BACKGROUND: Tumor necrosis factor-α (TNF-α) is one of important cytokines to promote the maturation of dendritic cells. Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1 (sTNFR1) may arrest dendritic cells in an immature state and induce stable, long-term tolerance. OBJECTIVE: To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells. METHODS: Total RNA of human peripheral blood mononuclear cells was taken as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned to the lentiviral vectors pXZ208, and ligated to the enhanced green fluorescent protein (eGFP) reporter gene to establish lentiviral vector, called pXZ9-sTNFR1. DNA sequencing was performed for lentiviral vector identification. Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1. Viral titer was determined by eGFP expression. C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4. On day 5 of culture, immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant, sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression by Western blot analysis. Following sTNFR1 gene modification and lipopolysaccharide stimulation, the phenotype characteristics of dendritic cells were observed. RESULTS AND CONCLUSION: Recombinant plasmid pXZ9-sTNFR1 was successfully constructed. Twenty-four hours after 293 FT cell transfection, eGFP expression was observed and viral titer was over 10<'6> U/L. RT-PCR demonstrated that pXZ9-sTNFRl-transfected immature dendritic cells showed sTNFR1 positive expression. Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection. On day 5 of culture, dendritic cells expressed low level of class Ⅱ major histocompatibility complex (MHC Ⅱ), as well as CD40, CD86, CD80, molecules. However, following lipopolysaccharide stimulation, dendritic cells expressed high level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules, exhibiting the phenotype characteristics of mature dendritic cells. But after sTNFR modification, the expression level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules was not altered obviously. Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed, and recombinant lentiviral plasmids with high titer were acquired. Following high efficacy of lentiviral gene transfection, immature dendritic cells stably express sTNFR1 mRNA and protein, which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.

Objective Evaluation of cerebral blood flow in patients with transient ischemic attack (TIA) using cerebral CT perfusion imaging.Methods CT perfusion scan was performed on a consecutive series of 20 patients with clinical definite TIA.Following their initial CT scan at acute stage of TIA, patients underwent two repeat CT perfusion scanning of region of interest at acute stage and one month after symptom remission.Results Mild to moderate decrease in regional cerebral blood flow (rCBF) and unchanged or mildly decrease in regional cerebral blood volume (rCBV) were observed at acute stage in the majority cases.Normal cerebral perfusion was found in 12 cases and mild to moderate decrease of rCBF in 8 cases one month after TIA.During the one-year follow-up period, all of 12 cases with normal cerebral perfusion did not have recurrence while among 8 cases with mild to moderate decrease of rCBF at initial scan, 6 cases had recurrent TIA or cerebral infarction and 2 cases did not have recurrence.Patients with more severe cerebral perfusion defects usually had a shorter interval time between two attacks.Conclusions Intensive intervention should be performed on patients with severe and long lasting decrease of cerebral perfusion.

Objective To study the inhibitory effect of curcumin on the proliferation,migration and invasion of non-small cell lung cancer cell A549,and to discuss further if it is closely related to the expression of c-Jun N-terminal kinase (JNK) and relative protein p38.Methods A549 cells were cultured by conventional method,and then treated with different concentration of curcumin (10,20,40,80 μmol · L-1).The proliferation,migration and invasion of A549 cells were measured by real-time cellular analysis (RTCA).The expression levels of JNK,p-JNK,p38 and P-p38 were detected by real-time PCR and Western blotting.Results Curcumin showed an antiproliferation effect against A549 cells with IC50 =40 μmol · L-1,and curcumin exhibited obviously inhibitory effect on the migration and invasion of A549 cells.Additionally,compared with control group,curcumin suppressed the expression of JNK and p38 at the gene level,and significantly inhibited the expression of JNK,P-JNK,p38 and p38 (P＜0.05) at the protein level.Conclusion These results demonstrated that curcumin can inhibit the proliferation,migration and invasion of A549 cells via reducing the level of JNK,p38 phosphorylation,and blocking JNK signal transduction pathway.

