ObjectiveTo investigate the protective effect of transfected microRNA-146a (miR-146a) on mice with sepsis-induced acute lung injury (ALI) in vivo.Methods Twenty-four healthy male BALB/C mice were randomly divided into sham group, sepsis group, transfection group and transfection control group, eachn = 6. Mice in transfection group were given miR-146a agomir loaded by in vivo-jetPEITM via airway before reproduction of model, and mice in transfection control group were given negative control loaded by in vivo-jetPEITM only via airway. The septic model was reproduced by cecal ligation and puncture (CLP) 12 hours after transfection , and the mice in the sham group underwent laparotomy and closure only without ligation and puncture of the cecum. The mice of each group were sacrificed at 24 hours post-operation. The expression of miR-146a in lung tissue was determined by real time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the quantity of tumor necrosis factor-α (TNF-α) in the bronchial alveolar lavage fluid (BALF) was determined with enzyme-linked immunosorbent assay (ELISA). The wet/dry ratio of lung (W/D) was determined. The pathohistological changes in the lung were observed and scored. Results The expression of miR-146a showed a significant increase in sepsis group, transfection group and transfection control group, which were (3.56±0.43), (27.64±3.46) and (3.72±0.54) folds of that in sham group, respectively (P< 0.05 orP< 0.01). The miR-146a expression in transfection group was significantly increased compared with sepsis group and transfection control group (bothP< 0.01), but no statistical difference in the expression was found between sepsis group and transfection control group (P> 0.05). Compared with the sham group, higher level of TNF-αin the BALF was found in the sepsis group, transfection group and transfection control group (ng/L: 511.65±43.47, 305.74±34.76, 492.27±42.21 vs. 50.72±7.23, allP< 0.01). The level of TNF-α in transfection group was significantly lower than that in sepsis group and transfection control group (bothP< 0.01). Compared with the sham group, the W/D ratio of lung in sepsis group, transfection group and transfection control group showed a significant increase (6.11±0.32, 5.02±0.29, 6.05±0.43 vs. 4.18±0.10, allP< 0.01). The W/D ratio of lung in transfection group was significantly lower than that of sepsis group and transfection control group (bothP< 0.01). The lung injury score of transfection group was significantly lower than that of sepsis group and transfection control group (6.12±0.75 vs. 10.53±1.52, 9.73±1.08, bothP< 0.01).Conclusions miR-146a agomir loaded by in vivo-jetPEITM instillation into airway was able to increase the expression of miR-146a in the lung tissue of septic mice. Up-regulation of miR-146a inhibit the release of the inflammatory cytokine TNF-α stimulated by sepsis, and alleviate inflammatory reaction and lung tissue injury in mice with sepsis-induced ALI.

Objective:To investigate the relationship between body mass index (BMI) and response time of cardioinhibitory type vasovagal syncope (VVS-CI) in children.Methods:The clinical data of 56 children with syncope or pre-syncope were retrospectively analyzed and they visited specialist clinic for syncope and were diagnosed as VVS-CI in the Second Xiangya Hospital, Central South University from December 2012 to September 2019.Based on height and weight, BMI was calculated, and divided into low BMI group (35 cases) and normal BMI group (21 cases). Between the 2 groups, baseline heart rate, head-up tilt test (HUTT) positive response heart rate, baseline head-up tilt test (BHUT) positive response time, and sublingual nitroglycerin-provocated HUTT (SNHUT) positive response time were compared.The correlation between BMI and positive response time was analyzed.SPSS 22.0 software was applied for statistical analysis.Results:There were no significant differences in age, sex, duration of disease and number of syncope between the 2 groups (all P>0.05). No significant differences were found in baseline heart rate and positive response heart rate between the 2 groups [(78.5±15.3) times/min vs.(72.8±8.7) times/min, t=1.223, P=0.230; (44.0±13.9) times/min vs.(47.0±10.0) times/min, t=-0.664, P=0.511]. Compared with normal BMI group, BHUT positive patients/SNHUT positive patients were higher in low BMI group (27/8 cases vs.9/12 cases, χ2=4.839, P=0.027), and the positive response time of BHUT was shorter [(13.1±4.6) min vs.(23.7±9.5) min, t=-2.691, P=0.023]. There were no significant differences in SNHUT positive response time between the 2 groups ( P>0.05). Low BMI was correlated with BHUT positive response time ( r=0.750, P=0.005). Normal BMI was not associated with BHUT positive response time ( r=0.316, P=0.217). There was no correlation between low BMI and normal BMI and SNHUT positive response time ( r=0.177, P=0.431; r=0.021, P=0.940). Conclusions:Low BMI is positively correlated with BHUT positive response time of children with VVS-CI.The time it takes for syncope occurrence was shorter in children with low BMI than that in normal BMI.

