Damage Repair And Evaluate The Prognostic Role Of Circulating Fibrocytes In Acute Lung Injury/Acute

Background:Myofibroblasts are the key effector cells that produce important extracellular matrix(collagen)substances during the repair of tissues and organs.They are considered to be key participants in the repair and regeneration of tissue arid organ damage and play an important role in the development of many fibrotic diseases effect.Circulating fibrocytes is one of the major sources of migration to damaged tissue into myofibroblasts and play an important role in the formation of pulmonary fibrosis.It is speculated that CF cells may play an important role in the repair and reconstruction of ALI/ARDS and the prognosis evaluation.The aim of this study was to investigate the role of CF cells in the repair of LPS-induced ALI and the prognosis of ALI/ARDS patients.To determine the reliable and valuable indicators of prognosis of patients with ALI/ARDS.Part 1 Damage Repair Role of Circulating Fibrocytes in LPS-induced Acute Lung InjuryObjectives:To establish the LPS-induced mouse ALI model,to explore the chemotaxis pathway,localization and the relationship between repair and damage,to clarify the role of CF cells in repairing damage in ALI.Methods:1)Kunming mice were randomly divided into two groups:Control group(tracheal instillation of equal volume of PBS);injury group(tracheal instillation LPS 5mg/kg).The peripheral blood mononuclear cells were isolated and cultured in vitro by density gradient centrifugation.The morphology of cells was observed by inverted phase contrast microscope,and CF cells were identified by flow cytometry;The number of CF cells in the peripheral blood of LPS-induced ALI were detected at different time(Id,3d,5d,7d,14d,21d and 28d);Gustavo Matute-Bello and Hubner Pathological Scoring Methods under light microscopy were used to observe lung injury and pulmonary fibrosis;Immunohistochemical method was used to observe the expression of COL-1 protein in lung tissue.2)Kunming mice were randomly divided into two groups:Control group(tracheal instillation PBS),injury group(tracheal instillation LPS 5mg/kg),10 days after the control group mice tail vein injection of PBS,and injury group were randomly divided into ALI-PBS group(tracheal instillation LPS + tail Intravenous injection of PBS)and blockade group(tracheal instillation LPS + tail vein injection AMD3100 2.5mg/kg).The levels of CXCL12 in peripheral blood of mice were detected by enzyme-linked immunosorbent assay(ELISA)at different time(1d,3d,5d,7d,14d,21d and 28d).Results:1.Morphological identification of mononuclear cells in ALI mice cultured in vitro:Peripheral blood mononuclear cells were round;Since the second day after separation,it can show obvious aggregation trend;The number of cell colonies and spindle cells increased significantly on the third day;On the 7th day,the cell colonies began to decrease and the spindle cells increased;On day 10,the cell colonies disappeared completely,and most of the adherent cells showed a typical spindle shape;On the 12th day,the cells showed a typical spindle shape.2.In vitro culture of ALI mice mononuclear cells surface markers identification:Flow cytometry can be seen to express CD45 + COL-1 + double positive cells,can be identified as CF cells.3.Histopathological changes of lung tissue:? HE staining showed that the lung tissue structure of the control group was normal,the alveolar cavity had no obvious exudation,the alveolar septum had no widening,no obvious edema,inflammation,no fibrosis and other special changes;The lung tissue of the injured group can be found at 3d and 7d.The alveolar wall thickening,alveolar septal fracture,alveolar cavity exudate filling,pulmonary interstitial inflammatory cell exudation and other cell changes;?Masson staining showed that the blue collagen fibers were positive in the lung tissue of the control group.In the lung tissue of the injured group,it was found that the blue collagen fibers appeared in the severe area of alveolar destruction At 14d and 28d,blue collagen fibers increased in the severe area of alveolar destruction.4.Correlation between the number of circulating fbrocytes and the degree