Effect And Mechanism Of Leptin Transfected Human Placenta-derived Mesenchymal Stem Cells On Oral Mucosa Repair Cells After Irradiation

Objective:Combined radiation and wound injury?CRWI?is mainly seen in patients with combined trauma such as tumor radiotherapy and nuclear radiation injury.Of all cancer patients,nearly two-thirds receive radiation therapy during treatment,which often results in acute skin and mucosal damage.In recent years,terrorist activities are still rampant,the most worrying of which is the occurrence of nuclear accidents.Therefore,it is necessary to study medical countermeasures against combined radiation and trauma to prevent radiation damage caused by bioterrorism and cancer treatment.The wounds caused by ionizing radiation are prolonged and difficult to heal,and the skin flaps become necrotic,which has become a clinical problem to be solved urgently.Previous studies showed that for the early and late complications of radiation exposure,stem cell therapy can improve the adverse effects of radiation damage on skin tissues and promote tissue healing.HPMSCs have become the main source of stem cells by virtue of their inherent advantages,such as non-invasive access,easy access,fewer ethical restrictions,pluripotency,low immunogenicity and immunomodulatory effects.Leptin is a pleiotropic cytokine,and it has been confirmed that Leptin also plays an important role in wound healing.Fei et al.found that HPMSCs expressing exogenous Leptin gene can significantly increase the survival rate of CRWI flaps,suggesting the potential research and application value of stem cell therapy combined with gene therapy in CRWI wound healing,but transfection of Leptin The effect of HPMSCs on skin or mucosal repair cells after radiation is still unclear.Based on the results of previous studies,this study established an in vitro model of oral gingival mucosal repair cell radiation-wound compound injury to further study the effects of HPMSCs/Leptin on the chemotaxis,proliferation ability and secretion of inflammatory factors of gingival mucosal repair cells.This is a follow-up experiment Research provides evidence.Methods:1.Experiment groupingThe experiment was randomly divided into HPMSCs/NC group?control group,Lv-pEB-copGFP?T2A?PURO?and HPMSCs/leptin group?experimental group,Lv-pEB-copGFP?T2A?PURO-leptin?,HPMSCs/NC and HPMSCs/ Leptin was cultured in DMEM-LG medium containing 10% serum and 1% double antibody,and placed in a CO₂ incubator?cultivation conditions were 5% CO₂,saturated humidity,37°C?.2.Irradiation treatment and Transwell detectionResuscitated HOK,HGnF and HUVEC cells were added to DMEM-LG medium containing 10% serum and 1% double antibody,and placed in a CO₂ incubator for culture.When the degree of cell fusion reached 90%,a single X-ray radiation was performed with a dose of 20 GY.Choose the Transwell system with 8.0um PET membrane,use serum-free DMEM to resuspend the irradiated HOK,HGnF and HUVEC,take 100 ul each?containing 1×105 cells?into the upper chamber of Transwell,and add 1× in the lower chamber 104 HPMSCs/NC or HPMSCs/leptin cells were incubated for 48 hours and discarded the upper chamber cells.The cells on the lower surface of the membrane were fixed with 4% paraformaldehyde,stained with crystal violet,observed and photographed under an inverted fluorescence microscope,randomly selected pictures and counted.3.Build a co-cultivation systemChoose 0.4um PET membrane Transwell system,take 400 ul of irradiated HOK,HGnF and HUVEC cell suspension?containing 1×105 cells?into the upper chamber,and add 200 ul each of HPMSCs/Leptin or HPMSCs/NC?containing Cells 1×104?into the lower chamber,placed in a CO₂ incubator for culture.Construct six groups of co-culture system,namely the experimental group: HGnF+HPMSCs/Leptin,HOK+HPMSCs/Leptin,HUVEC+HPMSCs/Leptin;control