Objective:To construct the prokaryotic expression system of open reading frame of KH gene (KH-ORF),an unknown gene in K562 cells.Methods:The 225bp KH-ORF cDNA was cleaved from plasmid pGEM-T-easy-KH-ORF with EcoRI and SalI and subcloned into the pCI-neo Mammalian Expression Vector cutted with the same restriction endonucleases.The recombinant expression plasmid was transfected into BL21 (DE3) plysS by hot-shock assay.Induced by IPTG, the recombinant protein product was tested by SDS-PAGE.Results:24kD KH-ORF product was obtained as inclusion bodies in BL21 (DE3) plysS after the expression vector had been induced by IPTG for 30 minutes Conclusion :The construction of prokaryotic expression system of KH- ORF is successful.

Objective:To construct the eukaryotic expression vector of open reading frame of KH gene(KH-ORF),an unknown gene obtained from K562 cells,and investigate its function.Methods:The KH-ORF cDNA was cleaved from plasmid expression vector cut with the same restriction endonucleases.pCI-KH-ORF,the pGEM-T-easy-KH-ORF with EcoRI and SalI and subcloned into the pCl-neo mammalian recombinant expression plasmid,was transfected into K562 cells with liposome and screened by G418.KH-ORF expression was tested by RT-PCR.The effect of expression product on cell proliferation was tested by growth-curve and LDH detection.Results:KH-ORF was expressed in K562 cells.The effects of increased proliferation were significant.Conclusion:The product of KH-ORF may be a functional protein that is related to the proliferation of K562 cells,and KH gene may be a functional unknown leukemia gene.

Objective To investigate the diagnostic value and safety of thoracoscopy routine pleural biopsy combined with frozen biopsy for pleural effusion. Methods A retrospective analysis was made on the pathological diagnosis rate of pleural effusion. Results 120 cases in thoracoscopy, 103 cases were confirmed with routine biopsy specimens (85.8%), 16 cases found in the lesions with conventional clamp not satisfactory tissue specimens, combined with frozen cut obtained satisfactory specimens, the diagnostic accuracy rate of 16 cases of cryobiopsy was 100.0%, and the total diagnostic accuracy rate of medical thoracoscopy combined with pleural biopsy and cryobiopsy was 95.0%. There was significant difference between conventional biopsy and cryobiopsy (P < 0.05). Conclusion Medical thoracoscopy combined with pleural biopsy and cryobiopsy can achieve a higher rate of pathological diagnosis, and the complications are mild, so it is worthy of clinical promoting.

Objective To investigate the effects of sevoflurane postconditioning on intestinal ischemiareperfusion (I/R) injury in rats.Methods Thirty-six adult male Sprague-Dawley rats,weighing 200-220 g,were randomly divided into 4 groups (n =9 each):sham operation group (group Sham),group I/R,ischemic postconditioning group (group Ipo) and sevoflurane postconditioning group (group Sevo).Intestinal I/R was induced by clamping the superior mesenteric artery (SMA) for 60 min followed by 120 min of reperfusion in groups I/R,Ipo and Sevo.In group Ipo the animals were subjected to 3 cycles of 30 min reperfusion-30 min ischemia starting from the beginning of reperfusion.The animals inhaled 1.15％ sevoflurane for 30 min starting from the beginning of reperfusion in group Sevo.The animals were sacrificed at 120 min of reperfusion and then the small intestines were removed for determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity (by colorimetric method) and caspase-3 protein expression in intestinal tissues (by Western blot).The density of apoptotic cells was calculated by TUNEL.Results Compared with group Sham,the intestinal injury score,density of apoptotic cells and MDA content were significantly increased,SOD activity was decreased,and caspase-3 protein expression was up-regulated in groups I/R,Ipo and Sevo (P ＜ 0.05).Compared with group l/R,the intestinal injury score,density of apoptotic cells and MDA content were significantly decreased,SOD activity was increased,and caspase-3 protein expression was down-regulated in groups Ipo and Sevo (P ＜ 0.05).There was no significant difference in the intestinal injury score,density of apoptotic cells,SOD activity,MDA content and caspase-3 protein expression between Sevo and Ipo groups (P ＞ 0.05).Conclusion Sevoflurane postconditioning can attenuate intestinal I/R injury through reducing lipid peroxidation and cell apoptosis in rats,and the protective effect is similar to that of ischemic post-conditioning.

