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.

AIM:To explore the association between single nucleotide polymorphism in exon 33 (E33SNP) of thyroglobulin gene and Graves ’ disease ( GD) relapse after antithyroid drug ( ATD) withdrawal .METHODS:The healthy controls (232 cases) and GD patients with discontinued treatment (243 cases) were selected.According to the time of re-lapse, the GD patients were divided into A, B and C subgroups.The A group contained 77 cases of relapse within 1 year, B group contained 86 cases of relapse 1~2 years after treatment and C group contained 80 cases without recurrence within 2 years.The genotypes of E33SNP were identified by RT-PCR.The genotype ratio of thyroglobulin between control group and observation group was comparatively analyzed , and the levels of thyroid-stimulating hormone ( TSH) , free triiodothyro-nine (FT3), free thyroxine (FT4) and thyrotropin receptor antibody (TRAb), ophthalmopathy and goiter size in A , B and C subgroups in different genotype GD patients were investigated .Moreover , cumulative efficiency for patients with different genotypes in the observation group after ATD treatment within 2 years were analyzed .RESULTS:The genotype of E33SNP between observation group and control group had no significant difference , but a significant difference between A , B and C subgroups was observed (P<0.05).The levels of TSH, FT3 and FT4, and goiter size of the patients with different geno-types had no significant difference , while the TRAb levels and ophthalmopathy presented a significant difference ( P <0.05).In addition, the cumulative efficiency within 2 years for GD patients with E33SNP T/T, E33SNP T/C and E33SNP C/C genotypes was 61.8%, 42.6% and 21.3%, respectively, all with significant differences (P<0.05).CONCLU-SION:The GD patients with E33SNP C/C genotype have significantly higher TRAb level and ophthalmopathy rate than those in the patients with E33SNP C/T and E33SNP C/C genotypes, and are more likely to relapse after ATD treatment . The GD patients with E33SNP T/T genotype show a lower recurrence rate .Therefore, combination treatment or other treat-ment modalities may be more reasonable for the GD patients with E 33SNP C/C genotype.

Objective:To explore the effect of self-designed fixed support in the postoperative nursing of abdominal flap repair.Methods:From January 2016 to December 2019, 47 patients with hand tissue defects undergoing abdominal skin flap repair in Shajing People′s Hospital, Guangzhou Medical University were divided into control group (23 cases) and observation group (24 cases) by random digits table method. The control group was treated with traditional methods, and the observation group was treated with self-designed fixed support. The wound healing of abdominal flap, the wound tearing after flap operation, pedicle ulcer after flap operation and the patients' nursing satisfaction were compared between the two groups.Results:There were 7 cases of primary healing and 16 cases of secondary healing in the control group, 18 cases of primary healing and 6 cases of secondary healing in the observation group. The difference between the two groups was statistically significant ( χ2 value was 9.368, P<0.01). The incidence rate of the wound tearing after flap operation was 26.1% (6/23) in the control group and 0 (0/24) in the observation group. The difference between the two groups was statistically significant ( χ2 value was 7.177, P<0.05). The incidence rate of pedicle ulcer after flap operation was 69.6% (16/23) in the control group and 33.3% (6/23) in the observation group. The difference between the two groups was statistically significant ( χ2 value was 6.170, P<0.05). The patients' nursing satisfaction was (76.8±8.4)points in the control group and (90.4±6.5) points in the observation group.The difference between the two groups was statistically significant (t value was -4.640, P<0.01). Conclusions:The use of fixed support after the operation of abdominal flap is helpful to the wound healing, reduce the occurrence of wound tear and pedicle ulcer, and improve the satisfaction of nursing, which is worth popularizing.

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 explore the clinical effect of the second toenail flap with the dorsal bone of the phalange in repairing the defect of fingernail bed.Methods:From January, 2012 to June, 2019, 10 patients with large area of fingernail bed defect were treated by the second toenail flap with bone on the back of the phalanx. The survival of the flap was observed after the operation, and the fracture healing, the shape of the nail and the flexion and extension function of the finger joint were observed in the outpatient follow-up.Results:All flaps of the second toenail survived. The average follow-up period was 8 (4-12) months. The fractured ends of 10 patients' phalanges healed well without nonunion, good appearance of toenail and deformity of toenail. The recovery of hand function was evaluated according to the evaluation standard of upper limb function of Hand Surgery Society of Chinese Medical Association, 9 cases were excellent, and 1 case was good.Conclusion:The second toenail flap with dorsal bone of the phalanx preserved is easy to cut, simple to operate, and has good clinical effect. It is a good method to repair the defect of the fingernail bed.

