Effects Of Human Umbilical Cord Mesenchymal Stem Cells Transfected With The Antimicrobial Peptide Gene On Staphylococcus Aureus

Objective In order to provide a new method for the treatment of staphylococcus aureus infection,we used a combination of antibacterial,immunomodulatory and infectious wound repair,with the combination of stem cells and antimicrobial peptides.Specifically,human umbilical cord mesenchymal stem cells（hUC-MSCs）were used as seed cells.The antimicrobial peptide LL-37 was infected to cells by transgenic technology.The gene expression of LL-37 in human umbilical cord mesenchymal stem cells（hUC-MSCs）was detected,and the exocrine substance was released from the stem cells after transfection of LL-37.The supernatant of cell culture had a strong antibacterial effect on staphylococcus aureus.The purpose of this paper is to lay a theoretical foundation for effective clinical treatment and rational application of non-antibiotic anti-infective drugs.Methods 1.HUC-MSCs were cultured by conventional methods such as passage,cryopreservation and resuscitation after tissue separation.The changes of cell morphology were observed by inverted microscope with fluid exchange.2.The extracted hUC-MSCs were identified by flow cytometry to verify whether they conform to the cellular immune phenotype of the stem cells.3.Lentivirus vector was constructed and packaged to carry LL-37,and the titer value of lentivirus was determined by fluorescence method.4.HUC-MSCs in the logarithmic growth stage were selected for cell grouping and labeled as 3 groups.HUC-MSCs transfected with LL-37 lentiviral vector were labeled as the experimental group.HUC-MSCs transfected with an empty lentiviral vector without LL-37 were labeled as an empty vector group.HUC-MSCs cultured in conventional fluid exchange instead of transfection were labeled as the control group.During operation,the cells of the experimental group and the empty vector group were transfected respectively,while the cells of the control group could be transfected without special treatment.During observation,the morphological characteristics of each group were observed with an inverted microscope.For the two groups requiring transfection,we performed observation of cell status and transfection efficiency using fluorescence microscopy.The target is to ensure that the transfection efficiency is greater than 80%before the subsequent antibacterial experiments can be carried out.The culture supernatant was collected during the liquid exchange and concentrated in an ultra-15 ultrafiltration tube at a volume ratio of 15:1.After the concentration is completed,the bacteria are filtered and removed by a filter and stored in the refrigerator at-80℃for bacteria-inhibiting experiments.5.The expression of LL-37 in each group was detected by Western blot.6.The concentration of LL-37 protein in the culture supernatant of was determined by ELISA.7.Cell scratch test was used to detect the movement and growth of cells.8.The antibacterial activity of hUC-MSCs with LL-37 was detected by microplate reader turbidimetry.9.The experimental data were classified and sorted out,and the statistical software was used for analysis:SPSS 23.0 and GraphPad Prism6.Result 1.The extracted cells were successfully cultured.2.The flow cytometry showed that cell surface CD90⁺100%,CD105⁺98.04%and CD73⁺100%were all>95%.CD34⁺,CD45⁺,CD11b⁺,CD19⁺and HLA-DR⁺were all<5%.It conforms to the immunophenotype identification criteria of hUC-MSCs.3.Lentivirus vector was successfully constructed.The LL-37 lentivirus vector was packaged and its titer was determined to be 1×10¹²TU/L.4.Lentivirus vector carrying LL-37 was successfully transfected into hUC-MSCs,and green fluorescence was observed after transfection for72 h,with transfection efficiency of 85.40%.5.Western blot showed that the hUC-MSCs could be enhanced after transfection with LL-37 in the experimental group,and the protein expression of LL-37 could be increased.6.ELISA showed that LL-37 transfection could enhance the hUC-MSCs and increase the LL-37 protein in culture supernatant.7.Cell scratch test showed that the movement and growth of cells in each group were good,and LL-37 transfection or empty vector transfection did not significantly affect the growth morphology of hUC-MSCs.8.The microplate reader turbidimetry showed that the hUC-MSCs were transfected with antibacterial peptide LL-37,and the collected culture supernatant had a significant inhibitory effect on staphylococcus aureus,with the inhibitory rate of 98.74%（P<0.05）.Conclusion 1.Under the mediation of lentivirus,LL-37 can be successfully transfected into hUC-MSCs,and the lentivirus has no obvious effect on the antimicrobial peptide LL-37 and stem cells.2.Compared with antibiotics,hUC-MSCs supernatant transfected with LL-37 also had a significant inhibitory effect on staphylococcus aureus.