Objective To investigate the protective role of Sestrin2 overexpression in hypoxia and re-oxygenation injury (H/R) injury of hippocampal neurons and its mechanism in rats.Methods Neurons were enzymatically isolated from hippocampi of newborn Sprague-Dawley rats (less than 24 h old) and culturedinvitro. These neurons were randomly assigned into 4 groups (n=20) using a random number table: control group, H/R group, vector group and Sestrin2 overexpression group. The hippocampal neurons were seeded in 6-well plates at a density of 2×104 cells/mL; neurons in the latter two groups were transfected with lentiviruses containing empty vector andSestrin2overexpressed genes, respectively; the hippocampal neurons in the later three groups were subjected to oxygen-glucose deprivation (OGD) for 6 h followed by restoration of O2 supply for 20 h. The reactive oxygen species (ROS) content was detected by Reactive Species Assay Kit, and the ATP concentration was detected by ATP Assay Kit. Cell apoptosis rates were measured by flow cytometry. The levels of Sestrin2, dynamin-related protein 1 (Drp1), Fis1, and apoptosis-related proteins Bcl-2, Bax and cytochrome C (Cyt C) were measured by Western blotting. The ratio of Bcl-2 to Bax was calculated. The ultrastructure of mitochondria was observed by transmission electron microscopy.Results As compared with control group, H/R group had significantly lower ATP concentration, Bcl-2 protein expression and ratio of Bcl-2 to Bax ([11.15±0.42] nmol/mg proteinvs. [5.30±0.39] nmol/mg protein; 2.20±0.26vs. 0.91±0.02; 6.46± 0.41vs. 1.04±0.05), statistically higher average fluorescence intensity of ROS and cell apoptosis rate (152.41±17.38vs. 1530.00±14.69; 3.77％±0.74％vs. 56.57％±1.35％), and significantly higher protein levels of Sestrin2, Drp1, Fis1, Bax and Cyt C (0.66±0.06vs. 1.11±0.20; 0.48±0.03vs. 1.16±0.07; 1.14± 0.09vs. 2.47±0.09; 0.34±0.03vs. 0.88±0.04; 0.17±0.03vs. 0.30±0.03,P<0.05); what's more, the structure of mitochondria was obviously destroyed in I/R group. As compared with H/R group, Sestrin2 overexpression group had significantly increased ATP concentration, Sestrin2 and Bcl-2 protein expressions and ratio of Bcl-2 to Bax ([8.95±0.27] nmol/mg protein; 2.67±0.07; 1.80±0.19; 3.95±0.28), significantly lower average fluorescence intensity of ROS and cell apoptosis rate (337.27±15.32; 10.33％±2.60％), and statistically lower protein levels of Drp1, Fis1, Bax and Cyt C (0.43±0.02; 1.11±0.08; 0.45± 0.02; 0.17±0.02,P<0.05); the structure of mitochondria was relatively completed in Sestrin2 overexpression group.Conclusion Sestrin2 overexpression can inhibit mitochondrial fission, reduce accumulation of reactive oxygen species, improve mitochondrial energy metabolism and block mitochondria mediated apoptosis pathway, thereby alleviating I/R injury of rat hippocampal neurons.

Objective To investigate the influence of curcumin and its analogue H8 on glucose and lipid metabolism disorder in db/db mice. Methods The type 2 diabetes mouse model (db/db mice) was intragastrically administrated with curcumin and analogue H8 for 8 weeks.The blood biochemical indexes were measured.The expression of PEPCK and G6Pase mRNA was detected by real-time PCR in liver tissues.The expression of PEPCK and G6Pase protein was detected by Western blotting. Results Curcumin analogue H8 reduced blood glucose and lipids in db/db mice (P<0.01) and improved liver function related enzymes significantly.The levels of PEPCK and G6Pase mRNA in db/db mice were significantly decreased (P<0.01) and the expression levels of PEPCK and G6Pase protein were significantly decreased(P<0.01). Conclusion Curcumin analogue H8 improves the glucose and lipid metabolism disorder in db/db mice,and it is related to inhibiting the expression of PEPCK and G6Pase gene and protein.

Objective To investigate the expression of soluble vascular endothelial growth factor receptor 1 (sFlt1) in human umbilical cord-derived mesenchymal stem cells (HUCMSC) under the condition of inflammatory cytokine and co-culture with rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs).Methods ① HUCMSC were cultured alone or stimulated by Tumour necrosis factor (TNF)-αt,interferon (IFN)-γor the combination of TNF-α and IFN-γ.The sFh1 levels of each group were detected by enzyme-linked immunosorbent assay (ELISA) at 24 h,48 h and 72 h respectively.② RA-FLSs and HUCMSC were cocultured in a transwell system with both of them cultured alone as control for 72 hours.ELISA was used to detect sFlt1 levels in each group.T test was used for comparison between two groups,and ANOVA was used to compare multi-group variables.Results ① The sFlt1 levels of TNF-α + IFN-γ group were significantly higher than those of the control group at 24 h and 48 h (24 h (5.4±0.4) ng/ml vs (2.8±1.7) ng/ml,t=0.942,P=0.026;48 h (7.2±0.8) vs (4.3±1.0) ng/ml,t=4.285,P=0.005),while that at 72 h was not significantly different [(9.1±1.7) ng/ml vs (7.0±1.4) ng/ml,t=0.683,P=0.52].And no significant difference in sFlt1 levels between control group and TNF-α group or IFN-γ stimulation group were observed.② Compared with RA-FLSs,the expression of sFlt1 in HUCMSC was significantly higher (8.19±3.64) ng/ml vs (0.19±0.08) ng/ml,t=7.280,P＜0.01].After co-cultured with RA-FLSs and HUCMSC,the sFlt1 concentration of the co-culture group was significantly higher than that of HUCMSC group [(17.26±6.92) ng/ml vs (8.19±3.64) ng/ml,t=3.985,P=0.000 7].Conclusion The sFlt1 expression of HUCMSC is up-regulated by inflammatory cytokine TNF-α combined with IFN-γ.RA FLSs may promote the co-cultured HUCMSC to increase sFlt1 expression by secreting inflammatory factors.

