Research On Fetal Bovine Lung Mesenchymal Stem Cells On Lipopolysaccharide-induced Acute Lung Injury In Mice

Objective:To establish a culture system of lungderived mesenchymal stem cells(LMSCs)in vitro.To study their biological characteristics,and to explore the therapeutic and repair effects of fetal bovine Lung mesenchymal stem cells(LMSCs)on lipopolysaccharidesinduced acute Lung injury in mice.Methods:(1)Isolation and culturing LMSCs: LMSCs were obtained from the lungs of aborted 3-4 months old bovine cattle by enzymatic digestion and tissue block adhesion method,and then subcultured.(2)Study on the biological characteristics of LMSCs: morphological observation,cell growth curve drawing,population doubling time calculation;Specific surface markers were detected by RT-PCR and immunofluorescence,and multidirectional differentiation potential was identified: osteoblasts,chondrocytes and adipocytes were induced by LMSCs,and the induced cells were identified by specific staining and RT-PCR.(3)The mice model of acute lung injury was established by direct intraperitoneal perfusion of Lipopolysaccharide(LPS).LMSCs were used to inject the mice,and the degree of lung injury and prognosis of the mice were evaluated by observing the histological section and serological test.Results:(1)Fetal bovine LMSCs were successfully separated by type II collagenase,cultured in vitro and to generation 22.The cell morphology was mostly long fusiform or fusiform,with large nucleo-cytoplasm ratio and irregular edge.(2)The growth curve of LMSCs showed a typical "S" shape,and the proliferation process went through the incubation,logarithmic,plateau and decline phases.The population doubling time of LMSCs was31.60h、35.18 h and 39.23 h,respectively.The results of RT-PCR showed that the LMSCs were positive for CD29,CD44,CD166 and CD73,but no response to CD31.Immunofluorescence results showed that the LMSCs of fetal bovine were positive for CD29,CD73,CD44 and CD166.LMSCs can successfully induce osteoblasts,chondrocytes and adipocytes.(3)Mice treated with LMSCs comparing with injured mice was observed pathological lung damage area and damage degree was significantly reduced,inflammatory cells infiltration and interstitial thickening and a small amount of collagen deposition,serological detection shows that group can effectively reduce the damage degree of serum TNF alpha,involved in regulating the secretion of IL-1β,IL-6,IL-10 and immune response.Conclusions:In this study,fetal bovine LMSCs were successfully isolated and cultured in vitro,and the cultured LMSCs were used in the LPS-mediated mouse model of acute lung injury.The results verified the therapeutic effect of LMSCs on acute inflammatory response to a certain extent,providing further experimental exploration for regenerative medicine and organ transplantation medicine.