Objective To determine kinetics of TNF-α and miR-146a (microRNA-146a)expressions in lipopolysaccharide (LPS)-induced NR8383 alveolar macrophages (AM) at different intervals and their relationships in order to explore regulatory effect and mechanism of miR-146a on alveolar macrophages inflammatory responses.Methods NR8383 alveolar macrophages were seeded in a 6-well plate,and stimulated with 1 μg/ml of LPS for 0 h,3 h,6 h and 12 h separately after 90 min.Cells were harvested and supernatant were collected 0 h,3 h,6 h and 12 h after incubation.The expressions of miR146a and TNF-α mRNA in cells were detected by real-time qPCR and the levels of TNF-α protein in the supematant of cells were assayed by enzyme-linked immunosorbent assay ( ELISA ).Pearson correlation analysis was used to analyze the correlation between miR-146a and TNF-α mRNA.Results ①The level of TNF-α protein in the supernatant of cell was significantly increased 3 h after LPS challenge ( 359.80 ±57.54) pg/ml (P ＜0.01 ),and peaked 12 h later (729.22 ±50.40) pg/ml (P＜0.01 ) ; ②the expression of TNF-α mRNA peaked 3 h after LPS challenge (67.48 ±24.52) fold,P ＜0.01 ),and then decreased gradually; ③the expression of miR-146a mRNA increased continuously until 6 h or 12 h after LPS challenge 6 h:(5.33 ±0.81) fold,12 h:(8.21 ±1.19) fold,(P＜0.01),and it showed an upward tendency;④ the expression of miR-146a mRNA was negatively correlated with TNF-α mRNA ( r =-0.895,P ＜0.01).Conclusions The miR-146a mRNA showed a negative correlation with TNF-α mRNA present in lipopolysaccharide-stimulated alveolar macrophages,suggesting miR-146a mRNA involved in regulating the inflammatory response of alveolar macrophages.

Objective To establish a convenient and rapid method for extracting DNA from bone.Methods Fifteen long bone samples were washed and sterilized.The skeletal fragments were obtained by electric drill,and lysed by PrepFiler Express BTATM lysis buffer.DNA was then manually extracted by silicon microbeads for further analysis.Results STR genotyping was successfully obtained in 14 out of the 15 samples,and the detection rate was 93.33％.Conclusion The method for DNA extraction from bone established in present study is convenient,quick,effective,and with a strong applicability,which is worth spreading and applying.

