Objective To establish a HEK293 cell line stably and highly expressing sense and antisense Alu-Sx.Methods According to Alu subfamily Sx sequence,a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized.PCR of the total DNA extracted from HEK293 cell line was performed,the products of which were cloned into a highly efficient eukaryotic expression vector pcDNA3.1/myc-His A.The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the HEK293 cell line by lipofectamine2000.The stable transfectants were screened by G418.Cell subclones were isolated by gradient dilution.The highly expressing clones were identified by Northern blotting.Results Eukaryotic expressing vectors stably and efficiently expressing sense and antisense Alu-Sx were constructed and cell subclones stably and efficiently expressing sense and antisense Alu-Sx were established.Conclusion Cell subclones stably and efficiently expressing sense and antisense Alu-Sx can used for our further study.

Objective To examine the effect of fibronectin connecting segment-1 (CS1) peptidefacilitated blockade of inflammatory cells-fibronectin adhesion on a rat liver transplantation model of prolonged ex vivo cold ischemia.Methods A model of liver transplantation in Wistar→Wistar rat was established.The donors of the CS1 treatment group received CS1 peptides through the tail vein for 3 days before operation.Another two doses of CS1 peptides were administered into the liver intraportally during procurement and before transplantion.Recipients received an additional 3-day course of CS1 peptides after transplantation.Rats in control group received scrambled peptides.Rats were sacrificed at 6,24 and 72 h after transplantion,and plasma transaminase activity and hepatic pathological changes were studied.The inflammatory cells and liver sinusoidal endothelial cells were visualized histochemically.Real-time PCR was used to detect tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF) mRNA expression in the liver.Results The plasma transaminase activity and hepatic necrosis areas in CS1 treatment group were significantly lower than in control group (P＜0.05).CS1 peptides treatment significantly decreased the number of Kupffer cells after transplantation and greatly inhibited the recruitment of neutrophils to the graft liver as compared with control group (P＜0.05).After prolonged cold ischemia,only a few hepatic endothelial cells exhibited positive staining of hepatic sinusoidal endothelial cell biomarker SE-1.Lots of hepatic sinusoidal endothelial cells positive for SE-1 staining could be detected in CS1 group at 72 h after transplantation,while much less SE-1 positive cells presented in the control goup.Prolonged cold ischemia caused a significant increase of TNF-α,IL-1β and VEGF mRNA expression in the graft liver of control group after transplantation.The expression of TNF-α mRNA at 6 and 24 h and VEGF mRNA expression at 24 h were significantly lower in CS1 group than in control group (P＜0.05).Conclusion Peptide-mediated blockade of inflammatory cells-fibronectin interaction decreased the mRNA expression of inflammatory cytokines,prevented hepatic sinusoidal endothelial cells from injury and subsequently protected against severe ischemia/reperfusion injury of the graft liver after transplantation.