Objective: To investigate the efficacy and safety of rapamycin in children with tuberous sclerosis complex (TSC) associated renal disease. Methods: A prospective self-control study was conducted. The clinical data of 92 children diagnosed with tuberous sclerosis complex associated kidney disease at the People′s Liberation Army General Hospital from January 2011 to January 2019 were collected. The long-term rapamycin treatment for all patients initiated at 1 mg/(m²·d), which was gradually adjusted to reach a blood concentration of 5-10 μg/L. The changes of the maximum diameter of renal lesions in children after rapamycin treatment were observed and analyzed with Wilcoxon test. Results: Ninety-two children, including 52 males and 40 females, who met the criteria were analyzed. Sixty patients had only renal angiomyolipoma(RAML), while 24 patients had only multiple renal cysts(MRC), and 8 patients had both lesions. The age of TSC diagnosis was 16.0 (7.0, 42.0) months, and the age of initial treatment with rapamycin was 63.5 (21.0, 103.0) months. The follow-up lasted for 12.0 (4.0, 23.0) months. Sequencing of TSC1 and TSC2 genes was performed in 54 children with TSC, including 3 patients (6%) with mutations in TSC1 gene and 51 patients (94%) with mutations in TSC2 gene. The maximum RAML diameter before treatment was 7.0 (4.0, 9.0) mm. The best effect reached at 3 months of treatment, with the diameter of 4.0 (0,7.0) mm. The maximum diameters at 6 months, 1 year and 1-2 years were 5.0 (0,9.8) mm, 5.0 (1.5, 8.5) mm, 5.5 (3.0, 9.0) mm, respectively, and were significantly different from the baseline (Z=-2.404,-2.350,-2.750,P=0.016,0.019,0.006, respectively). The maximum diameter after 2-3 years, and ≥3 years were 5.0 (3.9,7.0) mm and 6.0 (1.0, 11.0) mm, without significant difference from the baseline (Z=-0.856,-0.102,P=0.393,0.919, respectively).The maximum diameters of MRC after 3 months, 6 months, 1 year,1-2 years, 2-3 years, and ≥3 years were 11.0 (5.0, 14.0) mm,3.0 (0.0,11.0) mm,5.0 (0,21.0) mm,0 (0,14.0) mm,0 (0,10.0) mm, and 0 (0,18.3) mm, respectively, but were not significantly different rom the baseline (7.0 (5.0, 15.7) mm)(Z=-0.944,-1.214,-1.035,-1.896,-1.603,-1.214,P=0.345,0.225,0.301,0.058,0.109,0.225, respectively).Twenty-nine patients (32%) had oral ulcers during the entire treatment period, and no serious adverse reactions were observed. Conclusions Rapamycin could decrease the diameter of TSC-related RAML, but could not inhibit the growth of cysts. It is well tolerated in the treatment of renal diseases associated with tuberous sclerosis complex.

