| Backgrounds and objective Acute lung injury(acute lung injury,ALI)is a medical condition characterized by widespread inflammation in lung,which results in a large scale of pulmonary capillary endothelial cells and alveolar epithelial cells damage,and a variety of etiological factor may lead to this damage,including extra-pulmonary factors or pulmonary factors.Hyaline membrane formation,loss of pulmonary surfactant,diffuse pulmonary interstitial and alveolar edema,alveolar collapse and permeability of alveolar membrane increase could occur in patients with ALI/ARDS on histology.Acute respiratory distress syndrome is the result of the progression and development of acute lung injury.In spite of the progression of mechanical ventilation strategies,the emergence of extracorporeal membrane oxygenation,the mortality rate of ALI/ARDS remains high.In pursuit for a better therapy,scholars began to gaze in tissue repair and regeneration of BM-MSCs.As a newly approach to treat ALI/ARDS,BM-MSCs has its great of prospect.Research has shown that BM-MSCs play a role in cellular immune regulating inflammatory microenvironment in the damaged area by homing to the site of damage through chemokines released by inflammatory injury,to decrease endothelial cell permeability and alveolar edema and to reduce the effect of inflammation.What's more,BM-MSCs could play a role against pathogen through autocrine of antimicrobial peptides,reinforcement of phagocytosis of macrophage cell and regulation T cells.In addition,recent studies have shown that MSCs could transfer their mitochondrion across to damaged alveolar epithelial cells in ALI/ARDS animal models,saving the damage of alveolar epithelium and reducing the lung edema.Because of multipotent roles of BM-MSCs played on anti-inflammation and pulmonary diseases caused by acute lung injury,which has significant efficiency,however,there is still controversy to that pulmonary diseases caused by chronic inflammations.Understandablly,to explore inflammatory cytokines variation and therapeutic effect by bone marrow mesenchymal stem cells of BM-MSCs on acute lung injury at various time points could provide a basic for the further clinically applications in the future.Methods1.By extracted bone marrow from SD rat,bone marrow mesenchymal stem cells were isolated and purified through the cell adherent method in vitro.2.BM-MSCs were induced to differentiate to adipocyte-oriented,chrondrocyte-oriented and osteocyte-oriented cells after passage 3.3.Labeled their cluster of differentiatio by immunofluorescence antibody,BM-MSCs were examined the positive or negative ratio of the cluster of differentiation in order to identify the cells by flow cytometry.4.Acute lung injury animal models were established by caudal vein injections of LPS and all of them were evaluated by pathological assessment and arterial blood gas analysis.The animal pathology score refers to the animal pathology of acute lung injury score published in 20115.120 SD rats were divided into three groups,N group(Normal group),L group(LPS group)and L+M group(LPS+MSCs group),40 rats in each group.Based on the time points of intervention of BM-MSCs,each group was divided into 5subgroups--2h,8h,24 h,48h,96 h.All the assays or test were examined at 24 h after the injection of MSCsResult1.Bone marrow mesenchymal stem cells were successfully isolated and purified through the cell adherent method in vitro.Isolate and purify the adherent cells,then it could be differentiated into adipocytes,osteocytes and chondrocytes.Flow cytometry(FCM)results indicate that the positive rate of CD90 and CD105 is 98.89%,97.37% respectively,,whereas the positive rate of CD44 and CD34 is 3.17%,1.41% respecitvely.The results above indicate that the cells which we isolate and purify is bone marrow mesenchymal stem cells.2.LPS significantly decrease the arterial partial pressure of oxygen(Pa O2),and interventions of BM-MSCs significantly increase Pa O2 at 2h Comparing to the N subgroups at the same time point,the Pa O2 of all L subgroups were significantly decreased(P < 0.01),and comparing to the L subgroups at the same time point,Pa O2 2 h L + M subgroup was significantly increased(P < 0.05),while the rest of the L + M subgroups have no significant difference(P > 0.05).3.LPS significantly increase the concentration of TNF-??IL-1??IL-10 in BALF,and early interventions of BM-MSCs could significantly decrease the concentration of TNF-??IL-1? and increase IL-10TNF-?:Comparing to the N subgroups at the same time point,the TNF-? of all L subgroups were significantly increased(P < 0.05),and comparing to the L subgroups at the same time point,the TNF-? of 2h L+M,8h L+M,24 h L+M,48 h L+M subgroups were significantly decreased(P < 0.05),while the rest of the L + M subgroups have no significant difference(P > 0.05).IL-1?:Comparing to the N subgroups at the same time point,the IL-1? of L subgroups at all time points were significantly increased(P < 0.05),and comparing to the L subgroups at the same time point,the IL-1?of2h L+M,8h L+M,24 h L+M subgroups were significantly decreased(P < 0.01),while the rest of the L + M subgroups have no significant difference(P > 0.05).IL-10:Comparing to the N subgroups at the same time point,the IL-10 of 2h L and8 h L subgroups were significantly increased(P < 0.01),and comparing to the L subgroups at the same time point,the IL-10 of L+M subgroups at all time points were significantly increased(P < 0.05).4.LPS significantly increase the PMN in BALF,and interventions of BM-MSCs could significantly decrease the PMN at 2h,8h.Comparing to the N subgroups at the same time point,the total PMN of L subgroups at all time points were significantly increased(P < 0.01),and comparing to the L subgroups at the same time point,the total PMN of 2h L+M,8h L+M subgroups were significantly decreased(P < 0.05),while the rest of the L + M subgroups have no significant difference(P > 0.05).5.LPS significantly increase the ratio of wet/dry of lung,and interventions of BM-MSCs could significantly decrease at 2h,8h and 24 h.Comparing to the N subgroups at the same time point,the lung wet/dry ration of L subgroups at all time points were significantly increased(P < 0.01),and comparing to the L subgroups at the same time point,the lung wet/dry ration of2 h L+M,8h L+M,24 h L+M subgroups were significantly decreased(P < 0.05),while the rest of the L + M subgroups have no significant difference(P > 0.05).6.LPS could induce acute lung injury,and early intervention of BM-MSCs significantly decrease the lung injury score at 2h,8h and 24 h Comparing to the N subgroups at the same time point,the acute lung injury score of L subgroups at all time points were significantly increased(P < 0.05),and comparing to the L subgroups at the same time point,the acute lung injury score of2 h L+M,8h L+M and 24L+M subgroups were significantly decreased(P < 0.05),while the rest of the L + M subgroups have no significant difference(P > 0.05).Conclusion1.Isolating adherent cells from bone marrow,and purifying it by attachment method,after that the cells could be differentiated into adipocytes,osteocytes and chondrocytes.The result of FCM indicates that the adherent cells which we isolate and purify is bone marrow mesenchymal stem cells.2.Early intervention with BM-MSCs could significantly reduce the expression of inflammatory cytokines and inflammatory cells recruitment to repair lung injury and to improve arterial oxygen pressure in rats with acute lung injury induced by lipopolysaccharide. |
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