Objective To observe the expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury (ALI) and explore the function of Slit2 and Robo4 in ALI.Methods Forty-eight Sprague-Dawley rats were randomly (random number) divided into control group (n =24) and ALl group (n =24).ALI model was reproduced by cecum ligation and puncture (CLP).The control group only experienced a simulated operation without CLP.Both groups were further divided into 3 subgroups with 8 rats in each subgroup:12 h,24 h,and 48 h subgroups.artery blood gas analysis,lung tissue wet/dry weight (W/D) ratio,lung histopathologic changes,pulmonary microvascular permeability were observed.The serum tumor nocrosis factor-α (TNF-α) was measured with enzyme linked immunosorbent assay (ELISA).The expression of Slit2 and Robo4 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR).The expression of Slit2 and Robo4 protein in lung tissues was assessed by immunohistochemistry.Date were analyzed by one-way ANOVA with SPSS version 13.0 software.Statistical significance was established at a P value of less than 0.05.Results Compared with the control group,in ALI rats at different time points,partial pressure of oxygen in arterial blood (PaO2) decreased significantly,lung W/D weight ratio and pulmonary microvascular permeability,the serum TNF-α increased significantly (all P ＜ 0.05),histopathology of lung revealed signs of injury.The expression of Slit2 mRNA in lung tissues was decreased markedly after CLP compared with control group [(0.56±0.13) vs.(0.87±0.05),F=41.39,P＜0.05,(0.42±0.10) vs.(0.85±0.07),F=93.54,P＜0.05,(0.26±0.08) vs.(0.89 ±0.09),F=227.05,P＜0.05].but there were no significant difference in expression of Robo4 mRNA in lung tissue between ALI group and control group [(0.86±0.07) vs.(0.83±0.05),F=0.695,P＞0.05,(0.82±0.05) vs.(0.89±0.08),F=2.061,P ＞ 0.05,(0.86 ± 0.08) vs.(0.86 ± 0.05),F =0.035,P ＞ 0.05].Immunohistochemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells,while lung epithelial cell nuclei and endochylema.Robo4 protein was only expressed on the extracellular surface of vascular endothelial cells.Compared with the control group,expression of Slit2 protein in lung tissue in ALI group decreased markedly [(0.37 ± 0.05) vs.(0.45 ± 0.07),F =6.82,P ＜ 0.05,(0.32±0.06) vs.(0.47±0.09),F=23.54,P＜0.05,(0.28±0.07) vs.(0.46±0.06),F=28.01,P ＜ 0.05].As good as RT-PCR,there were no significant difference in expression of Robo4 protein in lung tissue between two groups [(0.53±0.04) vs.(0.52±0.05),F=0.155,P＞0.05,(0.53± 0.09) vs.(0.50±0.05),F=0.498,P＞0.05,(0.55±0.06) vs.(0.56±0.07),F=0.073,P ＞ 0.05].Conclusions Lung tissues of control group rats express Slit2 and Robo4.The decreased Slit2 mRNA and protein expressions in the lung tissue of rat with ALI caused by CLP may be associated with the occurrence of ALI.

Objective To investigate the effects of inhalation of different concentrations of carbon monoxide (CO) on brain death (BD)-induced lung injury in rats. Methods Thirty-two pathogen free adult male Wistar rats weighing 250-300 g were randomly divided into 4 groups ( n= 8 each): group Ⅰ sham operation (group S);group Ⅱ brain death (group BD) and group Ⅲ and Ⅳ BD + CO 0.025% and 0.050% (group C1, C2 ). The animals were anesthetized and tracheally intubated. Fogarty catheter was inserted into the skull. BD was induced by inflating the balloon slowly at 20 μl/min until apnea developed. The animals were then mechanically ventilated (VT 10 ml/kg, RR 50 bpm, PEEP 2 cm H2O) with 40% O2 in N2 . In group Ⅲ and Ⅳ CO 0.025% and 0.050%were added to the air mixture respectively. In group S the balloon was not inflated. BD was confirmed by apnea,dilated pupils and flat EEG. In group BD,C1 and C2, MAP was maintained at 80-120 mm Hg by norepinephrine infusion. The arterial blood gas analysis was performed before (baseline) and immediately after BD was confirmed (T1) and at 30, 60, 90 and 120 min (T2-5) of CO inhalation. The animals were then sacrificed. The plasma concentrations of IL-6 and TNF-α and the activity of myeloperoxidase (MPO) in the lungs were measured. The W/D lung weight ratio and lung injury score (LIS) were recorded. Results BD significantly decreased PaO2/FiO2, BE and pH while increased plasma IL-6 and TNF-α concentrations, MPO activity in the lungs, the W/D ratio and lung injury score as compared with group S. CO inhalation ameliorated the deleterious effects induced by BD. The antiinfiammatory effect of 0.050% CO was better than that of 0.025 % CO. Conclusion Inhalation of 0.025 % or 0.050% CO can ameliorate BD-induced lung injury in rats, but there is no significant difference in the efficacy.

