| Part 1.Isolation and Identification of Extracellular Vesicles in Alveolar Lavage Fluid and Peripheral Blood Plasma of Patients with Early ARDSObjective: Bronchoalveolar lavage fluid(BALF)and extracellular vesicles(EVs)in peripheral blood plasma are related to lung injury and outcome in patients with ARDS.However,there is no uniform standard for the methods of separation and source identification of EVs in BALF and plasma.This experiment compares the different methods for separating and extracting EVs and identifying the cells from which EVs originates and aims to establish an effective separation and identification method for subsequent experiments.Method: In the first Part,ARDS patients admitted to the Department of Critical Care Medicine,Zhongda Hospital of Southeast University from August 2020 to April 2021 were enrolled.At 24h and 72 h after enrollment,the patient’s BALF and plasma were collected.Subsequently,the EVs were obtained by ultracentrifugation and size exclusion chromatography(q EV).Set up four different ultracentrifugation schemes under different conditions: U1=ultracentrifugation 100000g×2h,U2=120000g×2h,U3=187500×2h and U4=120000g×70min.The differences of EVs obtained by U1,U2,U3,U4 and Q(q EV)separation are compared through the following three aspects.(1)Nanoparticle Tracking Analyzer(NTA)compares different methods to separate the difference in particle size and concentration of EVs.(2)Use transmission electron microscopy(TEM)and Western blot(WB)to identify the morphology and markers of EVs separated by different methods.(3)The result of direct detection of the expression of CD31 and CD68 on EVs by nanoflow cytometry was used as the gold standard for identifying the source of endothelial cells and macrophages,and the comparison of using latex microspheres and streptomycin biotin affinity microspheres to capture the same EVs was carried out.Normal flow test results and nano flow test results.Results: 1.Compare different separation methods to obtain the particle size and concentration of EVs After the EVs pellets obtained by four different ultracentrifugation were resuspended in 50 u LPBS,the particle size and concentration of EVs detected by NTA were S1=(190.8nm,2.5E+12/m L),S2=(143.68 nm,1.2E+12/ m L),S3=(139.4nm,8.2E+10/m L)and S4=(121.6nm,1.0E+13/m L);the q EV method collects EVs through the chromatographic column,and the NTA detection result is Q=(146.7nm,3.2 E+7/m L).2.Identify different separation methods to obtain EVs by TEM and WB The TEM results of EVs obtained by the four different ultracentrifugation methods are similar,described under the microscope as a "cup-shaped" vesicle-like structure,and the particle size is like the NTA result.Compared with U1,U2,U3 and U4,the size and distribution of EVs separated by U2 are more uniform and the background is clearer;the background of EVs separated by Q is more cluttered than U2.WB detects the surface marker proteins CD9,CD63 and CD81 of EVs obtained by different separation methods,and their molecular weights are close to the reference range.3.Compare the ratio of EVs source cells detected by different flow cytometry methods The results of nano flow cytometry detection of EVs in the plasma of ARDS patients showed that the expression of CD31 endothelial cell-derived EVs accounted for about 20%,and the expression of CD68 macrophage-derived EVs accounted for about 9.4%.After the same sample was combined with EVs by latex microspheres and streptomycin biotin affinity microspheres,the expression ratio of CD31 and CD68 was detected by ordinary flow cytometry.It was found that the ratio of CD31 detected by latex microsphere method(21%)was not significantly different from that of streptomycin biotin affinity method(19.5%),and the result was similar to that of nanoflow detection;the ratio of CD68 detected by latex microsphere method(9%)is higher than the streptomycin biotin affinity microsphere method(5.8%).Conclusion: The identification results of TEM,NTA and WB showed that ultracentrifugation at 120,000 g×2h has the best effect in separating EVs from BALF and plasma in patients;ordinary flow detection latex microspheres capture EVs better than streptomycin biotin microspheres.Part 2: Characteristics of EVs in BALF and Plasma in Early ARDS PatientsObjective: Compare the differences in the concentration and source of EVs in BALF and plasma between early ARDS patients and non-ARDS patients.Analysis the correlation between total EVs concentration and the cause of ARDS and the severity of lung injury.To clear whether EVs can be used as a potential biomarker or therapeutic regulation target for ARDS.Method: The ARDS patients admitted to the Intensive Medicine Department of Zhongda Hospital of Southeast University from August 2020 to April 2021 were included as the experimental group,and non-ARDS patients receiving invasive mechanical ventilation were included as the control group.Collect the demographic information of the enrolled patients,the source of the patient,the underlying disease,the severity of the disease,the site of infection,the results of laboratory examinations,and the support of organ function,and other electronic medical records.At the 24 h and 72 h after enrollment,the