Objective To explore the role and mechanism of phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin kinase(PI3K/AKT/mTOR)signaling in autoimmune thyroiditis(AIT).Methods 24 female C57BL/6 mice were randomly divided into four groups:a normal control(NC)group,an experimental autoimmune thyroiditis(EAT)group,and two groups treated with LY294002(25 mg/kg or 50 mg/kg LY294002).The degree of thyroiditis was observed by hematoxylin and eosin staining.The percentage of Th 17 cells in the spleen mononuclear cells(SMCs)was determined by flow cytometry.Enzyme-linked immunosorbent assay was used to measure the concentrations of thyroglobulin antibody(TgAb)and interleukin-17A(IL-17A)in the serum.Western blotting was conducted to detect the protein levels of IL-17A,p-AKT(Thr308),p-AKT(Ser473),p-mTOR(Ser2448),S6K1,and S6K2 in the different groups.Results Compared with the NC group,the infiltration of Th17 cells and the expressions ofIL-17A,p-AKT(Ser473),p-AKT(Thr308),p-mTOR(Ser2448),S6K1,and S6K2 rose remarkably in EAT mice.After the PI3K pathway was blocked,the degree of thyroiditis was significantly alleviated,followed by the proportion of Th17 cells,and the expression of IL-17A and PI3K pathway-related molecules decreased in a dose-dependent manner.Conclusion PI3K/AKT/mTOR signaling pathway participates in thyroid autoimmune jnjury of EAT mice by regulating Th17 cells differentiation.

Chronic refractory wound (CRW) is one of the most challengeable issues in clinic due to complex pathogenesis, long course of disease and poor prognosis. Experts need to conduct systematic summary for the diagnosis and treatment of CRW due to complex pathogenesis and poor prognosis, and standard guidelines for the diagnosis and treatment of CRW should be created. The Guideline forthe diagnosis and treatment of chronic refractory wounds in orthopedic trauma patients ( version 2023) was created by the expert group organized by the Chinese Association of Orthopedic Surgeons, Chinese Orthopedic Association, Chinese Society of Traumatology, and Trauma Orthopedics and Multiple Traumatology Group of Emergency Resuscitation Committee of Chinese Medical Doctor Association after the clinical problems were chosen based on demand-driven principles and principles of evidence-based medicine. The guideline systematically elaborated CRW from aspects of the epidemiology, diagnosis, treatment, postoperative management, complication prevention and comorbidity management, and rehabilitation and health education, and 9 recommendations were finally proposed to provide a reliable clinical reference for the diagnosis and treatment of CRW.

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.

Time series model was developed to investigate the effect and contributions of related biomarkers on the cerebral endothelial dysfunction induced by beta amyloid(Aβ). HCMEC/D3 was incubated with 2. 5 μmol/L Aβ for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h, and biomarkers including cytosolic calcium ion, mitochondrial membrane potential(MMP), endothelial nitric oxide synthase(eNOS)and cell viability were determined. Time series model was established to assess the dynamic relationship between the related biomarkers and cell viability and the contribution of different biomarkers to cell damage. Impulse response analysis indicated that after a positive impact on cytosolic calcium ion, cell viability decreased and this impact continued to decline; after a positive impact on endothelial nitric oxide synthase and mitochondrial membrane potential, cell viability increases, which increased rapidly in the early stage, and the rate decreased in later stage. The result of variance decomposition showed that the cytosolic calcium ion played a major role in cerebral endothelial dysfunction induced by Aβ. Combined with the model study, it is concluded that the intervention on the level of cytosolic calcium ion at the early stage may be the possible way to slow the disease progression.

