Experimental Study On The Effect Of HIF-1α Gene Modification On The Osteogenic Differentiation Of Beagle Bone Marrow Mesenchymal Stem Cells

Objective To analyze the changes of related osteogenic after the expression of hypoxia inducible factor-1α(HIF-1α)in beagle bone marrow mesenchymal stem cells by construcing lentiviral vectors,and explore its influence mechanism in the process of bone remodeling,which provided foundation for the follow-up study on the effect of HIF-1α gene modified BMSCs on the osseointegration of dental implants in the jaw defect area.Methods 1.Obtaining and culturing beagle BMSCs,and measuring their proliferation activity.2.Construction of HIF-1α gene lentivirus vector and transfection of BMSCs.3.Alkaline phosphatase staining and semi-quantitative detection of ①Lenti-HIF-1α group and ②Lenti-NC group after 7d,10d and 14d of osteogenic induction HIF-1α transfected BMSCs.4.Alizarin red staining and semi-quantitative detection of ①Lenti-HIF-1α group and ② Lenti-NC group after 14d and 21 d of osteogenic induction HIF-1α transfected BMSCs.Results 1.The primary cells were cultured and observed under a microscope,showing that the cells were scattered and most of them were short-spindle or granular.The subcultured cells showed radial growth,and the cell shape changed from round to spindle.The cell proliferation curve was "S" type,with the increase of time,the number of cells gradually increased,reached the highest value on the 6th day,and basically maintained on the 7th to 10th day.2.After PCR amplification,enzyme digestion,and sequencing comparison,the inserted sequence was consistent with the target sequence,and a lentiviral vector carrying HIF-1α was successfully constructed,and a green fluorescent reaction was visible after 72 hours of transfection.3.The results of alkaline phosphatase staining showed that the staining area and number of Lenti-HIF-la group were significantly more than those of Lenti-NC group;semi-quantitative results:the ALP activity of Lenti-HIF-1α group was higher than that of Lenti-NC group.There was no statistical difference between the 7th day groups(P>0.05),but there was a statistical difference between the two groups on the 10th and 14th day(P<0.05).4.Alizarin red staining results showed that the area and number of calcium nodules in the Lenti-HIF-1α group were significantly more than those in the Lenti-NC group;semi-quantitative results:the absorbance of the Lenti-HIF-la group was higher than that of the Lenti-NC group.There was no statistical difference between the 14th day groups(P>0.05),but there was a statistical difference between the two groups on the 21th day(P<0.05).Conclusion 1.The lentiviral expression vector carrying HIF-1α was successfully constructed by lentiviral packaging.2.Related osteogenic genes were up-regulated after transfection of canine BMSCs with HIF-1α lentivirus expression vector,indicating that HIF-1α can promote osteogenic differentiation of BMSCs.