The overexpression of EGFR and the typeⅠcAMPdependent protein kinase(PKAⅠ) has been found in most cancer tissue and tumour cells.The blockade of EGFR activation by using anti-EGFR monoclonal antibodies(MAbs) and inhibition of PKAⅠ expression by specific pharmacological agents such as the selective cAMP analogue 8-Cl-cAMP has been proposed as a potential anticancer therapy.We have shown that an interaction between EGFR and PKAⅠ occurs through direct binding of the RⅠsubunit to the Grb2 adaptor protein.We have demonstrated that the functional interaction between the EGFR and the PKAⅠ pathways could have potential therapeutic implications.In fact,the combined interference with both EGFR and PKAⅠ with specific pharmacological agents,has a cooperative antiproliferative effect on human cancer cell lines in vitro and in vivo.Studies on the antitumor activity of this combination are under human clinical trial evaluation.

Objective To prepare ?-elemene solid lipid nanoparticles and investigate their particle size diameter and entrapment efficiency.Methods The ?-elemene solid lipid nanoparticles were prepared by film ultrasonic wave dissolving techniques.And the optimum formula was selected through orthogonal design test according to the entrapment efficiency.Results The optimizing technique was ?-elemene(20 ?L),stearic acid(90 mg),lecithin(90 mg),Tween80(2.5 %,5 mL) and Poloxamer 188(2.5 %,5 mL).Conclusion The technique of preparing ?-elemene by film-ultrasonic wave dissolving technique is feasible.

Objective:To clone and express the Der f 7 recombinant protein from Dermatophagoides farinae and prepare the Der f 7 monoclonal antibody.Methods: The Der f 7 recombinant protein was expressed in prokaryotic expression system of pET28a(+)-Der f 7.BALB /c mice were immunized with the recombinant protein.Myeloma cells and spleen cells were fused,and hybridoma cells were screened by ELISA.Hybridoma cells were injected into the mice abdominal cavity to obtain ascites.It was purified by protein A agarose medium ascites,and then to dentified the titer and purity of the antibody.The specificity of the antibody was identified by Western blot.Results: Three hybridoma cells which stably secret recombinant Der f 7 monoclonal antibody were obtained.The monoclonal antibody had high purity,the titer was higher than 1∶243 000.Western blot showed that Der f 7 recombinant protein could be recognized well.Conclusion: We successfully obtained Der f 7 monoclonal antibody,which can be used for the quantification and localization of Der f 7 allergen and the diagnosis and treatment of allergic diseases.

Objective:To compare structure and function of mite Der f 3,Der p 3 and Eur m 3.Methods: Obtained mite allergens amino acid sequences from the International Union of Immunological Societies nomenclature database .Then physiochemical characterization,sequence alignment,secondary structure,three dimensional (3D) structure and epitopes of three proteins were analyzed by bioinformatics methods.Results:According to results of alignment ,Der f 3,Der p 3 and Eur m 3 displayed 88.51%sequence iden-tity.Der f 3,Der p 3 and Eur m 3 all contained three active sites and two trypsin functional domains ,which showed high identity of amino acid residues.Active sites of three proteins ,which closing to each other in three dimensional (3D) structure,constituting the active center of the enzyme.Secondary and 3D structure of three proteins all contains α-helices,β-sheets and random coils.Epitopes analysis revealed that Der f 3,Der p 3 and Eur m 3 all have 5 main potential epitopes located in random coils.Epitope sequences of Der f 3,Der p 3 and Eur m 3 overlapping in three domains (peptides of 79-81aa,129-135aa and 172-174aa),but the residues in these three domains were not identical.Conclusion:These studies lay the foundation for biochemical and genetic analysis of these 3 allergens,and may contribute to vaccine development for allergen-specific immunotherapy.