Objective To investigate the mechanism of reverse redistribution (RR) on dipyridamole 201Tl myocardial perfusion studies in the patients with coronary artery spasm. Methods Twenty-six patients with coronary artery spasm and presented as RR on dipyridamole 201Tl myocardial perfusion studies were enlisted as RR group, while other 16 patients with no coronary artery stenosis nor RR were enlisted as control group. Dipyridamole test was repeated during coronary angiography. Corrected thrombolysis in myocardial infarction (TIMI) frame count (CTFC) and TIMI myocardial perfusion grade (TMPG) were measured at RR related and non-RR related coronary arteries before and after dipyridamole infusion respectively.All of the data were analyzed by Student's t-test orχ2-test and correlation analysis. Results Coronary artery angiography showed slower blood flow and lower myocardial perfusion in RR related vessels when compared with non-RR related vessels in RR group, but there was no significant difference among the main coronary arteries in control group. The perfusion defects of RR area at rest were positively related to slowerblood velocity at corresponding coronary arteries ( r = 0.79, t = 10.18, P ＜ 0.001 ). In RR related vessels,CTFC were (36 ±6) frames and (26 ±7) frames (t =4.15, P ＜0.01 ), while TMPG were (2.02 ±0.39)grades and (2.92 ± 0.12) grades ( t = 2.25, P ＜ 0.05 ) before and after dipyridamole infusion, respectively.In non-RR related vessels, CTFC were (29 ±7) frames and (25 ±5) frames (t =2.31, P ＜0.05), while TMPG were (2.56 ± 0.31 ) grades and (2.96 ± 0.06) grades ( t = 2.17, P ＜ 0.05 ) before and after dipyridamole infusion, respectively. However, there were no significant changes of CTFC and TMPG before and after dipyridamole infusion in control group ( t = 0.932, 0.867, respectively, both P ＞ 0.05 ). Conclusion RR is related to the decreased blood flow and myocardial perfusion induced by coronary artery spasm at rest,which may be improved by stress test such as intravenous dipyridamole infusion.

Objective To observe the effect of microRNA-155 (miR-155) on the inflammatory response of rat alveolar macrophages induced by lipopolysaccharide (LPS). Methods The alveolar macrophages NR8383 of rat were cultured in vitro, the macrophages in logarithmic growth phase were harvested to conduct experiment. ① The 1 mg/L LPS was used to stimulate the rat alveolar macrophages for 3, 6, 12, and 24 hours, a phosphate buffer solution (PBS) control group was also set up. Enzyme linked immunosorbent assay (ELISA) was used to detect the dynamic changes of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant, and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the dynamics expression of miR-155 in the cells, which confirmed the optimal time for LPS stimulation was 12 hours. ② Carboxyfluorescein (FAM) labeled mimic (FAM mimic) and inhibitor (FAM inhibitor) were used to transfect the alveolar macrophage, and the transfection effect was observed under inverted fluorescence microscope 6 hours later to confirm the optimal transfection concentration of mimic was 20 nmol/L, and the optimal transfection concentration of inhibitor was 100 nmol/L. miR-155 mimic and miR-155 inhibitor were transfected to alveolar macrophages respectively at the optimal transfection concentration for 24 hours, and 1 mg/L LPS was used to stimulate the cells for 12 hours. A mimic negative control + LPS group and an inhibitor negative control + LPS group were set up. The expressions of IL-1β and TNF-α in the supernatant were determined by ELISA to observe the regulation of miR-155 on inflammatory response of alveolar macrophages. Results ① After stimulation of 1 mg/L LPS on alveolar macrophages, the contents of IL-1β and TNF-α in the supernatant and the expression of miR-155 in the cells were increased gradually with time prolongation, IL-1β and TNF-α contents peaked at 12 hours, and the expression of miR-155 peaked at 24 hours [as compared with PBS control group, IL-1β (ng/L): 910.43±36.09 vs. 22.66±7.84, TNF-α (ng/L): 3 138.39±394.10 vs. 233.92±8.84, miR-155 (2-ΔΔCt): 7.82±0.30 vs. 1, all P < 0.05]. ② Under inverted fluorescence microscope, after 20 nmol/L FAM mimic or 100 nmol/L FAM inhibitor transfected alveolar macrophages for 6 hours, a large number of cells showed green fluorescence, indicating that the transfection was successful. The expression of miR-155 in the cells transfected with 20 nmol/L miR-155 mimic was up-regulated by (236.73±46.49) times as much as that in the negative control group (P < 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly lower than those in the negative control group [IL-1β (ng/L): 324.37±36.59 vs. 799.31±39.44, TNF-α (ng/L): 1 554.01±342.48 vs. 3 020.49±418.30, both P < 0.05]. The miR-155 activity was significantly inhibited in the cells transfected with 100 nmol/L miR-155 inhibitor, and the expression of miR-155 was decreased by (4.00±3.26)% as compared with the negative control group, but the difference was not statistically significant (P > 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly higher than those in the negative control group [IL-1β (ng/L): 1 358.98±212.04 vs. 878.68±53.42, TNF-α (ng/L): 4 225.57±281.11 vs. 2 881.32±286.08, both P < 0.05]. Conclusion In LPS induced inflammatory response of alveolar macrophages, miR-155 plays an obvious inhibitory role.

