| BACKGROUNDThe scar healing is the main way of rotator cuff tear repair.It is difficult to restore the original fibercartilage structure of bone tendon junction?BTJ?,thus,the mechanical strength and maximum load are significantly reduced,and the rate of re-tear is relatively high.BTJ is located at the end of the tendon which is an area lacks vessels.The healing of BTJ after injury is scar healing mainly participated by fibroblasts.The formation of fiber cartilage structure of BTJ requires multipotential stem cells,especially those can differentiate into tendons and fibrous cartilage.Researchers have reported that the use of Scx-expressing mesenchymal stem cells in the rotator cuff tear model can improve the repair of BTJ.As one of well recognized tendon specific markers,Scx has a critical role on differentiation and development of tendon.Overexpression of Scx in mesenchymal stem cells regulates cell differentiation and development.However,the mechanism of Scx regulating mesenchymal stem cells to promote proliferation and differentiation remains unknow.The aim of this study was to investigate the mechanism of Scx regulation of mesenchymal stem cells in promoting tendon bone healing.We observed the role of Scx in mesenchymal stem cells by overexpressing Scx in rat bone marrow mesenchymal stem cells.And further explore the specific mechanism of Scx promoting the Proliferation and Differentiation of Mesenchymal Stem Cell.We hope this study can provide shoulder injury patients on postoperative rehabilitation with a theoretical basis and potential treatment.METHODS1.Animals and cell culturePrimary rat bone marrow mesenchymal stem cells?BMSC?were from 4 weeks SD rats,The rats were sacrificed after removal of tibia and femur,75%alcohol disinfection,To collect bone marrow cells,bone marrow were removed by centrifugation,resuspended,filtering and washing.complete medium,the adherent single gram long bone marrow mesenchymal stem cells,primary cells,the ratio of 1:3.2.Construction of lentivirus and cell transfectionUsing the classical method to complete the construction,amplification and titer determination of lentiviral vector,Scx was transfected into blot by lentivirus,and the transfection efficiency was detected by qRT-PCR and Western method.3.Western blot analysisWestern blot was used to detect the expression of Flag tagged protein and GAPDH protein in the transfected Lv-Scx cells.The cell samples were extracted by SDS-PAGE lysis,and the separation was selected according to the molecular weight of the target protein.electrophoresis,wet transfer film,closed after incubation with a resistance,TBST washing,incubation of the two resistance,after washing the film color.The detection and analysis of protein bands were performed by using the 4200 automatic chemiluminescence imaging system.4.Quantitative real-time polymerase chain reaction?qRT-PCR?analysisTotal RNA extraction reagent BMSC in rats and the use of synthetic primers was reverse transcribed into DNA qPCR SYBR Green Mix reaction were standard method of target gene and reference gene of each sample,using2-??C?,calculate the relative expression.5.Cell morphology and colony formation abilityAfter 2 passages,rats BMSC transfected with Scx were inoculated in polystyrene slides.Cultured cell morphology were observed after 24 h by normal phase difference microscope;cells were seeded in the density of 10 cells per cm2 in the 10 cm culture dish,.After 12 days with 0.5%crystal violet staining,the formation of methanol/colony was observed.Observing the changes of colony formation ability.6.Cell proliferation ability?CCK8?Cells were collected after transfection,cell culture plate,0,24,48,72 and 96 h after treated with CCK-8,microplate 450nm od.7.Differentiation ability of stem cellBMSC-Scx and BMSC-GFP were directional differentiation into osteoblasts,adipocytes and cartilage respectively.Then the cells were stained with alizarin red,oil red O and alcian blue to observe their differentiation ability.8.RNA high throughput sequencing technologyExtraction of total RNA cell samples for quality inspection,After quality inspection,RNA were reverse transcripted and amplificated to bulid a library using Illumina HiSeq2500 sequencing good library.Sequencing strategy was PE150.9.Cell immunofluorescenceTransfection of lentivirus BMSC after inoculation of rats in 24 well plates slide,after fixation,cleaning,sealing,incubation,incubation,second anti nuclear staining and mounting,photographed under fluorescence microscope camera..10.Statistical analysisData are expressed as mean±standard deviation?SD?from at least three separate experiments.SPSS 18 software was used for statistical analysis the differences between groups were analyzed using two-tailed unpaired Student's t-test.Differences were deemed statistically significant at P<0.05.RESULTS1.Ranscription factor Scx inhibits BMSC proliferationThe expression of Scx mRNA was increased by 16 times compared with control group after over-expression of Scx in rat BMSC?n=3,P<0.01?.Western blot results showed that the expression of Flag tag protein was increased.The growth rate of BMSC was significantly decreased in CCK8 test.Compared with the control group,the clone formation ability of BMSC was significantly decreased compared.Microscopic observation found that BMSC-Scx cells became fusiform,and the control group was still polygonal.2.Transcription factor Scx promotes the ability of BMSC to differentiate into tendon cellsThe expression of tendon associated gene in the mesenchymal stem cells after overexpression of Scx,the results showed that Scx promoted the expression of partial tendon genes in the rat compared with the control group.qRT-PCR results showed that the expression level of Col1a1,Col3a1 and Col5a1 increased about 6 times?n=3,P<0.05?,increased the expression of Tnmd and Dcn,inhibited the expression of Tnc,Fmod,Bgn change was not statistically significant,immunofluorescence results further confirmed this point.The results showed that the ability of BMSC-Scx and BMSC-GFP to differentiate into chondrocytes and adipocytes was significantly decreased and there was no significant difference in osteoblast differentiation ability.The further analysis of qRT-PCR showed that Sox9 expression was significantly affected by BMSC-Scx and decreased by 67%compared with the control group,and there was no significant difference between the expression of Runx2 and Taz.3.The mechanism of transcription factor Scx on proliferation and differentiation of mesenchymal stem cellsWe used RNA high-throughput sequencing to investigate the possible role of Scx in the regulation of BMSC proliferation and differentiation.The results showed that there were 174 significant up-regulated genes and 167 down regulated genes,screening and possible proliferation related gene Igf1,Hgf,Rasgrp4 and differentiation related genes Lgals3,Etv1,Cd36,Bglap,Apold1.the results of qRT-PCR showed that Scx promotes the expression of Murc in BMSC of rats.Inhibited the expression of CD36 and Igf-1,the difference was statistically significant?P<0.01,n=3?.CONCLUSION1.Transcription factor Scx can reduce the proliferation of mesenchymal stem cells.2.Transcription factor Scx promotes the differentiation of mesenchymal stem cells into tendon cells by reducing the expression of tendon associated genes,and reduces the pluripotent nature of mesenchymal stem cells.3.Scx inhibited the expression of Proliferation regulatory gene?Igf1?and adipocyte differentiation gene?CD36?,and increased the expression of Murc,a regulator of myogenic differentiation.. |
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