Objective To discuss combined detection of pleural biopsy under medical thoracoscopy and pulmonary serum tumor markers in diagnosis of pleural effusion with unknown reason.Methods 76 patients with pleural effusion caused by unknown reason from January 2014 to March 2016 were retrospectively analyzed. Pleural biopsy was conducted under medical thoracoscopy and sent for pathological examination, and 10 ml venous blood was collected from these patients upon admission for testing serum tumor markers (CEA, SCC-AG, ProGRP and CYFRA21-1).Results Among the 76 patients, there were 32 cases with benign lesions (14 with pulmonary tuberculosis, 9 with inlfammatory lesions, 6 with granulomatous inlfammation, 2 with empyema and 1 with hamartoma) and 44 cases with malignant lesions (18 with adenocarcinoma, 13 with squamous carcinoma, 6 with small cell lung cancer, 3 with adeno-squamous carcinoma, 2 with mesothelioma, 1 with large cell carcinoma and 1 with thymoma). The detection of serum tumor markers showed statistically significant differences in the levels of CEA, SCC-AG, ProGRP and CYFRA21-1 in serum between the malignant pleural effusion group and benign pleural effusion group (P = 0.021,P = 0.006,P = 0.003 andP = 0.010). The levels of various serum tumor markers in the malignant pleural effusion group were obviously higher than those in the benign pleural effusion group. According to the pathological results, patients with pleural effusions not caused by lung cancer (2 with mesothelioma and 1 with thymoma) were eliminated from 44 patients with malignant pleural effusions. The rest 41 patients with pleural effusions caused by lung cancer were divided into non-small cell lung cancer and small cell lung cancer according to the pathological types. The results showed that there were statistically signiifcant differences in the levels of CEA, ProGRP and CYFRA21-1 between non-small cell lung cancer and small cell lung cancer (P = 0.036,P = 0.005 andP = 0.008), while there was no statistically signiifcant difference in the level of SCC-AG (P = 0.811).Conclusions Due to high detection rate and high accuracy in detecting pleural effusions caused by unknown reason, medical thoracoscopy is of great signiifcance, especially for the diagnosis of malignant pleural effusions of pleural metastases. However, serum indicators may provide important reference values for us before the pathological results are available. Thus, it is an important means of diagnosing malignant pleural effusions caused by lung cancer and should be promoted in clinic.

Objectives To study the effect of VIM in Enterovirus 71 (EV71) infection of (human brain microvascular endothelial cells (HBMEC) and elaborating the mechanism of EV71 infection in the nervous system. Methods Knocked out the VIM by CRISPR technology , the differences in EV71 absorption , replication , release between wild VIM and VIM knocked-out (VIM-KO) HBMEC were detected by fluorescence quantitative PCR. Results 4 ℃ absorption experiment conformed that EV71 adsorption in VIM- KO is 40% less than in the normal HBMEC. After EV71 infect HBMEC for 48 h (48 h p. i.), the quantitative PCR result showed intracellular viral RNA in VIM-KO was only 1/12 of that in the normal HBMEC. Also the extracellular viral RNA was quantified, and the number of cells in VIM-KO had been reduced 1.4 times compared with the normal HBMEC. Conclusions Once VIM knocking out, EV71 attachment has been obviously reduced. Meanwhile, the level of viral RNA replication and release are decreased compared with the normal HBMEC. VIM may be an attachment receptor of EV71 in HBMEC , when the virus invades HBMEC with the binding of VIM. Moreover , VIM plays an important role in the replication and release of EV71.