of lung injury and the score of pulmonary fibrosis:There was a positive correlation between the number of CF cells in the peripheral blood group and the degree of lung injury(r =0.815,P<0.01);There was a positive correlation between the number of CF cells in the peripheral blood group and the fibrosis score after lung injury(r =0.904,P<0.01).5.Expression of COL-1 in lung tissue:There was a small amount of expression of type I collagen(COL-1)in lung tissue of control group;The expression of type I collagen(COL-1)protein in the lung tissue was significantly higher than that in the control group at the 7th,14th and 28th day(P<0.05).6.Changes of cytokine CXCL12 in peripheral blood:The content of CXCL12 in the peripheral blood of the injured group was significantly higher than that of the control group(5843,23 vs 1826.49,P<0.05);The content of CXCL12 in the peripheral blood of the blocking agent group was significantly lower than that in the injury group(2913.70 vs 5843.23,P<0.05),but still higher than the control group5843.23 vs1826.49,P<0,05).Conclusions:1.Circulating fibrocytes are released from the bone marrow into peripheral blood and migrate to damaged lung tissue in acute lung injury,which CXCL12/CXCR4 plays an important role.2.The number of circulating fibroblasts was positively correlated with the degree of lung injury and the score of pulmonary fibrosis,suggesting that circulating fibroblasts may play an important role in the repair and reconstruction of fibrosis in acute lung injury.Part 2 Prognostic Evaluation of Circulating Fibrocytes in Acute Lung Injury/Acute Respiratory Distress Syndrome PatientsObjectives:To study the relationship between the number of circulating fibroblasts in peripheral blood of ALI/ARDS patients and the degree of lung injury and mortality,and to determine the reliable value of circulating fibroblasts as a prognostic evaluation for ALI/ARDS patients.Methods:The CD45 + Col-1 + circulating fibrocytes in peripheral blood of 32 ALI I ARDS patients and 23 healthy subjects were counted by whole blood dissolution by flow cytometry;X-ray chest observation of alveolar collapse,atelectasis,the index of PaO2/FiO2,the level of PEEP,and the lung compliance were detected to calculate the Murray lung injury score;To evaluate the correlation between the number of CF cells in peripheral blood and the degree of lung injury and mortality in ALI/ARDS patients.Results:1.Object data:There was no significant difference in age and sex between the healthy group,the ALI group and the ARDS group,but the lung injury score and the 28-day mortality rate in the ARDS group were higher than those in the ALI group(P<0.05).2.The number change of ALI/ARDS patients peripheral blood fibrocytes:The number of peripheral blood CF cells in ALI/ARDS group was significantly higher than that in control group(6.2%vs 0.9%,P<0.05),The number of peripheral CF cells in ARDS group was significantly higher than that in ALI group(16.5%vs 2.9%,P<0.01);There was a positive correlation between the increase in the number of peripheral blood CF cells and the severity of lung injury in ALI/ARDS patients(P<0.01);During the 28-day observation period,the number of peripheral blood CF cells in the death group was approximately five times the survival group(14.8%vs 2.9%,P<0.01).The peripheral blood CF cells of ARDS patients in the death group and survival group were higher than those in ALI patients(18.2%vs 6.1%,P<0.01;5.7%vs 2.8%,P<0.05);3.Kaplan-Meier survival curvedrawing:ALI/ARDS patients survived 14 people during the 28-day observation period and killed 18 people.ALI group survived 12 people,the average survival time was 24.5 days,ARDS group survived 2,the average survival time of 13.8 days.4.ALI/ARDS mortality evaluation predictive value ROC curve:Peripheral blood of CF cells>5.85%predicted that the 28-day mortality rate of ALI/ARDS patients had the best predictive power(sensitivity:0.889,specificity:0.928).Conclusions:The number of peripheral blood fibroblasts in ALI/ARDS patients is closely related to the pulmonary function index and the degree of fibrosis of lung injury,and can be used as a reliable and valuable indicator for the prognosis of ALI/ARDS patients.