Repair Of Lipopolysaccharide-induced Acute Lung Injury In Mice By Transplantation Of Endothelial Progenitor Cells And In Combination With Simvastatin

PART Ⅰ:Isolation and culture of endothelial progenitor cells from murine bone marrowBackgroundIn vitro induce murine endothelial progenitor cells(EPCs)and establish a culture approach for murine EPCs which are foundation of the following research.MethodWe chose 4 weeks old mice and obtained femur and tibia.And then we washed the bone marrow and obtain the bone marrow suspension.And then suspension was added upon the lymphocyte separation liquid and centrifuged using the horizontal centrifuge.Then the buffy coat that containing monocytes was aspirated,centrifuged and washed.Then the cells were resuspended in EGM-2 MV SingleQuots supplemented with recombinant mouse growth factors.The suspension was then seeding in the culture plate coated with fibronectin.The half of the medium was refreshed in the second day.The whole medium was refreshed in the fourth day.After that,the whole medium was refreshed every other day.In the 14th day,we identified the EPC by ingesting DiI-Ac-LDL,binding FITC-UEA-1 and surface antigens detecting by flow cytometry.ResultsThe bone marrow-derived cells presented dense and little rounded immediately after seeding.Then the cells gradually grew up along with the culture time.Early EPCs were obtained in the7th day presented a spindle-shaped morphology.Late EPC was obtained in the 14th day present a cobblestone-like morphology.The identification of EPC indicated that almost all cells presented red fluorescent(DiI-Ac-LDL)and green fluorescent(FITC-UEA-1).The surface antigen detecting by flow cytometry showed that double positive rate for CD34 and Flk-1 was 84.85%.ConclusionMorphology and identification of EPC indicated that the cultured cells were EPCs.Part Ⅱ:Effects of Lipopolysaccharide on function of endothelial progenitor cells from bone marrow in miceBackgroundPrevious studies have shown that Lipopolysaccharide(LPS)is a cause of acute lung injury(ALI)and endothelial progenitor cells(EPCs)can participate in tissue repair.We hypothesize that LPS whether influences the number and function of EPCs directly and the influence is one aspect of the mechanism in LPS-induced ALI.MethodLPS was diluted to 10 pg/ml,100 pg/ml,1 ng/ml,10 ng/ml and 100 ng/ml and then act on the EPCs which were cultured for 7d which called early EPCs.Co-cultured for 4 h,8 h,12 h and 24 h,we tested the proliferation,senescence and adhesiveness of EPCs.In other side,mice were administered LPS(2.5mg/kg)via trachea and then after 4 h,8 h,12 h,and 24 h,we cultured the EPCs for 7 days and tested the proliferation,senescence and adhesiveness of EPCs in the 7th day.ResultAdhesiveness and senescence rate of EPC decreased in 100ng/ml,but proliferation was increased in 100 ng/ml in vitro while proliferation in vivo was decreased in 8 h and 12 h,senescence rate in vivo decreased in 12 h and 24h.Adhesiveness of EPC increased in 8 h and decreased in 12 h and 24 h.ConclusionThe reactions of EPCs influenced by LPS in vivo and in vitro were distinct.100ng/ml of LPS is sensitive for EPCs in vitro study.In the course of ALI induced by LPS,the EPCs became active in 8h and after then gradually recovered.Part Ⅲ:Repair of lipopolysaccharide-induced acute lung injury in mice by endothelial progenitor cells,alone and in combination with simvastatinBackgroundEndothelial progenitor cells(EPCs)are involved in endothelium repair of acute lung injury(ALI).Numerous studies have demonstrated that statins can promote EPCs function in vitro and in vivo;therefore,the purpose of this study was to determine whether simvastatin enhances the function of EPCs participating in repair of LPS-induced ALI and evaluate whether EPC transplantation combined with simvastatin can further repair LPS-induced ALI.MethodsBALB/C mice were initially pretreated with simvastatin by intraperitoneal administration 24 h before,and again at the time of,intratracheal instillation of lipopolysaccharide(LPS),and subsequently treated with EPCs by intravenous transplantation 2 h later.The effects of capillary permeability,endothelium repair,and inflammatory cytokines were measured.ResultsThis study revealed that both simvastatin administration and EPCs transplantation can reduce the severity of LPS-induced ALI in mice,and the effect can be further improved by combining the 2 therapies.ConclusionThe administration of simvastatin and EPCs transplantation can reduce the severity of LPS-induced ALI in mice,and improvement is moderately enhanced in some respects when EPCs transplantation is combined with simvastatin administration.The beneficial role of simvastatin on EPCs may be a component of its pleiotropic effects.Although the exact mechanism remains unknown,the combined administration of simvastatin and EPCs transplantation may be a potentially important cell-based,inflammatory-mediated therapy for patients with ALI/ARDS.