Overexpression Of TGF-β1 Regulates Autophagy Between Human Umbilical Cord Mesenchymal Stem Cells And Human Dermal Fibroblasts

Objectives:The research of stem cell and regenerative medicine has shown great clinical application value and brought hope to the treatment of patients with large area and deep skin burn.However,because there are many conditions in the wound environment after burn that are not conducive to stem cells to perform the function of wound repair,it has become one of the problems to be solved urgently.Transforming growth factor β(TGF-β)plays an important role in embryonic growth and development,inflammation regulation and tissue repair.Recent studies show that TGF-β plays an important role in vascular remodeling,cell proliferation and differentiation as well as immune regulation.In addition,TGF-β1 belonged to the transforming growth factor family,which accounted for the highest proportion and was abnormally down-regulated after thermal damage,and the content of TGF-β1 was correlated with the wound healing speed.At present,the effect of TGF-β1 on Stem Cells was mainly focused on the differentiation of stem cells,while the biological function of TGF-β1 on human Umbilical Cord Mesenchymal Stem Cells(hUC-MSCs)was rarely studied.The role of autophagy in physiology and pathology has attracted the attention of researchers.Transgrowth factors can regulate autophagy.However,the relationship between autophagy activity of TGF-β1 and umbilical cord mesenchymal stem cells remains unclear.In this study,gene modified TGF-β1 was used to investigate the biological effects of hUC-MSCs and further investigate whether the mechanism is regulated by autophagy,so as to investigate the effects of gene modified hUC-MSCs on Human Dermal Fibroblasts(HDFs).To explore the application value of wound healing.Methods:1.Umbilical cord mesenchymal stem cells were obtained from umbilical cord tissue by tissue block adhesion method and identified in terms of morphology,immunophenotype and multidirection differentiation ability.Human dermal fibroblasts were obtained from preputial tissue by tissue block adhesion method and their biomarkers were identified by immunofluorescence.2.Lentvirus-overexpressed human umbilical cord mesenchymal stem cells TGF-β1 were used to detect cell proliferation from day 1 to day 5 in the hUC-MSCs overexpression group and no-load group by CCK-8 assay,and the migration ability of hUC-MSCs overexpression group and noload group at 16 h was detected by transwell assay.Flow cytometry was used to detect cell cycle and apoptosis,and western blot assay was used to verify autophagy marker proteins and AMPK/mTOR pathway.3.Umbilical cord mesenchymal stem cells and fibroblasts were co-cultured and the co-culture time was determined.The proliferation of HDFs after 24 h co-culture with hUC-MSCs in overexpression group and no-load group was detected by CCK-8 assay.transwell assay was used to detect HDFs migration ability after 24 h co-culture with hUC-MSCs in overexpression group and no-load group.The apoptosis of HDFs cells was detected by flow cytometry after 24 h coculture with hUC-MSCs in overexpression and no-load groups.The protein levels of wound healing were detected by Western blot.Results: 1.hUCMSCs were extracted by tissue block adhesion method,and the cell morphology was long spindle shape and polygon shape.When the cells are fully fused,they grow in a vortex shape.Flow cytometry was used to detect hUCMSCs surface markers,and the expression rate of negative markers CD34 was close to 1.3%,negative markers CD45 was only 0.1%,and positive markers CD90 and CD105 were close to 100%,which was consistent with the characteristics of hUCMSCs.The multidirection differentiation ability of hUCMSCs was identified,and the results showed that intracellular fat droplets fused with oil red O to become obvious red,showing typical lipid differentiation.Calcified cell nodules combined with alizarin red dye showed a red color,showing typical osteogenic differentiation.The chondrocytes were blued by Alcian blue staining,which demonstrated the presence of chondroitin sulfate and colloidal sulfate proteins and showed typical chondroblast differentiation.2.HDFs was extracted by tissue block adherent method.After the complete fusion of P1 generation cells,it could be seen that the cells were closely arranged,uniform in size,stretched out in a long spindle shape,arranged like a school of fish,and grew in a vortex shape.Conforms to HDFs features.cytokeratin 15(CK15)and αsmooth muscle actin(α-SMA)staining were negative.Cytokeratin 15(CK15)and αsmooth Muscle Actin(α-SMA)staining were positive.3.MOI=30 and 24 h of infection were selected as the optimal infection conditions for overexpression of lentvirus.After three days of infection,the gene expression level of overexpression of TGF-β1 cells was 2.2 times higher than that of no-load group,while the expression level of TGF-β1 protein was significantly increased.4.The effect of overexpression of TGFβ1 on the proliferation capacity of hUCMSCs,the absorbance of the overexpression group TGFβ1 was lower at 450nm;The hUC-MSCs overexpressed with TGFβ1 migrated more cells at 16 h and 18 h.After TGFβ1 overexpression,the G1 phase of hUC-MSCs group was higher than the control group,and the S phase was lower than the control group,and the difference was statistically significant.However,the apoptosis rate of overexpression group was significantly higher than that of control group.5.Beclin1 and LC3BⅡ of hUC-MSCs in TGFβ1 overexpression group were increased,AMPK was activated as p-AMPK,and the expression level of p-mTOR was increased,while the expression level of P-Mtor was decreased.6.hUC-MSCs overexpressing TGFβ1 were co-cultured with HDFs.On the fourth to fifth day of CCK8,compared with the Huc-MSCs-NC group,the absorbance at450 nm in the overexpressing TGFβ1 group was lower,and the cell migration in the Huc-MSCs-TGfβ1 group was significantly increased compared with the control group after 16-hour migration test.In the cell apoptosis experiment,the apoptosis of the hUCMSCs-TGFβ1 group was significantly increased.WB results showed that the secretion of HDFs collagen Ⅰ and collagen Ⅲ was increased after co-culture with hUC-MSCsTGFβ1 compared with the no-load group.Conclusions: 1.Human umbilical cord mesenchymal stem cells obtained from umbilical cord tissue have good morphology,high purity and multidirectional differentiation ability;The cells obtained from the preputial tissue were consistent with the characteristics of human dermal fibroblasts.2.The overexpression of TGFβ1 with lentivirus promoted the vertical migration of hUC-MSCs,inhibited the proliferative activity of stem cells,blocked the cell cycle,and promoted cell apoptosis.3.Overexpression of TGFβ1 activated the autophagy of hUC-MSCs through AMPK/mTOR pathway.4.Co-culture of hUC-MSCs overexpressing TGFβ1 with HDFs promoted the proliferation,migration and apoptosis of HDFs,promoted the secretion of collagen Ⅰand collagen Ⅲ,and may accelerate wound healing.