Objective To evaluate in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against Exophiala dermatitidis (E.dermatitidis).Methods The minimum inhibitory concentrations (MICs) of itraconazole and terbinafine against 12 strains of E.dermatitidis were determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M38-A2 Document).A broth microdilution checkerboard method was used to evaluate the in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against E.dermatitidis.Results The MIC ranges of terbinafine and itraconazole against E.dermatitidis were 0.060-0.125 mg/L and 0.5-1 mg/L,respectively.The combination of tacrolimus with terbinafine showed synergistic inhibitory effects against 5 strains of E.dermatitidis,while the combination of tacrolimus with itraconazole revealed synergistic effects against 10 strains of E.dermatitidis.No antagonism was observed in either of the two combinations.Conclusion In vitro combination of tacrolimus with itraconazole or terbinafine can enhance the antifungal activity of itraconazole or terbinafine against E.dermatitidis.

The peptides,proteins and other biological molecules in transudative pleural effusion correlate directly or indirectly with specific physiological and pathological state,reflecting the information regarding the lungs or other parts of the body.In the present study,the peptide fraction in transudative pleural effusion was isolated by uhrafiltration.After desalted and enriched by C18 tips,the peptide mixture was analyzed by nano LC-MS/MS.The results showed that 314 peptides,which were originated from 52 proteins,in pleural transudate were identified.More than half of the peptides were derived from fibrinogen.Many peptides were characterized as displaying ladder sequences.In addition,a large number of proline oxidation modifications were detected in the peptides derived from collagen and fibrinogen.Gene ontology enrichment analysis showed that the most of the proteins extracellular properties of pleural transudate polypeptide components were protein with exocytosis.The study provided a rapid and efficient separation and analysis methods for lung disease markers related peptide compounds in pleural fluid leakage.Also this research provided a rapid and effective method for screening peptide biomarkers related to lung diseases from transudative pleural effusion.

Objective To verify whether iron can accelerate the process of liver fibrosis in rats. Methods The rats were divided into control group, high fat diet group, high iron group, high fat diet and high iron group, high fat diet and de?iron group, each with 24 rats. The rats were allowed to freely take normal diet and high fat diet, while the high iron rats, high?fat diet plus high iron rats received intramuscular injection of 50 mg/kg iron dextran every other day;high?fat di?et plus de?iron group rats received tail intravenous injection of 30 mg/kg deferoxamine one month before death, 3 times/week. 8 rats were selected at 4th, 5th, 6th month of intervention, to detect serum hyaluronic Acid (HA), collagen type IV ( COL?IV) , laminin ( LN) , procollagen III ( PC III) , and observe pathological changes in the liver with Masson staining. Results At 5th month of intervention, serum HA level of the high?fat diet plus high iron group was significantly higher than those of high fat diet group and control group. At 6th month of intervention, serum COL?IV and LN levels of the high?fat diet plus high iron group were significantly higher than those of the high fat diet group. At 6th month, serum PC III level was 1. 63 time of those of the high fat diet group. At the 6th month, liver tissue of high fat diet plus high iron group ap?peared collagen deposition revealed by Masson staining, which was not the case in other groups. Conclusions Iron can accelerate high fat?induced liver fibrosis.

