Objective:To reduce the immunogenicity of vaccinia virus vector by replacing the D8L region, which is a neutralizing antibody epitope in vaccinia virus, with an exogenous gene.Methods:A gene fragment encoding influenza virus hemagglutinin (HA) was inserted into the D8L region to replace it using homologous recombination technique. Then, a recombinant vaccinia virus influenza vaccine was constricted. A recombinant vaccinia virus vaccine with the TK region expressing HA was used as a control. The expression of HA was validated by Western blot. BALB/c mice were immunized with the vaccines and the serum antibody titers two weeks after each immunization were evaluated by ELISA and hemagglutination inhibition assay. The protective efficacy of the recombinant vaccinia virus was assessed through a challenge experiment.Results:Western blot confirmed the successful expression of HAD8L protein in the constructed recombinant vaccines. ELISA and hemagglutination inhibition assay showed that after the primary immunization, the anti-HA antibody titer induced by the recombinant vaccinia virus with D8L region mutation was slightly higher than that induced by the vaccine with TK region mutation, and the difference was statistically significant with the increase of immunization times ( P<0.05). The recombinant vaccinia virus with D8L region mutation showed significantly lower immunogenicity than the recombinant virus with TK region mutation after the primary immunization, but there was no significant difference between them with the increase of immunization times ( P>0.05). After H1N1pdm challenge, no virus was detected in the mice immunized with the recombinant vaccinia virus with D8L region mutation and the mice showed mild lung inflammation and less tissue damage. Conclusions:This study indicated that inserting exogenous genes into the D8L region of the neutralizing antibody epitope in the vaccinia virus vector could help to reduce the immunogenicity of the vector itself and enhance the immunogenicity of the exogenous genes. This provided a reference for the use of the vaccinia virus vector as a delivery tool in the field of vaccines or gene therapy.

Gingival pigmentation(GP) manifests as dark pigmentation spots, such as black or brown spots, in the gums. It is mostly caused by the deposition of melanin particles secreted by melanocytes on the gingival epithelium. The influencing factors may be divided into two categories, exogenous and endogenous. Exogenous factors include heavy metals, tattoos, smoking or drug use, and endogenous factors are related to certain diseases. The clinical grading of GP helps make a reasonable assessment of the necessity of treatment and prognosis. The Dummett-Gupta oral pigmentation index is a commonly used grading method, and the new grading method formed by combining the etiology and clinical manifestations described the patient’s situation more comprehensively. It is necessary to ask for a detailed medical history, complete examination, and correctly differentiate between physiological GP and GP caused by pathological state. Laser treatment is the currenttreatment with a better treatment effect and higher patient acceptance, and it is more comfortable and convenient, including diode laser, Er: YAG laser, and Nd: YAG laser, etc. This article summarizes the formation factors, clinical manifestations and treatment methods of GP to provide ideas for the clinical diagnosis and treatment of GP.

From June 2017 to September 2019, 5 patients who were diagnosed as having benign severe pyloric stenosis underwent fully covered stent placement using a new stenting method at the First Affiliated Hospital of Zhengzhou University. Five patients were performed successfully without serious complications. Postoperative barium meal radiograph revealed that the stents were in good location and the acontrast agent passed smoothly. The liquid diet was commenced 1-3 days after surgery. No vomiting, abdominal pain and diarrhea occurred. During the follow-up, all the patients had improved weight and nutritional status. Four patients underwent stent removal 3-4 months postoperatively. One patient was found that the stent had migrated to stomach 3 months after discharge. After removing the stent, balloon dilation and mucosal resection was performed for the mild pyloric stenosis. All 5 patients had an additional follow-up of 3 months, and no symptoms and restenosis occured. These preliminary results showed that the new stenting method of fully covered stent placement is feasible, safe and effective in the treatment of benign pyloric stenosis.