two groups of patients were collected BALF and plasma.The EVs were separated by ultracentrifugation at 120,000g×2h,the concentration of EVs was detected by NTA,and the cells from which the EVs originated were identified by the latex microsphere capture method.Results: A total of 33 ARDS patients and 10 non-ARDS patients were included in the study.1.Comparison of total EVs concentration in BALF and plasma between ARDS group and non-ARDS group 1.1 Baseline characterizes of ARDS patients and non-ARDS patients The age of patients in the ARDS group was 65.5±15.5 years old,of which 18 were male patients(54.4%).On the first day of enrollment,the APACHEII score was 22.8±7.4 points,and the SOFA score was 9.2±5.1 points.Invasive mechanical ventilation time is 11.0[6.5-23.0] days,ICU hospital stay is 16.0[8.0-26.5] days,total hospital stay is 18.0[11.0-26.5] days,the 28-day case fatality rate is 33.3%,and the 90-day case fatality rate It is 45.5%,and the average cost of ICU hospitalization is 16.1 [8.7-30.2] million yuan.The etiology of ARDS included 23 cases of intrapulmonary reasons and 8 cases of extrapulmonary reasons.According to pathogenic microorganisms,ARDS patients caused by infection were divided into 11 cases of bacterial infection,3 cases of fungal infection,5 cases of viral infection,and 3 cases of unknown pathogen.There were no statistically significant differences between ARDS group and nonARDS patients in gender,age,BMI,underlying comorbidities(including hypertension,diabetes,cardiac insufficiency,and central nervous system diseases)and liver and kidney function.The APACHEII score,SOFA score and procalcitonin of the ARDS group were significantly higher than those of the non-ARDS group when the patients were enrolled(P<0.05).The mechanical ventilation time of ARDS group was significantly longer than that of non-ARDS group(P=0.02),and the cost of ICU hospitalization was significantly higher than that of non-ARDS group(P=0.002).There was no significant statistical difference in the 28-day and 90-day mortality between the two groups(P=0.15).1.2 Total EVs concentration in BALF and plasma in ARDS patients and non-ARDS patients The average concentrations of total EVs in BALF and plasma in the ARDS group were 5.56E+10/m L and 4.63E+10/m L,respectively;the average concentrations of total EVs in BALF and plasma in the non-ARDS group were 1.31E+10/m L and 1.72E+10/m L m L.The concentration of EVs in BALF in ARDS group was significantly higher than that in non-ARDS group(P=0.02);the concentration of EVs in plasma in ARDS group was significantly higher than that in non-ARDS group(P=0.04).2.The distribution of the source cells of EVs in BALF and plasma in ARDS patients and non-ARDS patients In the BALF of ARDS patients,the EVs-derived cells are at most epithelial cells(23.0%),followed by endothelial cells,macrophages,CD4+ T lymphocytes and CD8+ T lymphocytes(21.8%,8.5%,3.9% and 1.0%)Endothelial cells(19.35%)are the highest percentage of EVsderived cells in plasma,followed by epithelial cells,macrophages,CD4+ T lymphocytes and CD8+ T lymphocytes(14.8%,6.1%,1.5% and 0.7%).In the BALF of non-ARDS patients,the most EVs-derived cells are epithelial cells(22.7%),followed by macrophages,endothelial cells,CD4+T lymphocytes and CD8+T lymphocytes(16.7%,9.35%,1.2% and 0.52%);The highest proportion of EVs-derived cells in plasma is endothelial cells(25.7%),followed by macrophages,epithelial cells,CD4+T lymphocytes and CD8+T lymphocytes(17.5%,6.7%,2.1% and 0.42%).3.Correlation between the concentration of total EVs in BALF and plasma in ARDS patients and the severity of lung injury According to the Berlin standard,ARDS patients were divided into three groups: mild,moderate,and severe.There was no significant difference in the EVs concentration in BALF between the mild ARDS group and the moderate ARDS group(P=0.09).The EVs concentration in the BALF in the mild ARDS group was significantly lower than that in the severe ARDS group(P=0.02);to varying degrees There was no significant difference in the concentration of EVs in the plasma of ARDS patients.According to P/F≥150,ARDS patients were divided into mild and moderate-severe two groups.The total EVs concentration in BALF in patients with moderate-to-severe ARDS was significantly higher than that in mild ARDS(P=0.01),and there was no significant difference in plasma EVs concentration between the two groups.There was no correlation between the total EV concentration in BALF of ARDS patients and the P/F level(r=-0.23,P=0.19),and the total EVs concentration in plasma had no correlation with the P/F level(r=0.10,P=0.57).The concentration of total EVs in BALF was significantly positively correlated with Murray score level(r=0.43,P=0.01).The total concentration of EVs in BALF has no correlation with the driving pressure level(r=0.33,P=0.06),and it has no correlation with the static compliance level(r=-0.32,P=0.07).4.Comparison of total EVs concentration in patients with ARDS caused by different causes The study found that the EVs concentration in the BALF of ARDS patients caused by intrapulmonary causes was significantly higher than that in the extrapulmonary cause group(P=0.02),and the EVs concentration in the BALF of ARDS patients caused by infection was significantly higher than that in the non-infected group(P=0.01),There was no significant difference between groups in EVs from plasma.