Objective To investigate the relationship between the education level of staff members and the risk factors for stroke in a university in China.Methods From January 2014 to May 2014,a total of 659 staff members in a university in China were collected with a cluster sampling method.The basic information and the relevant data of the risk factors for stroke of the subjects were collected and recorded completely.The subjects were divided into 3 groups according to their education levels from low to high.The relationship between their education levels and the risk factors for stroke were analyzed.Results A total of 633 staff members were enrolled,including 426 men (67.3％) and 207 women (32.7％).The low,middle and high education level groups were 188 (29.7％),193 (30.5％),and 252 (39.8％),respectively.With the increase of the education level,the prevalence of hypertension,dyslipidemia,and stroke decreased,and the awareness rates of hypertension and dyslipidemia and the treatment rate increased.The proportion of drink-ing increased in the male group and that decreased in the female group.The proportion of regular exercise increased in the female group.Multivariate logistic regression analysis showed that after adjusting for age,per capita income,employment,drinking,smoking,regular exercise and other factors,compared with the high education level group,the risk of hypertension increased 2.55 times in the low education level group in males (odds ratio [OR] 2.55,95％ confidence interval [CI] 1.42-4.58;P =0.002);the prevalence risk of dyslipidemia increased 2.25 (OR 2.25,95％ CI 1.31-3.86;P =0.003) and 2.02 times in the low and middle education level groups (OR 2.02,95％ CI 1.23-3.33;P =0.006) respectively;the risk of smoking decreased 42％ in the middle education level group (OR 0.58,95％ CI 0.36-0.93;P =0.024);the risk of hypertension increased 6.27 times in the low level education group in women (OR 6.27,95％ CI 1.59-24.74;P =0.009);the risk of dyslipidemia increased 3.91 times in the middle education level group (OR 3.91,95％ CI 1.70-8.98;P =0.001);the risk of drinking increased 3.49 times in the low level education group (OR 3.49,95％ CI 1.12-10.92;P =0.032),and the weekly regular exercise decreased 65％ in the low level education group (OR0.35,95％ CI0.15-0.82;P =0.016).Conclusion The incidence of the risk factors for stroke in the low education level group was higher than the popuhtions of high or middle education level.

Objective To investigate the clinical significance of platelet activation markers CD62p and PAC-1 in patients with hypertension and coronary heart diease.Methods To select patients who were in the Second Affiliated Hospital of Dalian Medical University between January 2012 and September 2012,42 patients with hypertension,46 patients with coronary heart disease.Flow cytometry was used to test the positive percentage of the platelet surface activation markers CD62p and PAC-1 .To observe the difference among hypertension group,coronary heart disease group and health control group;hyper-tension and coronary heart disease group.Results The positive percentage of platelet activation markers CD62p and PAC-1 in hypertension (36.36±9.62,7.18±8.20)%,coronary heart disease group (42.74±14.60,8.81±12.53)% showed sta-tistically significant differences (t=4.150~5.853,P<0.01)compared with healthy control group (26.82±9.13 ,1.09± 1.05)%.The positive percentage of CD62p in coronary heart disease group (42.74±14.60)% was also higher than that of hypertension (36.36±9.62)%,and the difference between them was of statistical significance (t=2.444,P<0.05).Con-clusion The hypertensive patients and coronary heart disease were in pre-thrombotic state,the activation of platelet increase significantly.Molecular markers should be measured in hypertensive patients and coronary heart disease for prevention and treatment of thrombotic disease complication.

Objective To evaluate the relationship between endometriosis fertility index (EFI) and pregnancies after laparoscopic surgery in endometriosis-associated infertility.Methods From Jan.2005 to Jan.2010,medical documents of 350 infertile patients due to endometriosis undergoing laparoscopic surgery were studied retrospectively.Pregnancy outcomes were followed up by telephone.EFI was calculated by history factors,least function score and some aspects of the revised American Fertility Society (r-AFS) endometriosis stage.The cumulative pregnancy rate was calculated and compared by Kaplan-Meier survival analysis.Results Within 3 years after surgery,the cumulative pregnancy rates among patients with EFI score 8,9,10 were 62.5％,69.8％ and 81.1％,respectively.There was no significant difference in pregnancy rates among those three groups of patients ( P =0.24 ).The cumulative pregnancy rates among patients with EFI score 5,6,7 were 49.8％,43.9％ and 41.6％,respectively,which did not reach statistical difference ( P =0.83 ).The cumulative pregnancy rates of EFI score 8 - 10 was significantly higher than that of EFI score 5 -7 (71.8％ vs.44.4％,P =0.000).The patients with EFI score 0 -4 was quite small with only 33 cases,among which 15 cases were pregnant.Conclusions There is relationship between EFI and pregnancy in patients with endometriosis-associated infertility.EFI is meaningful to guide post surgical treatment.