OBJECTIVE：To preliminarily optimize the preparation technology of Ligustrazine pellicle ，and to study its in vitro percutaneous permeation characteristics. METHODS ：With the amounts of PVA- 124，ethyl alcohol ，glycerin，tween-80 and azone as factors ，single factor experiment was used to optimize the Ligustrazine pellicle matrix formulation ；modified scoring standard was used to evaluate the film formation time ，film formation ability ，ductility，uniformity and the presence of bubble. On the basis of the optimal matrix formulation ，the pellicle with different loading amount of ligustrazine （300，250，200，150，100，50 mg/mL） was prepared and its maximum loading amount was investigated. HPLC method was adopted to determine the content of ligustrazine，and methodology investigation was conducted. Isolated back skin of rats were collected ，the percutaneous permeation test was conducted for high ，medium and low loading amount （100，75，50 mg/mL）of Ligustrazine pellicle. At 15，30，45，60， 75，90，120，150，180 min，the sample was taken and the permeation rate of ligustrazine was calculated. RESULTS ：When the amounts of PVA- 124，ethyl alcohol，glycerin，tween-80 and azone were 2.5 g，7.0 mL，1.97 mL，0.07 mL，0.28 mL（in terms of 50 mL formulation amount ），the optimal matrix formulation of Ligustrazine pellicle was obtained. The maximum drug loading amount of ligustrazine was 100 mg/mL. The linear ranges of ligustrazine was 3.125-100 μg/mL. The specificity，precision， reproducibility，recovery and stability investigation of content determination method of ligustrazine were all in line with the requirements（RSD＜2％）. The permeation rate of high ，medium and low loading amount of Ligustrazine pellicle were 608.42， 384.19，158.20 μg（/ cm2·h）. CONCLUSIONS ：According to the optimized formulation ，the prepared Ligustrazine pellicle had a short film forming time ，stable and re liable quality ； the drug-loading amount was up to 100 mg/mL. The pellicle with drug-loading amount of 75 mg/mL had reached the penetration rate range of effective plasma concentration of ligustrazine treatment.

Objective: To investigate the relationship between gut microbiota structure and biochemical changes in patients with different types of nonalcoholic fatty liver disease (NAFLD), in order to provide evidence for clinical diagnosis and prevention of NAFLD. Methods: Forty-eight NAFLD cases (NAFLD group), 40 NAFLD cases with type 2 diabetes mellitus (NAFLD combined with type 2 diabetes mellitus group) and 30 healthy cases (healthy group) were randomly enrolled, and their body mass index, serum alanine aminotransferase, aspartate aminotransferase, total bilirubin, total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein and uric acid were measured. Serum levels of TNF-alpha and fasting insulin were measured using ELISA, and then insulin resistance index was calculated. The gut microbiota of three groups of subjects was detected using 16S rDNA-based high-throughput sequencing. Lastly, the correlations between the various factors were analyzed. The comparison among groups was conducted by 2 test, and one-way ANOVA was used for comparison among groups with normal distribution and homogeneity of variance. Furthermore, the LSD method was used to compare the two groups. K-W rank sum test was used for comparison among groups without normal distribution or homogeneity of variance. Results: Body mass index, aspartate aminotransferase, triglyceride, total cholesterol, low density lipoprotein, uric acid, tumor necrosis factor-alpha, fasting insulin and insulin resistance index of NAFLD group were higher than healthy group, while the high-density lipoprotein was lower in the healthy group, and the difference was statistically significant (P< 0.05). Compared with NAFLD group, the life expectancy, fasting blood glucose and insulin resistance index of NAFLD combined with type 2 diabetes mellitus group were higher, while the body mass index, aspartic acid aminotransferase, total cholesterol and HDL levels were decreased, and the difference was statistically significant (P< 0.05). NAFLD group (P= 0.016) had decreased abundance of firmicutes than healthy group, and the abundancy of the firmicutes in the NAFLD combined with type 2 diabetes group was significantly lower (P< 0.001). The abundance of bacteroidetes in NAFLD combined with type 2 diabetes group was higher than healthy group, and the difference was statistically significant (P= 0.006). At the "genus level," the abundance of Roseburia and Subdoligranulum in the NAFLD group was decreased, while the Roseburia in the NAFLD group with type 2 diabetes group was significantly lower (P< 0.05). In addition, the abundance of Faecalibacterium, Blautia, Anaerostipes and Fusicatenibacter in NAFLD combined with type 2 diabetes group was lower than healthy group, and the difference was statistically significant (P< 0.001). Fusicatenibacter, Blautia, Anaerostipes, Faecalibacterium, and Roseburia were negatively correlated with fasting blood glucose and insulin resistance index levels (r< 0,P< 0.05), and positively correlated with high-density lipoprotein levels (r> 0,P< 0.05). Fusicatenibacter was negatively correlated with tumor necrosis factor-alpha (r= -0.211,P= 0.044), and Lachnoclostridium was positively correlated with body mass index, alanine aminotransferase, aspartate aminotransferase levels (r> 0,P< 0.05). Fusobacterium was positively correlated with aspartate aminotransferase level (r= 0.245,P= 0.019). Escherichia-shigella was positively correlated with fasting blood glucose, low-density lipoprotein, alanine aminotransferase, aspartate aminotransferase levels (r > 0,P< 0.05). Megamonas was negatively correlated with high-density lipoprotein levels (r= -0.231,P= 0.027). Conclusion A structural change of gut microbiota had occurred in patients with NAFLD, suggesting changes in some of these bacterial genuses had relation to insulin resistance and inflammatory response, which may become a new target for the treatment of NAFLD.

