Effects Of EGR1 And PRP Related Growth Factors On The Repair Of Rabbit Tendon After Rotator Cuff Injury

ObjectiveTo study the effect of EGR1 on the ability of inducing differentiation of tendon stem cells,and the effect of EGR1 and PRP related growth factors on tendon repair after rotator cuff injury in rabbits.Methods(1)By enzyme digestion of rabbits were taken the middle part of the patellar ligament tendon tissue after treatment and cultured after isolated and purified enzyme cut tendon stem cells(TSCs),after immunofluorescence staining of 4,6-two amidino-2-phenylindole(DAPI),TNMD and SCX antibody,and the fluorescent microscope under the observation further confirmed the identification of TSCs cells.(2)The experimental group TSCs was transfected with plasmid(pCDNA-EGR1)with expression of EGR1 gene,and the control group was transfected with blank plasmid TSCs.Expression of BMP12 and Smad1 genes in RNA TSCs microparticles by short hairpin lentiviral(shRNA).TSCs in the experimental group and the control group under the action of EGR1 were cultured for 14 days to induce tendon cell differentiation,purified tendon cells by immunofluorescence staining and quantitative PCR were used to identify the tendon cells,and the expression of related genes were detected.(3)By RNA extraction and real-time quantitative PCR detection(qRT-PCR),comparing the experimental group and control group TSCs differentiation ability formation of tendon cells in gene level,and in the TSCs culture and differentiation process(0h,12 h,1D,3D,7d,14d)extraction of protein and the application of Western blotting detection by protein products BMP12/Smad1/5/8 pathway is generated,and the products were compared with normal tendon healing.(4)To establish a rabbit model of rotator cuff injury(40 rats),each of the experimental animals were cut off the right shoulder joint muscle tendon,and the left shoulder joint was treated with sham operation to form a control group.6 weeks after surgical repair of rotator cuff injury model established,and the experimental animal were divided into three groups(n = 10): A group,B group,simple repair surgery;repair surgery +TSCs implantation(tendon defects);group C: surgical repair of +EGR1-TSCs implantation(tendon injury site)+PRP injection(tendon bone interface).After 8 weeks,all the animals were sacrificed and the bilateral humeral large nodules of each animal were obtained.Each group took 3 samples were used for mRNA extraction and protein extraction(for Western blotting);the remaining tissue samples in each group(including from the repair parts of the supraspinatus tendon and greater tuberosity)made of 5 m thick paraffin sections,HE staining,immunohistochemical analysis and to evaluate the formation of type I collagen the healing of the tendon,evaluation.Results(1)The TSCs tendon stem cells culture was successful,and the third generation TSCs labeled DAPI nuclei showed bright blue fluorescence(Fig.3),the labeling rate was 100%,and the cell activity was higher.SCX and NMD staining showed green fluorescence,and the whole cell morphology and distribution of the cells could be observed.(2)The expression of EGR1 gene in the experimental group TSCs,and no expression in the control group TSCs.Expression of BMP12 and Smad1 gene in short hairpin lentiviral(shRNA)RNA particles targeted to TSCs.Under the action of EGR1,the experimental group was successfully differentiated into the tendon cells,and the cells were isolated and purified,and immunofluorescence staining and quantitative PCR were performed to identify the tendon cells.(3)RNA and real-time quantitative PCR detection(qRT-PCR),the experimental group showed that TSCs differentiate into tendon cells,and the application of Western blotting detection: BMP12/Smad1/5/8 pathway protein products generated and the same with normal cells.(4)The rabbit rotator cuff injury model was established successfully.HE staining and immunohistochemical analysis showed that the formation of type I collagen was good,and the tendon healing was good.ConclusionEGR1 has a good ability to induce differentiation of tendon stem cells.EGR1 and PRP related growth factors have positive effects on tendon repair after rotator cuff injury in rabbits.