Simultaneous Activation Of TGF?1 And VEGFA Genes Of The MC3T3-E1 Cells To Promote Bone Defect Regeneration Based On CRISPRa System Research

Background:The development of cutting-edge technologies such as tissue engineering and regenerative medicine has provided new ideas for the use of tissue-engineered bone in the treatment of clinical bone defects,while bone replacement materials loaded with growth factors and controlled delivery of cells can enhance and accelerate functional bone formation.Transforming growth factor ?1(TGF?1)and vascular endothelial growth factor(VEGFA)play important roles in bone growth,development and healing.Here we aim to co-activate TGF?1 and VEGFA genes in seed cells through the CRISPRa system to regulate seeds Osteogenic differentiation and angiogenic potential of cells.And by preparing injectable hydrogel scaffold materials in combination with seed cells that have the potential to promote angiogenesis and bone formation,promote tissue engineering bone vascularization and osteogenesis.Also,explore gene editing seed cells Effects and mechanisms on vascularization and osteogenesis.Purpose:The purpose of this article is to co-activate TGF?1 and VEGFA genes in seed cells through the CRISPRa system,to study the effects of gene editing seed cells on osteoblasts and angiogenic genes,proteins,and cell staining.At the same time,through the preparation of new injectable hydrogel materials and gene editing Seed cells were explored to explore the mechanism of their effects on vascularization and osteogenesis during bone defect repair after implantation in vivo.Method:1.Chemical synthesis of pAsp(MTAS-NLS-co-DMH),a biodegradable polyaspartic acid(cellular microtubule and nuclear localization signal fusion peptide-dimethylhistidine)with high transfection efficiency for plasmids Polyamino acid carrier and characterization verification.2.Effectiveness identification of pAsp(MTAS-NLS-co-DMH)vector transfected cells:laser confocal and flow quantification analysis were used to detect pAsp(MTAS-NLS-co-DMH)vector transfected with eGFP plasmid into MC3T3-E1 cells and select the best transfection N/P ratio.3.Construct sg-RNA plasmid expression vectors of TGF?1 and VEGFA,and select the best expression sequence and expression bar pieces.4.Real-time quantitative PCR was used to detect the effects of dcas9 plasmid and sg-RNA plasmid transfected with TGF?1/VEGFA on the expression of TGF?1/VEGFA genes and osteogenic differentiation-related genes.At the same time,Western blot experiments and Elisa experiments were used to further verify.5.Chemical method is used to prepare double-crosslinked hydrogel,which is characterized and tested,and mtt method is used to detect cytotoxicity.6.Transfection of the transfected cells into hydrogel and implantation into the mouse skull bone defect model.The animals were sacrificed at 4w and 8w,respectively.Micro-CT was used to detect the results of new bone formation,HE staining,Masson staining,OCN and CD31 groups.Staining,analysis of new bone,collagen and angiogenesis.Result:1.Verification of NMR characterization verified the successful synthesis of the pAsp(MTAS-NLS-co-DMH)plasmid transfection vector.2.The eGFP expression after transfection of the eGFP plasmid proved the effectiveness of the vector construction.According to the results of flow cytometry and confocal analysis,N/P?10:1 was selected as the optimal transfection ratio.3.The sg-RNA recombinant plasmid of TGF?1/VEGFA was verified by sequencing,and the optimal sequence and expression conditions were screened.4.In the TGF?1/VEGFA co-transfection group,the expressions of TGF?1 and VEGFA at the RNA level were significantly higher than those of the control group.Western blot detection of TGF?1 protein was significantly higher than that of the control group,and there was no significant difference in VEGFA protein expression.Elisa detected TGF?1 protein extracellular there was no significant difference in expression,and the expression of VEGFA protein increasedsignificantly.5.Infrared spectroscopy indicated that the double-crosslinked hydrogel was successfully prepared.Different concentrations of hydrogel were non-toxic to cells.6.Micro-CT results showed that the new bone formation area,volume,and new bone density at 4w and 8w of the TGF?1/VEGFA co-transfection group were significantly higher than those of the control group.HE staining and OCN staining confirmed that the new bone formation was the same as that of Micro-CT results.Masson staining showed that the amount of collagen formation in the co-transfection group was significantly higher than the other groups,and the angiogenesis detected by CD31 indicated that the number of new blood vessels in the co-transfection group was significantly higher than the other groups.Conclusion:Successfully synthesized pAsp(MTAS-NLS-co-DMH)vector with high transfection efficiency for plasmids.Sg-RNA expression vector of TGF?1/VEGFA was used to overexpress TGF?1 and VEGFA genes in MC3T3-E1 cells.Bone-related factors are expressed,and cell hydrogel scaffolds can effectively promote bone defect repair after implantation in vivo.