Objective:To construct and identify the monoclonal antibody of ATP synthase beta subunit (ATP5B) with high purity, and to lay foundation for further study.Methods:The ATP5Bgene was amplified by PCR and cloned into the pET28avector and transformed into E.coli BL21 (DE3) .The protein expression was induced by IPTG and then the fusion protein was purified by nickel affinity chromatography column.The protein purity was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .Three female Balb/C mice were immunized with purified fusion protein and the tail vein blood was taken to detect the titer of ATP5Bantibody by indirect enzyme-linked immunosorbent assay (ELISA) .The spleen cells from the immunized mice with the highest serum titer were mixed with the SP2/0cells to establish the hybridoma cells and the fused cells were screened by indirect ELISA and monoclonally cultured.Karyotype analysis were performed in the positive cells.The hybridoma cells were intraperitoneally injected into 12weeks old BALB/C mice to estabilish the ascites models.The titer of ascites was detected by indirect ELISA.The purity of the antibody was detected by SDS-PAGE.The antibody subtype was detected by ELISA.Results:After PCR amplification, a specific band of 1 455bp was obtained, and the pET28aempty vector was ligated to obtain a recombinant pET28a/ATP5Bvector.The target protein was expressed in the IPTG-induced bacteria solution;the SDS-PAGE results showed that the protein band was found at51 000.The indirect ELISA results showed that the serum titer of the venous blood of immunized mice was up to1:64 000.In karyotype analysis, the total number of chromosomes in hybridoma cells was about the sum of myeloma cells and normal mouse spleen cells.The mouse ascites was prepared with the hybridoma cell line, and the highest titer of the antibody was 1:240 000.The subtype of the monoclonal antibody produced by the hybridoma cells was IgG1.Conclusion:The monoclonal antibody against ATP5Bprotein is successfully prepared by cloning, expressing and purifying the recombinant protein.

Objective To study the incidence of acute renal injury ( AKI), related clinical risk factors and recent prognosis in critically ill neonates. Methods The study was a retrospective analysis from January 2016 to December 2016 in the neonatal care department of General Hospital of Shenyang Military Region and the First Hospital Affiliated to China Medical University. We collected the perinatal data,clinical data,biochemical indexes and short-term prognosis. Logistic regression analysis was used to identify the risk factors. Results The incidence of AKI in critically ill neonates was 13. 11%(32/244),and the probability of poor prognosis was 62. 50%(20/32). Gestational age,birth weight,1 min and 5 min Apgar score,con-sciousness,mechanical ventilation,blood pH,blood glucose,blood urea nitrogen,white blood cell and red cell distribution width were the related factors for AKI in critically ill neonates. Logistic regression analysis showed that consciousness(OR=4. 542,95%CI 1. 176-17. 539,P=0. 028),mechanical ventilation(OR=0.267,95%CI 0.101-0.705,P = 0.008),5 min Apgar score(OR = 0.750,95% CI 0.605-0.930,P =0. 009),blood urea nitrogen value(OR=1. 074,95%CI 1. 006-1. 146,P=0. 030)were identified as the inde-pendent risk factors of AKI. ConclusionThe incidence of AKI is high in critically ill neonates. Consciousness,mechanical ventilation,5 min Apgar score,blood urea nitrogen value are identified as independent risk factors for AKI.

Objective To evaluate the curative effect of compound nutrient assisted phototherapy on neonatal jaundice.Methods Neonatologists at seven hospitals participated in the study.A total of three hundred and twenty full-term newborns with high indirect bilirubin admitted to hospital from September 2017 to January 2018 were selected.One hundred and sixty-six cases in the observation group,and one hundred and fifty-four cases in the control group,all enrolled neonates were given single-sided,conventional intensity phototherapy.Observation group took compound nutrient at the same time.The average gestational age,age,birth weight of two groups before treatment were not significantly different.Serum total biilirubin,indirect bidirubin,liver function (ALT,AST) and phototherapy time were monitored before treatment and 3 days after treatment.Results The serum total bilirubin in the observation group was significantly lower than that of the control group after 3 days of treatment[(196.7 ± 57.2) μmol/L vs (216.5 ± 54.6) μmol/L],(t=3.17,P＜0.01).The indirect bilirubin in the observation group was significantly lower than that of the control group after 3 days of treatment [(176.3 ± 54.3) μmol/L vs (197.2 ± 52.9) μmol/L],(t=3.50,P＜0.01).The time of phototherapy of the children in the observation group was significantly shorter than that of the control group[(19.8 ± 14.4)d vs (22.9 ± 13.3) d],(t =2.00,P ＜ 0.01).Rash,fever,bronze disease,spilled milk,vomiting,abdominal distention,diarrhea,constipation,liver damage etc.were no significant difference the observation group and the control group(P ＞ 0.05).Conclusion Compound nutrients had good efficacy and safety in adjuvant phototherapy for neonatal high indirect bidirubin.

