Lentivirus-mediated TRPC4 Gene Scilencing Canine Progenitor Cells And Its Funtionn Cffection Study

Objective Distraction osteogenesis(DO)is a widely used surgical technique.Controlled mechanical force is intermittently applied to the fractured bone ends to enhance the regeneration process and induce new bone formation in the distraction space.Since bone is a highly vascularized organ,we believe that the process of angiogenesis is an important part of the bone formation process.The involvement of EPCs in angiogenesis is regulated by angiogenic substances and by inhibition of angiogenic substances.By detecting TRPC mRNA and protein,it was found that various types of TRPC family members were expressed in cultured vascular endothelial cells and intact vascular endothelium.TRPC4 is an important member of the TRPC family and is mainly present in vascular endothelial cells.TRPC4 participates in The physiological processes also mainly focus on the cardiovascular system.In this experiment,EPC was used as seed cells to construct a lentiviral vector-mediated si RNA silencing of TRPC4 gene.The siRNA with the best silencing effect was selected and the siRNA with the best silencing effect was transfected into EPCs.To study the molecular regulation of TRPC signal transduction pathway in the distracted neovascularization,and to provide a theoretical basis for further elucidating the mechanism of neovascularization in the distraction osteogenesis model.Methods 1.Isolation,culture and identification of canine endothelial progenitor cells(EPCs)in vitro: Take a 3-week-old healthy mongrel dog and collect the bone marrow of the humerus by bilateral patella puncture.Separate the dog bone marrow by density gradient centrifugation combined with special induction culture adherent culture method.Endothelial progenitor cells were observed by inverted phase-contrast microscopy;cell surface markers were detected by flow cytometry;the ability of canine endothelial progenitor cells to form lumens was detected by Matrigel tubule formation assay;and TRPC4 gene was detected by immunohistochemistry to determine whether the TRPC4 gene was expressed in endothelial progenitors cells.2.Lentiviral-mediated transfection of EPCs with TRPC4-siRNA: Construction of TRPC4 interference lentiviral expression vector,determination of virus titer and detection of plasmid expression;EPCs of second-generation or third-generation EPCs from dogs and lentiviral vectors as mediators The TRPC4 interference gene was transfected into EPCs in dogs and the optimal MOI was determined.The transfection condition of the cells was observed under fluorescence microscope.The mRNA expression of TRPC4 in EPCs after transfection of lentivirus was detected by fluorescent quantitative PCR.3.Effect of silencing TRPC4 gene on the biological behavior of EPCs: CCK-8 assay was used to detect the effect of TRPC4 on EPCs proliferation;Transwell chamber was used to investigate the ability of EPCs to transendothelial migration after TRPC4 was silenced;Adhesion experiments were performed to examine the effects of silencing TRPC4 on endothelial cells.Effect of adhesion;Matrigel tubule formation experiment to observe the effect of the lumen formation ability of EPCs after TRPC4 silencing;ELISA method to detect the expression of downstream signaling molecules VEGF and SDF-1 of TRPC signal after transfection of EPCs.Results 1.Morphology of endothelial progenitor cells: A small number of cells exhibit spindle-like morphology after adherence to endothelial progenitor cells.A large number of attached spindle cells grow radioactively from the middle to the periphery.The cells present typical island-like structures and peripheral spindle cells.The final fusion formed a typical "paving stone"-like structure growth;flow cytometry detected coexistence of the two markers CD133,CD34 that the cell is EPCs;Matrigel tubule formation experiments showed that the cell can form a capillary-like lumen structure,EPCs showed the ability to form new blood vessels.Immunohistochemistry experiments showed positive expression of TRPC4 antibody.2.The TRPC4 interference lentiviral expression vector was successfully constructed.The MOI value of the canine endothelial progenitor cells infected with lentivirus was 10;detected by fluorescent quantitative PCR.The relative expression of TRPC4 mRNA was significantly decreased after transfection to lentivirus(P<0.05),and the relative expression level of one of the designed targets was the lowest.This group was considered to have the best silencing effect.The subsequent experiments used the designed target lentivirus to The infected cells silence the TRPC4 gene.3.CCK-8 assay detected silent TRPC4 gene J to reduce the proliferation of EPCs(P<0.05);Silencing TRPC4 gene reduced the transendothelial migration ability of EPCs(P<0.05),and decreased the adhesion of EPCs to activated endothelial cells(P< 0.05),reduced EPCs tube-like structure formation.The results of ELISA detection of Lentivirus transfected EPCs showed that the expression of VEGF and SDE-1 downstream TRPC signal was decreased(P<0.05).Conclusion 1.EPCs stably expressing low TRPC4 gene were successfully obtained by lentivirus-mediated gene transfection.2.The silencing of TRPC4 gene reduced the proliferation of EPCs,reduced the adhesion,transendothelial migration,and luminal-like structure of activated endothelial cells,and decreased the expression of VEGF and SDF-1 proteins downstream of TRPC signaling.This eventually leads to a decrease in blood vessel formation capacity.