To study the effects of sodium valproate (VPA) on human myelodysplastic syndrome cell line MUTZ-1. The cell proliferation was determined by MTT assay, apoptotic morphological features were observed by light microscopy and transmission electronmicroscopy, cell apoptosis and cell cycle shift were analyzed by flow cytometry (FCM). The results showed that VPA could inhibit the growth of MUTZ-1 cells in dose-and time-dependent manners. The typical apoptotic morphological features appeared in MUTZ-1 cells treated with 4 mmol/L VPA for 72 hours. Pyknosis of cells and nuclei, disintegration of nuclear chromatin and apoptotic body could be observed by light microscopy. Aggregation and margination of nuclear chromatin, concentration of plasm, increment of density and chromatin mass of irregular size could be observed by transmission electronmicroscope. The flow cytometric analysis indicated that the VPA could induce cell apoptosis, apoptosis rate increased in dose-dependent manner, ratio of cells at G(0)/G(1) phase increased and ratio of cells at S phase decreased in dose-dependent manner, the cells were arrested at G(0)/G(1) phase. It is concluded that the VPA can induce apotosis and inhibite proliferation of MUTZ-1 cells via arresting cells at G(0)/G(1) phase.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Humans ; Myelodysplastic Syndromes ; pathology ; Valproic Acid ; pharmacology

Objective To investigate the clinical therapeutic effects of lung protective ventilation and sequential recruitment maneuver (RM) on treatment of patients with severe acute pancreatitis (SAP) complicated with acute respiratory distress syndrome (ARDS). Methods Sixty patients with SAP complicated with ARDS admitted to the Department of Critical Care Medicine of the First Affiliated Hospital of Nanchang University from April 2014 to March 2016 were enrolled. They were divided into control group and experimental group by random number table, 30 patients in each group. On the basis of comprehensive treatment, the patients in control group were treated with lung protective ventilation mode: low tidal volume ventilation (6 mL/kg) + optimal end-expiratory positive pressure (PEEP) ventilation mode, when the intra-abdominal pressure (IAP) was essentially returned to a normal level (Ⅰ grade intra-abdominal hypertension), the patients in experimental group were treated by the combination with RM therapy, and the rest treatment was the same as the control group. Under the two types of ventilation strategies, the clinical effects, respiratory mechanics, hemodynamics and arterial blood gas indexes were compared between the two groups. Results The mechanical ventilation time (days: 13.82±4.40 vs. 19.87±7.40), the length of ICU stay (days:22.67±4.40 vs. 26.43±5.39) and incidence of ventilator associated pneumonia [VAP: 16.67% (5/30) vs. 26.67% (8/30)] of the experimental group were lower than those of the control group (all P < 0.05), the mortality rate of the experimental group was slightly lower than that of the control group [26.67% (8/30) vs. 30.00% (9/30)] without statistical significance (P > 0.05). Plateau pressure (Pplat) and the peak airway pressure (PIP) at each time point were decreased after treatment in both groups, while the static lung compliance (Cst), the arterial partial pressure of oxygen (PaO2) and oxygenation index (PaO2/FiO2) were increased compared with those before treatment, especially the changes at 72 hours after recruitment in the experimental group were more significant than those in the control group [Pplat (cmH2O, 1 cmH2O = 0.098 kPa):15.6±4.0 vs. 21.2±5.6, PIP (cmH2O): 18.3±5.0 vs. 25.1±5.4, Cst (mL/cmH2O): 41.2±4.8 vs. 31.2±6.0, PaO2 (mmHg, 1 mmHg = 0.133 kPa): 90.93±6.45 vs. 80.27±4.51, PaO2/FiO2 (mmHg): 238.33±18.31 vs. 185.83±11.14]. Heart rate [HR (bpm): 110.23±7.92 vs. 98.23±8.44] and the central venous pressure [CVP (mmHg): 8.62±1.52 vs. 6.32±1.42] were significantly higher than those before treatment, the mean arterial pressure [MAP (mmHg): 86.74±7.65 vs. 94.92±10.93] and cardiac output [CO (L/min): 5.32±1.36 vs. 6.42±1.32] were significantly reduced compared with those before treatment (all P < 0.05). The values of HR, MAP, CVP, CO at 5 minutes after recruitment were (97.87±5.77) bpm, (94.54±6.87) mmHg, (6.33±1.44) mmHg, (6.32±1.41) L/min, respectively. The changes of these parameters were not significant when compared with those of the basal conditions (P > 0.05) Conclusions Based on the lung protective ventilation in the early stage, sequential RM is applied in treatment of patients with SAP complicated with ARDS, after the IAP is essentially returned to a normal, which is beneficial to improving lung compliance, promoting oxygenation, shortening the time of mechanical ventilation, reducing the length of ICU stay, and decreasing the incidence of VAP without any obvious hemodynamic influence.

