after transfection cultured continuously for 7 days; ALP activity detection kit was used for detecting the activity of ALP in the three consecutive days cell supernatants of each group which were collected at the 3rd day, the 7th day, the 14th day; Calcium nodules of the cells after transfection with the 14 days continuous culture was stained by the alizarin red. Results:1, The hBMP-2 gene was amplificated by PCR. After transformation and amplification, the recombinant plasmid was abstracted for BamHI/EcoRI digestion, and got two fragments:1200bp and 5400kbp approximately; DNA sequencing results matched with the sequence of hBMP-2 gene reported in Genbank.2, The transfection of pcDNA3.1-hBMP-2 and pcDNA3.1-EGFP can be mediated by LipofectamineTM 2000 successfully, and after calculation, it was obtained that the transient transfection efficiency was (18.0±0.42)%. With the screen of G418 at the concentration of 400μg/ml, the stable transfected cells were harvested. The cell growth curve of each group drawn by MTT colorimetry indicated that:there was no significant effect on the growth and proliferation of adipose-derived stem cells with gene transfection mediated by LipofectamineTM 2000. The hBMP-2 levels in the cell supernatants quantificated by ELISA showed that hBMP-2 expression of hBMP-2 groups at the 3rd day, the 7th day, the 14th day, the 21th day were all higher than the EGFP groups'and non-transfected groups'in the corresponding period (between groups p<0.05), and the cells of hBMP-2 groups could provide a steady hBMP-2 expression (intra-group p>0.05). During the above training periods, according to the standard curve calculation, the hBMP-2 content of the hBMP-2 group in cell supernatants was about 12.36ng (the cells were inoculated at concentration of 8×104pcs/holes).4, Indicators of osteogenic differentiation:collagenⅠimmunocytochemical staining showed collagenⅠexpressed by cells of the hBMP-2 group was much higher than the EGFP group's and the non-transfected group's (p<0.05). After detecting the ALP activity of each group by the ALP activity detection kit at different times, it was found that the ALP activity of the hBMP-2 groups was higher than those of the EGFP groups and the non-transfected groups (between groups p<0.05). With the incubation time carrying on, the ALP activity of hBMP-2 groups was a gradual increase (intra-group p<0.05). After the transfected cells cultured for 14 days stained by 2% alizarin red, the orange-red calcium nodules were found more in the hBMP-2 group, while there was no obvious stained nodule found in the EGFP group and the non-transfected group. Conclusion:The hBMP-2 eukaryotic expression vectors---the recombinant plasmid pcDNA3.1-hBMP-2 is constructed successfully; The transfection of hBMP-2 gene into ADSCs can be mediated by LipofectamineTM 2000 successfully, and there is no significant effect on the growth and proliferation of the transfected cells. With the screening of G418, the stable 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.