Objective ① To Screen for the miRNAs differently expressed in the peripheral blood mono-nuclear cells (PBMCs) of rheumatoid arthritis (RA) by microarray experiments.② To further evaluate the expression of miR-155 in PBMCs of RA.③ To determine the relevance between the expression of miR-155 and clinical as well as laboratory features.④ To test whether inflammatory mediators can induce miR-155 expression in PBMCs of RA.Methods ① Total RNA was isolated from peripheral blood mononuclear cells obtained from 5 patients of RA and 5 normal controls.Expression profiling of miRNAs was performed in a microarray analysis.② MiR-155 was identified for further study by stem-loop real-time RT-PCR based on SYBR-Green.PBMC from 26 patients of RA and 23 normal controls were collected.③ Association between miR-155 and the clinical and laboratory features of RA was evaluated.④Induction of miR-155 following stimulation with TNF-α, IFN-γ and LPS of cultures of RA PBMCs was examined by real-time RT-PCR.Statistical analysis was done with student's t test, paired t test, and ANOVA, Spearman correlation.Results ① Expression profiling of miRNAs revealed significant differential expression of 14 miRNAs, of which signal intensity changed over two times.MiR-155 was up-regulated in PBMCs of RA than in normal controls (t=9.218, P=0.001).② The expression level of miR-155 had a positive correlation with serum CRP level (r=0.57, P=0.002).③ Expression of miR-155 was markedly up-regulated in PBMCs of RA after stimulated with TNF-α,IFN-γ and LPS, especially with TNF-α.Conclusions The expression of miR-155 is induced by stimulating with TNF-α, IFN-γ and LPS.MiR-155 may be a regulator in RA pathogenesis.Further studies are required to elucidate the function of miR-155.

Objective To analyze the efficacy of the combination of MALDI-TOF and immunoadsorption to detect new biomarkers for lupus. Methods Twenty lupus patients at active stage (SLE group), 10 SLE patients in remission (SLE control group), 10 RA patients and 10 PSS patients (other rheumatic disease control group) and 20 healthy volunteers (healthy control group) were enrolled. The serum samples before and after immunoadsorption from SLE group and those from the control groups were co-incubated with activated chitosan copper derivative nano material. The adsorbed nano material was spotted onto the matrix used in MALDI-TOF for analysis by the Axima-CFR plus MALDI-TOF mass spectrometer. T-test was used for statistical analysis. Results MALDI-TOF MS screening showed that three potential protein biomarkers of mass-to-charge (m/z) ratio 3136, 3264, 3326 were found to be very specific for lupus patients: All of them were expressed before immunoadsorption in high quantity and none of them could be detected both after immunoadsorption and in all the three control groups. None of them (<10 000) were in the molecular weight range of the biomarkers used nowadays such as auto antibodies and complement (>50 000). Conclusion The combination of MALDI-TOF and immunoadsorption is effective in the detection of new serum protein biomarkers for lupus and it may be helpful in the screening of SLE patients at active stage from healthy people.

Objective To analyze fungal isolates from patients with superficial fungal infections during 1960-2006.Methods Fungal strains isolated from patients with superficial (mucocutaneous and cutaneous)fungal infections and identified in the Medical Mycology Clinical Laboratory,Department of Dermatology and Venereology,Union Hospital,from 1960 to 2006 (data from September 1991 to July 1992 were unavailable),were subjected to a classification and statistical analysis.Clinical samples for mycological examination were taken from outpatients or inpatients of different departments in hospitals of Hubei province and surrounding areas.Morphological,physiological and biochemical methods were applied for species identification.Results A total of 11 989 Candida strains were isolated,which belonged to 23 species and 16 genera.They fell into 3 groups,i.e.,dermatophytes,Candida and yeasts (including Malassezia),and non-dermatophyte moulds.Since 287 strains of moulds were suspected to be contaminating fungi,11 702 residual isolates were analyzed.Of the analyzed isolates,Candida species (5642/11 702,48.2％ )and dermatophytes (5279/11 702,45.1％ )predominated,followed by yeasts (449/11 702,3.8％) and Malassezia species (332/11 702,2.8％).The most frequently isolated species was Trichophyton rubrum (3865/11 702,33.0％),Candida albicans (3110/11 702,26.6％ ) and non-albicans Candida species (2532/11 702,21.6％ ).Dermatophyte strains were mostly isolated from lesions of smooth skin with an exception of palmoplantar and interdigit regions (1787/5279,37.7％).The most common dermatophyte species was Trichophyton rubrum,followed by Trichophyton violanceum.Candida was mainly isolated from mucous membrane lesions (4099/5642,72.7％),with Candida albicans being the predominant species.Conclusions Candida species and dermatophytes predominate in patients with superficial fungal infections during 1960-2006,with Trichophyton rubrum being the most common species.