Objective:To investigate the effect of arthroscopic superior capsular reconstruction (ASCR) of irreparable massive rotator cuff tears (RCTs) using the "Sandwich" patch graft (autologous fascia lata + LARS artificial ligament + autologous fascia lata).Methods:The patients with irreparable massive RCTs who underwent ASCR by using "Sandwich" patch graft were retrospectively evaluated between December 2016 and October 2018. All cases were followed up more than two years. The pain and function of the shoulder were evaluated by visual analogue scale (VAS), active forward elevation (aFE), American Shoulder & Elbow Surgeons score (ASES), University of California Los Angeles (UCLA) score, and Constant-Murley score. The acromiohumeral distance (AHD) and patch healing were assessed by shoulder X-ray and MRI scan.Results:Twenty-nine patients (12 males and 17 females) were enrolled for final analysis. The average age was 66.0±5.88 years (range 55-77 years). The average length of follow-up was 35.3±7.20 months (range 24-46 months). The tendon of the subscapularis muscle was intacted in 13 cases and repairable in 16 cases. All patients' teres minor muscles were intact and the function of deltoid muscles was all complete. Based on Hamada classification of massive RCTs, 11 cases with type 2, 14 cases with type 3, and 4 cases with type 4. At the last follow-up visit, the AFE of the surgical shoulder was 158.45°±23.87° (range 70°-180°), which was significantly higher than before the surgery 92.59°±45.99° (range 20°-160°, t=6.190, P<0.001). The ASES score was 92.92±9.08 (range 64-100), UCLA score was 31.62±3.93 (range 19-35), and Constant-Murley score was 85.8±8.44 (range 68-94), which were higher than those before the operation 30.69±12.99 (range 68-95), 10.93±3.43 (range 4-17), 39.62±12.68 (range 14-55). There were significant differences between them, respectively ( t=21.145, P<0.001; t=21.348, P<0.001; t=16.333, P<0.001). The VAS was 0.41±0.57 (range 0-2 points), which was significantly lower than that of 4.90±1.05 (range 3-7 points) before operation ( t=20.267, P<0.001). At the last follow-up visit, MRI showed that the AHD increased from 3.31±1.57 mm (range 1.1-6.6 mm) to 6.94±1.76 mm (range 3.0-10.8 mm) significantly ( t=12.195, P<0.001). Radiological outcomes were evaluated according to MRI, the total healing rate was 89.7% (26/29). There were two cases of complete tears, which including one case of infection and 1 partial tears. Conclusion:ASCR of irreparable massive RCTs using "Sandwich" patch graft showed the high healing rate in the short-term follow-up, which is possible to restore the shoulder functions early. It is an effective method for the treatment of irreparable massive RCTs.

Objective Collagenous gastritis is a rare entity, characterized by the deposition of a subepithelial collagen band with an inflammatory infiltrate in the mucosa.This report describes the first case of collagenous gastritis occurring in a young Chinese woman and reviews the literatures.Methods The patient underwent the gastroscopy screening, and the biopsy specimens were treated with HE staining, Masson staining, Congo red staining and Warthin-Starry staining.Patients' clinical data was discussed and followed up.Results A twenty-year-old girl had intermittent epigastric pain for 4 years, abdominal distention, hiccup and weight loss for two months.The gastric endoscopy revealed diffuse white nodular appearance of the mucosa in angular incisure and antrum.Pathologic examination of the gastric biopsies from the antrum and angular showed a subepithelial collagen deposition with moderate infiltrates of lymphoplasma cells and eosinophils of the lamina propria.The collagen band measured up to 120.3 μm (mean 43.8 μm).Prednisone 20 mg/d for 4 weeks led to clinical remission and weight gain.Conclusion There are about 40 cases in literatures to date, and the cause and pathogenesis of collagenous gastritis remain unknown.According to the clinical and pathological characteristics, the patient in this article is the subtype of collagenous gastritis that occurring in children and young adults.Specific therapy has not been established, the gluten-free diet and glucocorticosteroid may be helpful to relieve symptoms in collagenous gastritis patients.

Most chemical medicines have polymorphs. The difference of medicine polymorphs in physicochemical properties directly affects the stability, efficacy, and safety of solid medicine products. Polymorphs is incomparably important to pharmaceutical chemistry, manufacturing, and control. Meantime polymorphs is a key factor for the quality of high-end drug and formulations. Polymorph prediction technology can effectively guide screening of trial experiments, and reduce the risk of missing stable crystal form in the traditional experiment. Polymorph prediction technology was firstly based on theoretical calculations such as quantum mechanics and computational chemistry, and then was developed by the key technology of machine learning using the artificial intelligence. Nowadays, the popular trend is to combine the advantages of theoretical calculation and machine learning to jointly predict crystal structure. Recently, predicting medicine polymorphs has still been a challenging problem. It is expected to learn from and integrate existing technologies to predict medicine polymorphs more accurately and efficiently.

