Effect Of Bone Marrow Mesenchymal Stem Cells On The Secretion Of Inflammatory Cytokines In Early Acute Lung Injury

Extensive burns are usually associated with inhalation injury, shock and infection, the body under the influence of trauma, shock, infection and other factors, led to excessive systemic inflammatory response, called systemic inflammatory response syndrome (SIRS). Lung is a target organ of SIRS, gathered a large number of inflammatory cytokines in the lung, vascular endothelial cells, resulting in pulmonary edema, and pulmonary edema among a wide range of ventilation and ventilation affect lung function, leading to acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), causing the body stubbornly persistent hypoxemia, so that the body organs of oxygen, dysfunction, leading to treatment failure in patients.ALI is a exudative lesions and the physiological characteristics is lung volume reduction, decreased compliance, serious ventilation/perfusion imbalance for the pathological, clinical manifestations were progressive hypoxemia and respiratory distress. The alveolar structure destruction, neutrophils, macrophages, local inflammatory cytokine release, cause lung inflammation. Mainly involved in cell ALI process:alveolar macrophages, neutrophils, pulmonary vascular endothelial cells, alveolar epithelial cells and lymphocytes, alveolar macrophages, which play an important role in the process of ALI.Present study suggests that, in the whole process of macrophages in ALI is not a participant, but the initiator and regulator. Macrophages are thought to be a multifunctional cell, its clearance, involved in nonspecific killing and functional immune responses to establish specific outside, more important is the production and secretion of hundreds of kinds of immune factors, such as tumor necrosis factor alpha (TNF-α), interleukin10(IL-10), interleukin6(IL-6), interleukin1(IL-1). The TNF-α is one of the earliest cytokine release, it is a kind of multifunctional cytokine, plays a key role in regulating inflammation. IL-10is a cytokine synthesis inhibitory factor, plays its role through inhibition of proinflammatory cytokines IL-1, IL-12, TNF-α and IL-2.Bone marrow mesenchymal stem cells (BMSCs) is a mature adult stem cells in recent years research. Studies showed that BMSCs have the effect of immunomodulatory,and the immunomodulatory effect plays a role by regulates the inflammatory cytokines of macrophages and reduces the degree of inflammation.Studies have shown that, BMSCs is the synthesis or secretion induced the secretion of cytokine synthesis with inflammatory regulation role of macrophage to realize its immunomodulatory effect, can inhibit the excessive inflammatory reaction, but the specific mechanism needs further study.The experimental study on the application of BMSCs in the animal model of acute lung injury early, by in vitro and in vivo experiments to explore the problems:1Could BMSCs influence the macrophage inflammatory factor release.2Could BMSCs function in macrophages, thereby reducing the body’s inflammatory response intensity.3Could BMSCs relieve the injury animal models of inflammation reaction strength acute lung, lung injury, improve the pulmonary ventilation function. For the application of BMSCs in large area burn, serious injury and combined injury caused by the body of SIRS, provide a theoretical basis for the clinical treatment of immune function disorder.Part1The research of BMSCs regulates the TNF-α、IL-10of macrophages mediated by lipopolysacharideObjective:To investigate the effect of bone marrow mesenchymal stem cells of the secretion of TNF-α、IL-10of macrophages mediated by lipopolysacharide (LPS)Methods:(1)Adherent culture method to obtain macrophages,adding1μg/mlLPS, respectively, when1,3,5,7,12,24h culture supernatant was collected and to content ELISA kit for TNF-α.(2)Bone marrow culture BMSCs, adherent culture method to obtain macrophages,the two co-cultured cells,add1μg/mlLPS,respectively1,3,5,7,12,24h culture supernatant collected fluid, ELISA kits to detect the content of TNF-α, IL-10.Result:(1)Content of TNF-α in Mφ+LPS group was significantly higher than the PBS group and Mφ group, P<0.001; Mφ group and PBS group had no significant difference between levels of TNF-α.(2)Content of TNF-α in Mφ+LPS group was significantly higher than the Mφ+BMSCs+LPS group and BMSCs+LPS group.Content of IL-10in Mφ+BMSCs+LPS group was significantly higher than the Mφ+LPS group and BMSCs+LPS group.Conclusion:(1)LPS stimulates macrophages produces more TNF-α, IL-10.(2)When LPS-stimulated macrophage, BMSCs can negatively regulates the secretion of TNF-α and positive regulates the secretion of IL-10by macrophage.Part2The research of BMSCs regulates the TNF-α IL-10in early Acute lung injuryObjective:To study the influence of TNF-α、IL-10by tail vein injection of BMSCs on acute lung injury in rats, to explore the repair effect of BMSCs on acute lung injury in SD rats.Methods:(1)Separate and culture of BMSCs,mark the cells by Brdu when P2cells at logarithmic growth phase.The experiments were performed in48hours.(2)Estab-lishment of rat model of acute pulmonary injury induced by intratracheal infusion of LPS. To determine the concentration of LPS to establish acute lung injury model in the pre experiment.(3)SPF grade male SD rats, were randomly divided into blank control group(NS+PBS), control group(LPS+PBS), experimental group:(LPS+B-MSCs),(LPS+BMSCs (early lh)).Each group in after the success of modeling,1h,7h,12h,24h at different time points in rat lung tissue specimens (n=5in each time point). Lung tissue of the left upper lobe of lung wet to dry weight detection; right middle lung tissue homogenate, ELISA content in the homogenate were detected inflammatory cytokine TNF-a, IL-10, IL-6; right lower lobe were stained with HE, Brdu immunostaining detection.Result:(1)Intratracheal instillation of5mg/kgLPS concentration in lung tissue of rats with NS50ul, serious congestion, edema, pulmonary interstitial.(2)Compared with NS+PBS group, LPS+PBS group showed significantiy difference in the W/D and the secretion of IL-10and TNF-α after1hours points (p<0.05). Compared with LPS+PBS group, LPS+BMSCs and LPS+BMSCs (early1h) group showed significantiy difference in the W/D and the secretion of IL-10and TNF-α (p<0.05). Compared with LPS+BMSCs group, LPS+BMSCs (early1h) group showed no significantiydifference in the W/D and the secretion of IL-10and TNF-α after1hours points (p>0.05)。Pulmonary edema, reached the peak at7h; TNF-α reached the peak at7h; IL-10reached the peak at12h.(3)Using Brdu labeled BMSCs were injected into the tail vein and acute lung injury in rats in vivo, Brdu by immunohistochemical staining of lung tissue, expression of labeled cells were positive for Brdu.Conclusion(1)BMSCs could reduce the extent of edema in early acute lung injury by macrophages to reduce the release of TNF-α and increase the release of IL-10.(2)BMSCs may play a role in the phenomenon gathered by homing in acute lung injury by tail vein injection.