Objective To investigate the correlation between NRAMP1 gene polymorphisms and susceptibility to tuberculosis ( TB) in Tibetan people in Qinghai. Methods A case-control study was con-ducted in this study, involving 99 Tibetan patients with TB and 89 healthy Tibetans. The single nucleotide polymorphisms of NRAMP1 gene at rs17235409 and rs3731865 sites were detected by using TaqMan probe method. Gene cloning and sequencing typing were performed to analyze the single nucleotide polymorphisms of NRAMP1 gene at the rs17235416 site. SPASS20. 0 software was used to statistically analyze the correla-tion between NRAMP1 gene polymorphisms and susceptibility to TB in Tibetan people. Results No signifi-cant difference in the genotype frequencies of rs3731865 and rs17235409 was found between the two groups (χ2=0. 852, P=0. 356;χ2=0. 279, P=0. 597). The genotype frequencies of TGTG/TGTG and TGTG/del+del/del at the rs17235416 site were 70. 7% ( 70/99 ) and 29. 3% ( 29/99 ) in patients with TB and 86. 5% (77/89) and 13. 5% (12/89) in healthy subjects. There were significant differences in the geno-type frequencies of TGTG/TGTG and TGTG/del+del/del between the two groups (χ2=6. 870, P=0. 009). The genotypes of TGTG/del and del/del at rs17235416 were risk factors for TB ( OR=0. 376; 95%CI:0. 178-1. 794 as compared with the TGTG/TGTG genotype in Tibetan people in Qinghai. Conclusion This study suggested that the NRAMP1 gene polymorphisms at rs3731865 and rs1723409 sites had no correlation with the susceptibility to TB in Tibetans in Qinghai. However, the NRAMP1 gene polymorphisms at rs17235416 site were correlated with the susceptibility to TB. The TGTG/del alleles at the rs17235416 site might be the risk factors for tuberculosis in Tibetans in Qinghai.

Objective To observe the clinical effect of hemoperfusion in the treatment of patients with critical severe organophosphorus poisoning. Methods Sixty-two patients with critical severe organophosphorus poisoning admitted to the Department of Critical Care Medicine of Jincheng People's Hospital from August 2016 to August 2018 were enrolled, and they were divided into a routine treatment group and a hemoperfusion group according to whether hemoperfusion or not, 31 cases in each group. The routine treatment group was treated with western drugs combined with continuous gastric lavage, while the hemoperfusion group was additionally treated with hemoperfusion for consecutive 3 days on the basis of the routine emergency regimen. The changes of the dosage of penehyclidine hydrochloride used, recovery time of consciousness, recovery time of cholinesterase (ChE) activity, off-line time of mechanical ventilation, hospitalization time, poisoning rebound and mortality were observed in the two groups after treatment; Glasgow coma scale (GCS) was used to assess the prognosis of patients. Results The dosage of penehyclidine hydrochloride used in hemoperfusion group was less than that in the routine treatment group (mg: 3.1±1.2 vs. 5.8±1.3), and the time of consciousness recovery (hours: 3.3±1.7 vs. 13.4±2.4), recovery time of ChE activity (days: 7.7±1.5 vs. 17.9±3.3), off-line time (days: 2.1±0.9 vs. 7.5±2.6), hospitalization time (days: 12.3±1.5 vs. 19.8±3.6) in hemoperfusion group were shorter than those in the routine treatment group (all P < 0.05); poisoning rebound [3.23% (1/31) vs. 16.13% (5/31)] and mortality [9.68% (3/31) vs. 25.81% (8/31)] in hemoperfusion group were lower than those in the routine treatment group (both P < 0.05). The Glasgow coma score (GCS) of the hemoperfusion group on 3, 4 and 5 days after treatment were all higher than those of the routine treatment group (9.9±2.9 vs. 5.7±2.6, 13.3±2.7 vs.7.8±3.2, 13.3±1.5 vs.9.3±2.6, all P < 0.05). Conclusion The conventional treatment, western drug and gastric lavage, combined with hemoperfusion in patients with critical severe organophosphorus poisoning can further reduce the hospital stay, improve the quality of life and reduce the mortality of such patients, therefore.