group:HGnF+HPMSCs/NC,HOK+HPMSCs/NC,HUVEC+HPMSCs/NC.4.CCK-8 was used to detect the proliferation activity of the three cells at 24 h,48h,72 h,96h after X-ray irradiation,and ELISA was used to detect the expression of inflammatory factors at 24 h,48h,72 h after X-ray irradiation?human IL-1?,IL-1?,IL-6,IL-8,GM-CSF and TNF-??levelsResults:1.Transwell test resultsHPMSCs/NC and HPMSCs/Leptin have different degrees of chemotaxis to HGnF,HOK and HUVEC after irradiation?Figure 2.3.1?.Compared with the HPMSCs/NC group,the HPMSCs/Leptin group is placed in the Transwell model In the lower chamber,more HUVEC can be moved to the lower chamber,indicating that HPMSCs/Leptin can promote the migration of HUVEC.It is speculated that HPMSCs/Leptin has an enhanced tropism for HUVEC;but compared with the HPMSCs/Leptin group,the HPMSCs/NC group When placed in the lower chamber of the Transwell model,more HGnF and HOK can migrate to the lower chamber,indicating that HPMSCs/Leptin inhibits the migration of HGnF and HOK.It is speculated that HPMSCs/Leptin has a weakened tropism for HGnF and HOK.2.CCK-8 test results of cell proliferation activityAs shown in Figure 2.3.3A,the OD450 value of HGnF+HPMSCs/Leptin mixed culture system is not significantly different from that of HGnF+HPMSCs/NC mixed culture system?P>0.05?.The proliferation ability of HGnF has little effect.Figure 2.3.2B shows that the OD450 value of the HOK+HPMSCs/Leptin mixed culture system is significantly higher than the OD450 value of the HOK+HPMSCs/NC mixed culture system?0.001<P<0.01?,indicating that HPMSCs/Leptin can promote radiation injury Proliferation of HGnF.As for 96 h,the OD450 value of HOK+HPMSCs/Leptin mixed culture system was significantly lower than that of HOK+HPMSCs/NC mixed culture system.The reason may be that the OD450 value of HOK+HPMSCs/NC mixed culture system at 0h was slightly higher than HOK +HPMSCs/Leptin mixed culture system.Figure 2.3.2C shows that the OD450 value of the HUVEC+HPMSCs/Leptin mixed culture system is significantly higher than the OD450 value of the HUVEC+HPMSCs/NC mixed culture system?P<0.001?,indicating that HPMSCs/Leptin can promote the HUVEC after irradiation injury proliferation.3.ELISA test resultsEnzyme-linked immunosorbent assay?ELISA?was used to determine the concentration of inflammatory cytokines secreted in the mixed culture system at 24 h,48h and 72 h after irradiation injury.Compared with the control mixed culture system?HGnF+HPMSCs/NC,HOK+HPMSCs/NC?,the experimental mixed culture system?HGnF+HPMSCs/Leptin,HOK+HPMSCs/Leptin,?in the medium supernatant of human IL-1?,The secretion of GM-CSF and TNF-? in each time period?24h,48 h and 72 h after irradiation?was significantly higher than that of the control mixed culture system?P<0.05?.72 h after irradiation injury,the secretion of human IL-6 in the supernatant of each mixed culture system was detected.There was no significant difference between HOK+HPMSCs/NC and HOK+HPMSCs/Leptin?P>0.05?,mixed culture The system HGnF+HPMSCs/Leptin was significantly higher than its control mixed culture system HGnF+HPMSCs/NC?P<0.001?.In addition,the secretion of human IL-1? and IL-8 in each mixed culture system was too small to be detected.In summary,the experimental results show that HPMSCs/Leptin can promote HGnF and HOK to secrete inflammatory factors human IL-1?,IL-6,GM-CSF and TNF-?.Conclusions:1.HPMSCs/Leptin can promote the migration of HUVEC and inhibit the migration of HGnF and HOK.2.HPMSCs/Leptin can enhance the proliferation of HOK and HUVEC after irradiation,but it has no obvious effect on the proliferation of HGnF.3.HPMSCs/Leptin can promote the secretion of inflammatory factors human IL-1?,IL-6,GM-CSF and TNF-? by HGnF,HOK and HUVEC,but has no significant effect on the secretion of human IL-1? and IL-8.