OBJECTIVEThis study was to explore the cytotoxic effect and the related injury mechanism of deoxynivalenol (DON) on articular chondrocytes in human embryo.

METHODSArticular cartilage cells were isolated from knees of human embryo and cultured in DMEM/F12 medium. The cells of the 4th generation were divided into five groups and incubated with varying concentrations of DON as the followings: control group and group with DON of 0.1, 0.2, 0.4, 1.0 µg/ml. The effects of DON were observed 72 hours after incubation. Cell apoptosis was assayed by flow cytometry (FCM) with Annexin V-FITC/PI staining; MMP-13 and PGE2 were detected by ELISA kits; NO was measured by Griess assay with spectrophotometer. Inducible nitric oxide synthase (iNOS) and collagen II in cells were detected by FCM. The expression levels of iNOS, mRNA and collagen II mRNA were measured with RT-PCR.

RESULTSThe rates of cell apoptosis in DON groups were 6.78% - 19.05%, which were significantly higher than that in control (1.20%, F = 174.761, P < 0.05). The levels of NO in DON groups were 20.8 - 40.7 µmol/L, which were significantly higher than that in control (10.2 µmol/L, F = 91.966, P < 0.05). The levels of MMP-13 in DON groups were 0.25 - 0.56 µmol/L, which were significantly higher than that in control (0 µmol/L, F = 78.420, P < 0.05). The levels of PGE2 in DON groups were 3.2-20.6 µmol/L, which were significantly higher than that in control (11.6 µmol/L, F = 276.453, P < 0.05). The proportions of cells with positive iNOS in DON groups were 14.8% - 56.8% which were significantly higher than that in controls (7.1%, F = 214.614, P < 0.05). The proportions of cells with positive collagen II in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 56.7% and 52.7%, which were significantly lower than that in control (62.2%, F = 5.134, P < 0.05). The relative absorbance values of iNOS mRNA in DON groups were 1.07 - 1.33, which were significantly higher than that in control (0.62, F = 8.358, P < 0.05). The levels of collagen II mRNA in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 0.83 and 0.82, which were significantly lower than that in control (1.14, F = 7.887, P < 0.05).

CONCLUSIONDON could promote anabolism of NO in articular cartilage cells by which up-regulated the expression of PGE2 and MMP-13, which both promoted resolution of articular cartilage matrix such as collagen II. DON induced apoptosis in articular cartilage cells.

Cartilage, Articular ; cytology ; embryology ; Cells, Cultured ; Chondrocytes ; drug effects ; metabolism ; Dinoprostone ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; metabolism ; Nitric Oxide ; biosynthesis ; Trichothecenes ; toxicity

OBJECTIVETo offer both the accurate three-dimensional anatomical information and algorithmic morphology of perforators in the lower leg for perforator flaps design.

METHODSThe cadaver was injected with a modified lead oxide-gelatin mixture. Radiography was first performed and the images were analyzed using the software Photoshop and Scion Image. Then spiral CT scan was also performed and 3-dimensional images were reconstructed with MIMICS 10.01 software.

RESULTSThere are (27 +/- 4) perforators whose outer diameter > or = 0.5 mm ( average, 0.8 +/- 0.2 mm). The average pedicle length within the superficial fascia is (37.3 +/- 18.6) mm. The average supplied area of each perforator is (49.5 +/- 25.5) cm2. The three-dimensional model displayed accurate morphology structure and three-dimensional distribution of the perforator-to- perforator and perforator-to-source artery.

CONCLUSIONSThe 3D reconstruction model can clearly show the geometric, local details and three-dimensional distribution. It is a considerable method for the study of morphological characteristics of the individual perforators in human calf and preoperative planning of the perforator flap.

Arteries ; Cadaver ; Humans ; Image Processing, Computer-Assisted ; methods ; Imaging, Three-Dimensional ; Leg ; blood supply ; Perforator Flap ; blood supply ; Surgical Flaps

Adolescent ; Adult ; Ambulatory Surgical Procedures ; instrumentation ; methods ; Child ; Child, Preschool ; Female ; Humans ; Male ; Middle Aged ; Radio Waves ; Tonsillectomy ; instrumentation ; methods ; Young Adult