ObjectiveTo investigate the value of peripheral blood long non-coding RNA-LET (lncRNA-LET) in the diagnosis of chronic hepatitis B (CHB) cirrhosis, and to provide a basis for early clinical diagnosis and treatment of liver cirrhosis. MethodsA total of 175 CHB patients who attended The Affiliated Hospital of Chengde Medical University from March 2017 to May 2019 were enrolled, among whom 52 patients with hepatitis B cirrhosis were enrolled as cirrhosis group and 123 patients without the pathological changes of liver cirrhosis were enrolled as non-cirrhosis group. A total of 40 healthy individuals who underwent physical examination in our hospital during the same period of time were enrolled as normal control group. Liver function parameters and the level of lncRNA-LET in peripheral blood were measured for all subjects. The t-test was used for comparison of continuous data between two groups; an analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The chi-square test was used for comparison of categorical data between groups, and the Kruskal-Wallis H test was used for comparison of ranked data. A Pearson correlation analysis was performed to investigate correlation. The receiver operating characteristic (ROC) curve was used to investigate the value of peripheral blood lncRNA-LET in predicting liver cirrhosis. Results Compared with the normal control group, the cirrhosis group and the non-cirrhosis group had significantly higher serum levels of the liver function parameters total bilirubin (TBil), total bile acid (TBA), albumin (Alb), and alanine aminotransferase (ALT) (all P＜0.05) and a significantly lower serum level of cholinesterase (ChE) (P＜0.05); compared with the non-cirrhosis group, the cirrhosis group had significantly higher serum levels of TBil, TBA, Alb, and ALT (all P＜0.05) and a significantly lower serum level of ChE (P＜0.05). Compared with the normal control group, the cirrhosis group and the non-cirrhosis group had significantly lower relative expression of lncRNA-LET in peripheral blood (P＜0.05), and the cirrhosis group had significantly lower relative expression of lncRNA-LET in peripheral blood than the non-cirrhosis group (P＜0.05). The relative expression of lncRNA-LET decreased significantly with the increase in liver fibrosis stage (P＜0.05). In the patients with CHB, the relative expression of lncRNA-LET in peripheral blood was negatively correlated with liver fibrosis stage, TBil, TBA, Alb, and ALT (r=-0.352,-0.372,-0.364, and -0.410, all P＜0.001) and was positively correlated with ChE (r=0.340, P＜0.001). The ROC curve was used to analyze the value of peripheral blood lncRNA-LET in predicting liver cirrhosis, and the area under the ROC curve was 0934, with an optimal cut-off value of 0.833, a sensitivity of 84.57%, and a specificity of 80.57%. ConclusionThe expression level of lncRNA-LET in peripheral blood decreases with the progression of liver fibrosis and has a good value in the diagnosis of CHB cirrhosis, and therefore, it can be used as a potential biological indicator for the diagnosis of liver cirrhosis.