Objective:To explore the application effect of video education combined with Teach-back in the treatment of patients with chronic periodontitis.Methods:Using non-simultaneous experimental research methods, From January to August in 2019, 41 patients with chronic periodontitis who underwent implant restoration in Shanghai Tenth People′s Hospital was selected by convenience sampling method as the control group, and implemented routine oral health education. From September 2019 to May 2020, 42 patients with chronic periodontitis who underwent implant restoration in Shanghai Tenth People′s Hospital were the observation group, and implemented video education combined with Teach-back. Compare the two groups of oral health care self-efficacy, periodontal clinical indicators, and the incidence of peri-implant mucositis.Results:Comparing 6 months and 12 months after implant restoration, the total scores of oral health self-efficacy and regular oral visits, correct brushing, and balanced diet in the observation group were 66.31 ± 4.32 and 67.19 ± 4.65, 22.04 ± 1.35 and 21.69 ± 1.82, 21.73 ± 1.65 and 22.64 ± 1.82, 22.54 ± 1.62 and 22.86 ± 1.74 respectively, which were higher than the control group 53.93 ± 4.78 and 54.09 ± 5.67, 17.02 ± 2.58 and 17.43 ± 2.16, 17.65 ± 1.74 and 18.54 ± 2.36, 19.14 ± 2.13 and 18.12 ± 2.58, the difference between the two groups at the two time points were statistically significant ( t values were 6.52-12.39, all P<0.05). And the PLI, mSBI, and PIS scores of the observation group were 0.80 ± 0.17 and 0.75 ± 0.14, 0.79 ± 0.19 and 0.81 ± 0.18, 2.08 ± 0.45 and 2.10 ± 0.53, respectively, which were lower than the control group 0.92 ± 0.19 and 0.99 ± 0.21, 1.03 ± 0.17 and 1.16 ± 0.21, 2.45 ± 0.68 and 2.62 ± 0.61, the difference between the two groups at the two time points were statistically significant ( t values were 2.93-8.16, all P<0.05). 12 months after, the incidence of mucositis around implants in the observation group was 7.14%(3/42), which was lower than 26.83%(11/41)in the control group ( χ2=5.73, P<0.05). Conclusions:Video education combined with Teach-back can improve the self-efficacy of oral health care during implant restoration treatment in patients with chronic periodontitis, improve oral health care behavior, thereby improving the periodontal condition around the implant and reducing the incidence of mucositis.

The purpose of the study is to explore the in vitro refolding protocol for humanized anti-CTLA4-scFv expressed in E. coli. The inclusion bodies are denatured and then diluted or dialyzed into a refolding buffer. We analyzed several factors affecting the refolding yield, including refolding time, temperature, and redox environment. The refolded target proteins are analyzed by non-reducing SDS-PAGE, and the concentration of refolded proteins are examined by Bio-Rad Dc Protein Assay kit. The result shows that a high yield of the protein with natural conformation can be acquired in the condition of 0.15 mol x L(-1) sodium chloride, 50 mmol x L(-1) Tirs-HCl, pH 8.0 buffer containing 1 micromol x L(-1) reduced glutathione and 3 micromol x L(-1) oxidized glutathione. The refolding time is 48 to 54 h at 4 degrees C. 28 mg refolded proteins are produced from 3.9g E. coli.
Antibodies, Monoclonal, Humanized ; chemistry ; CTLA-4 Antigen ; chemistry ; Escherichia coli ; metabolism ; Humans ; Inclusion Bodies ; chemistry ; Protein Folding ; Single-Chain Antibodies ; chemistry

Objective To analyze the serotypes of enteroviruses(EVs) isolated from patients with influenza-like illness in Beijing in 2017. Methods Oropharyngeal swab specimens were collected from pa-tients with influenza-like illness in eight districts of Beijing from July 2017 to October 2017. EVs were detec-ted by real-time PCR. Specific primers were synthesized and used to amplify the VP1 fragments of EVs. PCR products were sequenced and the results were compared with the reference sequences by using Basic Lo-cal Alignment Search Tool(BLAST) to identify the serotypes of isolated EVs. Results A total of 666 spec-imens were collected and 91 (13.66%) were positive for EVs. VP1 sequences of 66 EVs were successfully amplified and BLAST analysis revealed that these strains belonged to 14 serotypes,including seven serotypes of EV-A species,six serotypes of EV-B species and one serotype of Rhinovirus species. The predominant se-rotypes were CVA2 and CVA6. Eight out of 14 CVA6 strains that were collected in Shunyi District shared high homology. All seven CVB5 strains were collected in Shijingshan District and grouped into one cluster. Conclusion EVs causing influenza-like illness in Beijing in 2017 belonged to 14 serotypes and CVA2 and CVA6 were the predominant serotypes.

Objective: To investigate the status of immunization of National Immunization Program (NIP) and its adverse reaction rate in children with tuberous sclerosis. Method: Questionnaire survey was adopted to identify the vaccination coverage and its adverse events; 72 cases of children with tuberous sclerosis and 78 normal controls (healthy children completing age-appropriate NIP) admitted to Chinese People′s Liberation Army General Hospital from December 2014 to November 2015 were involved into this study. Result: The age-appropriate NIP coverage rate of tuberous sclerosis was 36%(26/72). The coverage rate of bacillus calmette-guerin (BCG), hepatitis B vaccine 1^st to 3^rd doses (HepB1-3), oral poliovaccine 1^st dose (OPV1), diphtheria, pertussis and tetanus 1^st dose (DPT1), DPT1-3, meningococcal polysaccharide vaccine group A (MPVA), measles amd rubella vaccine/measles vaccine 1^st dose (MRV/MCV1), and Japanese encephalitis vaccine 1^st dose (JEV1) were 100%(72 cases), 75%(51 cases), 97%(66 cases), 91%(62 cases), 82%(56 cases), 66%(45 cases), 69%(42 cases), and 61%(37 cases) respectively. The reasons why the children did not complete the vaccination plan were that parents were concerned about vaccination-induced seizures or seizures had not been controlled. Among 72 children with TSC, the rate of adverse events or suspected adverse events after vaccination was 17% (12 cases), which was higher than the normal control children (2 cases, 3%) (χ²=8.799, P<0.05). The main adverse events were seizure events, which accounted for 92%(11 cases). Conclusion The age-appropriate NIP coverage rate among children with tuberous sclerosis is low. The high incidence of adverse events may be associated with a fact that there are some nervous system abnormalities in cases with tuberous sclerosis. TSC children vaccination is relatively safe, with no serious adverse events.

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.