Objective To investigate the situation of subjective well-being feeling of type 2 diabetes patients and its influential factors.Methods One hundred and twenty-seven patients with type 2 diabetes were recruited as our subjects and all of them were performed the questionnaire investigation about well-being feeling.Influential factors were analyzed through several statistic methods.Results The index of subject wellbeing ranged from 3.70 to 13.70 and the average was 10.67 ± 2.10 of all type 2 diabetes patients.Of which The index of total affectation was 5.11 ± 0.92 and the range was from 2.63 to 6.63.The score of life satisfaction was 5.58 ± 1.22,and the range was from 1.10 to 7.70.Index of subjective well-being significantly associated with education,family income,therapy approach,HbA1 c level,complication,medicine burden.However there was no correlation with gender,age,diabetes duration.Conclusion The main types of subjective well-being feeling of patients with type 2 diabetes were positive effect and positive experience.Many factors could be the influence factor to well-being.Measures should be taken to improve Subjective well-being of type 2 diabetes patients.

Aim To investigate the effect of matrine on cytoskeletonof K562 cells. Methods Micropipette aspiration technique was adoptedto investigate the viscoelasticity of K562 cells, while the different ex-pression of cytoskeletal protein gene was analyzed by DNA microar-ray. Results In matrine-treated K562 cells, the viscoelastic propertiesKI, K2 and were decreased significantly from 726 ± 215 to 432 ±67,433 ±119 to 242±31, 72±38 to 50±15 respectively, and the geneexpression of prefoldin and ezrin was much stronger than that of controlcells. Conclusion The strueture and function can be changed in ma-trine-treated K562 cells.

Objective To evaluate whether immunization with △A146 Ply could confer protections against pneumococcal infections in murine models and to reveal the possible role of △A146 Ply-specific IgG in the protection elicited.MethodsBALB/c mice were immunized intraperitoneally with △A146 Ply or PBS plus alum.Fourteen days after the third immunization,mice were intranasally challenged with serotype 14 and 19F Streptococcus pneumoniae.Three days after inoculation,lungs were removed from mice and homogenized in PBS,followed by plated on red cell plates.Viable bacteria were counted after overnight incubation.As to the sepsis models,vaccinated mice were challenged intraperitoneally with different dosage bacteria of D39 and serotype 3 strain.The numbers of CFU log10 were compared by Mann Whitney U test and survival rates were analyzed using log-rank test.Passive protection was used to evaluate the role of △A146 Ply-specific IgG in the protection against otherwise lethal infection resulted from D39.Results ELISA analysis demonstrated higher titer specific antibody responses to △A146 Ply was produced after 3 times immunization.Mice boosted twice with △A146 Ply survived significantly longer than that for mice boosted once with △A146 Ply in Alum adjuvant,which were significantly longer than that of control group.Immunization with △A146 Ply was effective in reducing the numbers of pneumococcal strain 31614(serotype 14) and 31693(serotype 19F),which resulted in 50-and 20-fold decreases in bacterial load in the lungs respectively when compared to control protein-immunized mice.60% of vaccinated mice survived the infection with pneumococcal D39 of 1200 CFU.60% protection was achieved when mice intraperitoneally infected with D39 and received △A146 Ply-specific IgG,whereas no mice survived the infection when they were passively administered with △A146 Ply-specific IgG depleted antisera.Conclusion Immunization with △A146 Ply could confer protection against pneumococcal infections,and protection elicited was mediated by △A146 Ply-specific IgG.

cholesterol 7α-hydroxylase gene ( CYP7A 1 ) plays a key role in the catabolism of cholesterol into bile acids. To investigate whether the A-204C polymorphism in CYP7A1 gene affects the gene expression,using luciferase as the reporter gene, four recombinants were constructed by inserting forward or reverse sequence with A or C allele at the polymorphism site into the promoter-less vector pGL3-basic. The constructs were then transfected into four cell lines and the luciferase activity of each expression vector was examined by dual luciferase reporter gene assay system. The results showed that activities of the forward sequence of both genotypes were higher than that of reverse sequence. Promoter activity of the recombinants with A allele was about one third lower than that with C allele. According to the analysis with TRANSFAC database, there may exist a Zic3 binding site when there is the C allele at -204. Our study indicates that the A-204 C polymorphism in CYP7A1 promoter region decreases its promoter activity and thus represses the gene expression, possibly due to the lack of a potential Zic3 binding site.