OBJECTIVETo investigate the value of the intraoperative fine-needle aspiration cytology (IFNAC) in the diagnosis of pancreatic cancer.

METHODSThe IFNAC data of 70 pancreatic cancer patients were retrospectively reviewed. IFNAC had been done in our hospital only in a few patients before 2001, however, more and more patients have been examined by renovated method since 2002. But as the way of carrying out IFNAC improved by changing from the ordinary 10 ml syringe and 22 gauge needle (0.7 mm) before 2003 to 5 ml syringe and 25 gauge skin testing needle (0.5 mm), the panreas itself is properly exposed before IFNAC, the operator at frst fixes the suspected mass with his left hand and does the puncture with his right hand. Puncturing is done while the syringe is rotated, negative pressure is being kept at the same time. The needle is withdrawn under negative pressure, 6-8 syringe are used for each patient by puncturing at 6-8 points, then smears are made with the syringes. The whole process takes 20 min to accomplish.

RESULTSThe overall positive rate was 84.3%. The positive rate of the conventional IFNAC was 66.67%, while it was increased to 95.3% following the introduction of the renovated method (P = 0.002); No complication was observed in this series.

CONCLUSIONIntraoperative fine-needle aspiration cytology using 25 gauge skin testing needle and multi-point rotating maneuver during puncturing in the diagnosis of pancreatic cancer is safe and effective with few complications.

Adenocarcinoma ; diagnosis ; pathology ; surgery ; Biopsy, Fine-Needle ; Cytodiagnosis ; False Negative Reactions ; Female ; Humans ; Intraoperative Period ; Lymph Nodes ; pathology ; Male ; Middle Aged ; Pancreas ; pathology ; surgery ; Pancreatic Neoplasms ; diagnosis ; pathology ; surgery ; Predictive Value of Tests ; Retrospective Studies

This study was aimed to establish a cytokine-independent human myelodysplastic cells line from bone marrow of a patient with MDS-CMML. This cell line was incubated in mixed culture of RPMI 1640 and DMEM with 15% bovine serum, but without cytokines; its biological characteristics were identified by morphology, surface marker profiles, cell proliferation, differentiation and apoptosis. The results showed that the established cell line could not depend on cytokines for long-term survival and growth, and could differentiate into colony-forming unit-macrophage, colony-forming unit-megakaryocyte. In conclusion, a cytokine-independent human myelodysplastic syndrome cell line, named MDS-JSN04 (MDS Nanjing Jiansu 04), was established. Its partial biological characteristics were identified and clarified.
Antigens, CD19 ; analysis ; Antigens, CD20 ; analysis ; Apoptosis ; drug effects ; Bone Marrow Cells ; metabolism ; pathology ; ultrastructure ; CD79 Antigens ; analysis ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Culture Media ; pharmacology ; Cytokines ; pharmacology ; Flow Cytometry ; HLA-DR Antigens ; analysis ; Humans ; Lewis X Antigen ; analysis ; Male ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Middle Aged ; Myelodysplastic Syndromes ; metabolism ; pathology ; Sialic Acid Binding Ig-like Lectin 2 ; analysis ; Time Factors