Objective To compare the treatment of displaced intra-articular calcaneal fractures by homeopathic closed leverage anatomical plate with compression bolt through small posterior lateral approach vs.traditional open reduction and internal fixation.Methods A retrospective case control study was made on 98 cases of displaced intra-articular calcaneal fractures admitted from September 2012 to May 2015.According to the random number table,the subjects were assigned to homeopathic closed leverage anatomical plating with compression bolt through small posterior lateral approach (experiment group,58 cases,66 sides) and open reduction and internal fixation through L-shape approach (control group,40 cases,45 sides).Experiment group consisted of 50 male and eight females cases aging from 27-56 years (mean,41.9 years),and the Sanders classification was 40 cases of type Ⅱ,24 type Ⅲ and two type Ⅳ.Control group consisted of 36 male and four female cases aging from 25-58 years (mean,43.7 years),and the Sanders classification was 25 cases of type Ⅱ,18 type Ⅱ and two type Ⅳ.Operation time,bone reduction,postoperative Bohler's angle,width of the calcaneum,and incision healing were recorded.Functional outcomes were evaluated with Maryland hindfoot scoring system at last follow-up.Results Operation time was (52.6 ± 11.2) min in experiment group,significantly shorter than that in control group [(86.4 ± 14.1) min] (P ＜ 0.01).All cases were followed up from 18-50 months (mean,30.8 months).Reduction of the calcaneal posterior facet in 53 sides (80％) was graded as nearly anatomical in experiment group,and 38 sides (84％) in control group (P ＞ 0.05).Postoperative Bohler's angle was (28.0 ± 6.2) ° in experiment group,and (26.8 ± 7.0) ° in control group (P ＞ 0.05).Width of the calcaneum was (31.3 ±3.6)mm in experiment group and (34.9 ± 4.0)mm in control group (P ＜ 0.01).All cases presented satisfactory shape of the calcaneus without lateral-side impact syndrome.No case had wound infection and incision-edge necrosis in experimental group,while two cases of superficial wound infection and three cases of incision-edge necrosis were found in control group (P ＜ 0.01).At last follow-up,Maryland hindfoot score was (87.1 ± 7.6)points in experiment group and (84.9 ± 9.1)points in control group (P ＞ 0.05).Conclusion Homeopathic percutaneous leverage and anatomical plate with compression bolt through small posterior lateral approach is an effective method for treatment of displaced intra-articular calcaneal fractures,for it has advantages of minimal invasion,less operation time,good reduction and function,and less wound complications.

Objective To investigate the frequency of c-kit mutation and prognosis in t (8;21) acute myeloid leukemia (AML) patients with trisomy 4. Methods A total of 145 de novo t(8;21) AML patients from February 2005 to January 2013 were analyzed retrospectively. Detection of exons 8 and 17 mutation of c-kit by PCR and cytogenetic analysis by R-banding technologies were performed on bone marrow samples of all patients at diagnosis. Clinical data were collected and analyzed statistically. Results Among 145 t (8;21) AML patients, 12 cases (8.3 %) were trisomy 4, 91.7 % (11/12) of them were identified with c-kit mutation, which was significantly higher than that without trisomy 4 [26.3 % (35/133), P< 0.01]. The follow-up data showed that the patients with trisomy 4 were correlated with the lower overall survival (OS) rate (15 % vs 56 %, P< 0.01) and disease-free survival (DFS) rate (0 vs 51 %, P< 0.01) when compared with patients without trisomy 4. Furthermore, the subgroup of patients with both trisomy 4 and c-kit mutation had a worse OS and DFS (P< 0.05). Conclusions Trisomy 4 is associated with high frequency of c-kit mutation and demonstrates poor prognosis in t(8;21) AML patients. Trisomy 4 or it combined with c-kit gene mutation is the main influencing factor on the survival of the patients with t(8;21) AML.