Objective:To compare the application effect of domestic and imported intravenous radiofrequency closure system in the treatment of primary varicose veins of lower extremities.Methods:This single-center prospective, non-inferiority randomized controlled trial was performed in the Department of Vascular Surgery, the Fourth Affiliated Hospital, Zhejiang University School of Medicine from January 2021 to January 2022. Patients with primary varicose veins of lower extremities who met the ataxation criteria were randomly assigned to the experimental group(domestic novel venous radiofrequency closure system) or the control group(imported venous radiofrequency closure system) in a ratio of 1∶1. The two groups of subjects were compared in terms of target vein closure rate, technical success rate, system operation performance, incidence of adverse events and incidence of serious adverse events(SAE) within 6 months after surgery. Quantitative data were compared by Mann-Whitney U test, and categorical data were compared by χ2 test and non-inferiority test. Results:A total of 80 subjects were included in the trial (41 in the experimental group and 39 in the control group), including 27 males and 53 females, aged ( M(IQR)) 55(23) years (range:40 to 78 years). There were 48 cases of left lower limb and 32 cases of right lower limb. The technical success rate and system control performance between the groups were 100%.The incidence of adverse events (58.5% (24/41) vs. 61.5% (24/39), χ2=0.075, P=0.784), and the incidence of SAE (7.3% (3/41) vs. 5.1% (2/39), χ2=0.163, P=0.686) within 6 months after surgery in experimental group and control group had no statistical significance. There was one device-related adverse event in each of the two groups. In the experimental group, one patient developed endovenous heat-induced thrombosis after surgery and recovered after taking rivaroxaban tablets. One patient in the control group had pain in the upper right thigh for more than 1 day after operation, which was cured after using analgesic cream. No device-related SAE occurred. The venous closure rate of the experimental group was 100% (38/38) at 6 months after surgery, and that of the control group was 97.4% (37/38). The difference between the two groups was 2.63% (95% CI:-3.19 to 8.45, Z=4.865, P<0.01), and the 95% CI lower limit of the difference in target venous closure rate between two groups was greater than the non-inferiority threshold of -10.00%. Conclusion:The early application effect of the new domestic intravenous radiofrequency closure system in patients with primary varicose veins of lower extremities is in line with expectations, it is not inferior to the imported system.

Objective:To compare the application effect of domestic and imported intravenous radiofrequency closure system in the treatment of primary varicose veins of lower extremities.Methods:This single-center prospective, non-inferiority randomized controlled trial was performed in the Department of Vascular Surgery, the Fourth Affiliated Hospital, Zhejiang University School of Medicine from January 2021 to January 2022. Patients with primary varicose veins of lower extremities who met the ataxation criteria were randomly assigned to the experimental group(domestic novel venous radiofrequency closure system) or the control group(imported venous radiofrequency closure system) in a ratio of 1∶1. The two groups of subjects were compared in terms of target vein closure rate, technical success rate, system operation performance, incidence of adverse events and incidence of serious adverse events(SAE) within 6 months after surgery. Quantitative data were compared by Mann-Whitney U test, and categorical data were compared by χ2 test and non-inferiority test. Results:A total of 80 subjects were included in the trial (41 in the experimental group and 39 in the control group), including 27 males and 53 females, aged ( M(IQR)) 55(23) years (range:40 to 78 years). There were 48 cases of left lower limb and 32 cases of right lower limb. The technical success rate and system control performance between the groups were 100%.The incidence of adverse events (58.5% (24/41) vs. 61.5% (24/39), χ2=0.075, P=0.784), and the incidence of SAE (7.3% (3/41) vs. 5.1% (2/39), χ2=0.163, P=0.686) within 6 months after surgery in experimental group and control group had no statistical significance. There was one device-related adverse event in each of the two groups. In the experimental group, one patient developed endovenous heat-induced thrombosis after surgery and recovered after taking rivaroxaban tablets. One patient in the control group had pain in the upper right thigh for more than 1 day after operation, which was cured after using analgesic cream. No device-related SAE occurred. The venous closure rate of the experimental group was 100% (38/38) at 6 months after surgery, and that of the control group was 97.4% (37/38). The difference between the two groups was 2.63% (95% CI:-3.19 to 8.45, Z=4.865, P<0.01), and the 95% CI lower limit of the difference in target venous closure rate between two groups was greater than the non-inferiority threshold of -10.00%. Conclusion:The early application effect of the new domestic intravenous radiofrequency closure system in patients with primary varicose veins of lower extremities is in line with expectations, it is not inferior to the imported system.

Objective:To explore the effect of comprehensive follow-up mode in patients with chronic obstructive pulmonary disease (COPD) .Methods:From February to June 2019, a total of 80 COPD patients in The First Affiliated Hospital of Guangxi Medical University were enrolled and divided into the control group and the comprehensive follow-up group (40 cases in each group) . All patients were given home rehabilitation program guidance and health education. The control group was followed up using telephone, while the comprehensive follow-up group were followed up by telephone combined with Internet which lasted for one year. The differences of pulmonary function, motor function, compliance and recurrence between the two groups were compared.Results:One year after discharge, percentage of forced vital capacity to the predicted value (FVC%) , percentage of forced expiratory volume in one second to the predicted value (FEV1%) , 6 minutes walking distance, SF-36 quality of life and rehabilitation compliance rate in the comprehensive follow-up group were higher than those of the control group, while CAT score, mMRC score, number of patients with acute attack of COPD and recurrence rate were lower than those of the the control group, and the differences were statistically significant ( P<0.05) . Conclusions:For CPOD patients, the comprehensive follow-up mode can effectively reduce the 1-year recurrence rate, promote the recovery of respiratory and motor functions, and improve compliance and quality of life.