1.Jining Hospital;2.Jinan Hospital;3.Fang Dianji
Abstract: Objective: To construct an adenovirus-mediated stromal cell-derived factor-1 (SDF-1) and Nell-1 molecule (Nell-1) dual gene vector and transfect adipose stem cell ADSCs to observe and discuss the comparison of the dual genes A single gene enhances its ability of osteogenic differentiation in vitro. Method: 1: Carry out the in vitro amplification and subculture of ADSCs by enzymatic digestion method, and carry out the transformation of osteogenic and adipogenic direction. 2: Take the third generation ADSCs for experimental grouping, A: simple cell group; B: empty plasmid transfection group; C: SDF-1 simple transfection group; D: NELL-1 simple transfection group; E: SDF-1 /NELL-1 co-transfection group, and use adenovirus vectors carrying different target genes to transfect ADSCs according to groups and observe the transfection efficiency. Western blot detects the expression of SDF-1 and Nell-1 in ADSCs after target gene transfection level. 3: 14 days after osteogenic induction, by observing the formation of calcium nodules, alkaline phosphatase activity and RT-PCR in each group to detect the expression of related genes osteopontin OPN and osteocalcin OCN in each group, and do statistical analysis . Results: 1: After transfection, there was a large amount of SDF-1 secretion in the C and E groups, and a large amount of NELL-1 was secreted in the D and E groups. The SDF-1/NELL-protein level in the co-transfection group was compared with the single Compared with the gene transfection group, the difference was significant (P<0.05); 2: The formation of calcium nodules in the co-transfection group was found to be better than that of the other groups by Alizarin Red staining. ALP detection can get the alkaline phosphate of group E The enzyme activity is the highest. Among them, the ALP activity of group C and D is lower than that of group E but slightly higher than that of group A and B. Statistical analysis shows that the difference between co-transfection group and single gene transfection group is significant (P<0.05); 3: RT-PCR detection revealed that the expression of osteopontin OPN and osteocalcin OCN in the co-transfection group was significantly better than the other groups, with statistical significance (P<0.05). Conclusion: Both SDF-1 and NELL-1 can be transfected into adipose-derived stem cell ADSCs and obtain their protein expression. Adenovirus-mediated SDF-1 and NELL-1 dual-gene vector transfected with ADSCs has a more significant osteogenic effect than single-gene in vitro.