(1)The intrapulmonary cause of the highest EVs concentration in BALF of ARDS patients is microbial infection(7.7E+10/m L),followed by aspiration(3.8E+9/m L);the extrapulmonary cause of the highest EVs concentration is unknown cause(1.9E+10/m L),followed by pancreatitis,surgical stress,trauma,and burns.(2)The intrapulmonary cause of the highest EVs concentration in the plasma of ARDS patients is microbial infection(3.5E+10/m L),followed by aspiration(1.1E+10/m L);the extrapulmonary cause of the highest EVs concentration is surgical stress(1.5E+11/m L),followed by pancreatitis,trauma,unexplained cause,and burns.(3)The subgroup with the highest EVs concentration in the BALF of ARDS patients caused by microbial infection is bacterial infection(1.36E+11/m L),and the subgroup with the highest EVs concentration in plasma is also bacterial infection(4.8E+10 /m L).Conclusion: The concentration of total EVs in BALF and plasma in ARDS patients is higher than that in non-ARDS patients.EVs from different cell sources were different between the two groups.The concentration of EVs derived from BALF in ARDS patients showed an increasing trend with the severity of ARDS lung injury.The concentration of EVs in BALF and plasma of ARDS patients caused by bacterial infection is the highest.Part 3: Correlation of T cell-derived EVs with Lung Injury Severity and Outcome in Patients with Early ARDSObjective: T cells play an important role in the early development of ARDS,but there is no evidence that ARDS lung injury is related to EVs secreted by T cells.This experiment aims to explore the potential correlation between the severity and prognosis of lung injury in ARDS patients and T cell-derived EVs.Method: According to the Berlin diagnostic criteria,33 patients with ARDS admitted to the Department of Critical Care Medicine,Zhongda Hospital of Southeast University from August 2020 to April 2021 were included.At 24 h and 72 h after enrollment,the patient’s BALF and plasma were collected.The EVs were separated by ultracentrifugation at 120000g×2h,the concentration of EVs was detected by NTA,and the EVs were captured by the latex microsphere capture method,and then the CD4 and CD8 signals were detected by flow cytometry to identify the source of the cells.The main indicator for evaluating the severity of lung injury is the oxygenation index(P/F),and the secondary indicators are lung injury score(Murray score),lung driving pressure(DP)and lung static compliance(Cst).The main outcome indicator is 28 days.Case fatality rate,the secondary outcome indicators were ICU mechanical ventilation time and 90-day case fatality rate.Results: A total of 33 patients with ARDS were included in the study.1.The difference in the proportion of T cell-derived EVs between ARDS group and nonARDS patients CD4+T cell-derived EVs(CD4+T-EVs)in BALF and plasma of ARDS group were higher than CD8+T-cell-derived EVs(CD8+T-EVs).The ratio of BALF to CD4+T-EVs in plasma and the ratio of CD8+T-EVs in ARDS group were higher than those in non-ARDS group,but there was no significant statistical difference between the two groups.The ratio of the two in BALF in the ARDS group(CD4/CD8+T-EVs)was significantly higher than that in the non-ARDS group(P=0.016),and the ratio of the two in BALF in the ARDS group(CD4/CD8+T-EVs)was also It was significantly higher than the non-ARDS group(P=0.007).2.Correlation analysis between T cell-derived EVs and severity of lung injury in patients with ARDS CD4+T-EVs is negatively correlated with P/F(r=-0.09,P=0.61);CD8+T-EVs is positively correlated with P/F(r=0.19,P=0.30);but both has no statistical significance.CD4/CD8+T-EVs has a good negative correlation with P/F(r=-0.27,P=0.13).CD4/8+T-EVs in BALF was positively correlated with mechanical ventilation time,but there was no statistical significance(P=0.31).3.The predictive value of T cell-derived EVs for the prognosis of ARDS patients ROC curve assesses the predictive value of APACHEII score,SOFA score,and CD4/8+TEVs for 28-day prognosis and 90-day prognosis.Compared with CD4/8+T-EVs,the area under the ROC curve of the three is the highest.The 28-day prognosis AUC was 0.855 and the cutoff value was 3.1.The 90-day prognosis AUC was 0.86,and the cut-off value was 2.2.According to the cut-off value,ARDS patients were divided into two groups: CD4/8+T-EVs≥3.1 and CD4/8+T-EVs<3.1.The survival curve showed that the 28-day mortality rate in the CD4/8+T-EVs≥3.1 group was significant.Higher than CD4/8+T-EVs<3.1 group(P=0.026,HR=0.41[95%CI 0.18-0.94]).The 90-day mortality rate in the CD4/8+T-EVs≥2.2 group was also significantly higher than that in the CD4/8+T-EVs<2.2 group(P<0.001,HR=0.23[0.07-0.75]).Conclusion: The concentration of T-EVs in BALF and plasma in early ARDS patients is higher than that in non-ARDS patients.The CD4/CD8+T-EVs in the BALF of ARDS patients are positively correlated with the severity of lung injury in ARDS patients,and have a good predictive value for the prognosis of ARDS patients. |
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