Objective To investigate the risk factors for hypertriglyceridemia-induced acute pancreatitis,and to provide a reference for clinical prevention and treatment.Methods A total of 42 patients with hypertriglyceridemia-induced acute pancreatitis who were admitted to The People's Hospital of Guang'an from January 2014 to June 2016 were enrolled as study group,and 42 patients with non-hypertriglyceridemia-induced acute pancreatitis (biliary or alcoholic) were enrolled as control group.The two groups were compared in terms of sex,age,fatty liver,body mass index (BMI),type 2 diabetes,hyperlipidemia,family history of hyperlipidemia,serum triglyceride,serum cholesterol,low-density lipoprotein (LDL),high-density lipoprotein (HDL),whole blood low-shear viscosity,and D-dimer concentration at the time when they visited the hospital.The t-test was used for comparison of continuous data between groups,and the chi-square test was used for comparison of categorical data between groups.A multivariate non-conditional logistic regression analysis was performed for the statistically significant variables identified in the univariate analysis.Results The univariate analysis showed that there were significant differences between the two groups in sex,fatty liver,BMI,type 2 diabetes,hyperlipidemia,family history of hyperlipidemia,serum triglyceride level,whole blood low-shear viscosity,whole blood high-shear viscosity,and D-dimer concentration (all P ＜ 0.05).The multivariate non-conditional logistic regression analysis showed that type 2 diabetes (odds ratio [OR] =2.206,95％ confidence interval [95％CI]:1.125-4.263,P =0.024),serum triglyceride level (OR =5.253,95％ CI:2.502-9.568,P =0.001),BMI (OR =3.812,95％ CI:1.896-7.529,P =0.011),fatty liver (OR =4.255,95％ CI:2.185-8.236,P =0.009),and D-dimer concentration (OR =6.258,95 ％ CI:3.526-11.653,P =0.006) were independent risk factors for hypertriglyceridemia-induced acute pancreatitis.Conclusion It is of great clinical significance to provide intervention for risk factors for hypertriglyceridemia-induced acute pancreatitis and minimize its incidence rate.

Objective To determine the effects of Che A and CheY proteins of Campylobacter jejuni regulating the bacterial chemotaxis in vitro and colonization in vivo. Methods By using pET42a plasmid and E. coli BL21DE3 as expression vector and expression strain, respectively, prokaryotic expression systems of cheA and cheY genes of C. jejuni strain NCTC11168 was constructed. Rabbits were immunized with the target recombinant expression proteins, rCheA and rCheY, to prepare the antisera. rCheA-IgG and rCheY-IgG in the antisera were separated using DEAE-52 ion exchange column. pBluescript- II -SK was applied to construct suicide plasmid which used to generate cheA gene knock-out mutant (cheA-). A chemotaxis model in vitro of C. jejuni based on DOC-HAP, the chemotactic ability of cheA' mutant as well as the effect of rCheA-IgG and closantel inhibiting the bacterial chemotaxis were demonstrated. The phosphorylation levels of CheA and CheY after DOC treatment were examined by using either rCheA-IgG or CheY-IgG capture method. The difference of colonization ability between cheA- mutant and wild-type of C. jejuni in mice were checked and then compared. Results The constructed prokaryotic expression systems could efficiently express rCheA and rCheY. The data from PCR and sequencing confirmed the cheA gene knock out from cheA- mutant chromosome. cheA- mutant lost its chemotactic ability towards DOC. Both the rCheA-IgG and closantel could inhibit the chemotaxis of wild-type of C. jejuni (P < 0.05 ). When treatment of DOC, the phosphorylation levels of CheA and CheY in wild-type of C. jejuni rapidly decreased (P < 0. 05 ). The colonization ability in murine jejunum of cheA- mutant was also lower than that of the wild-type ( P<0.05 ) . Conclusion Chemotaxis-associated two-component signaling system (Che-TCSS) of C. jejuni are composed of CheA and CheY, and the two proteins are activated by dephosphorylation. CheA in the Che-TCSS play a critical role in chemotaxis in vitro and colonization in vivo of C. jejuni, and this protein can be used as a target for developing novel anti- C. jejuni drugs.

Objective To investigate the effects of different cell cycles and their regulating genes on apoptosis of mononuclear-macrophages induced by Leptospira interrogans. Methods The diversity and alteration of cell cycles of murine mononuclear-macrophage line(J774A. 1 ) and human monocyte line(THP-1 ) before and after infected with L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai were detected using Cell Cycle Stain Kit plus flow cytometer. The cell cycle synchronized J774A. 1 and THP-1 cells were generated and then identified by using different cell cycle blocking agents and flow cytometer. By using Annexin V/PI Detection Kit combined with flow cytometer, the rates of early-apoptosis and late-apoptosis/necrosis in the synchronized and non-synchronized J774A. 1 and THP-1 cells after infection with L. interrogans strain Lai were determined. Several real-time fluorescence quantitative RT-PCRs were performed to the changes of mRNAs levels of p21, p27, p53, c-myc and cycA genes that associated with cell cycle and apoptosis in J774A. 1 and THP-1 cells before and after infected with L. interrogans strain Lai. Results There were G1, S and G2/M phases in both the non-infected normal J774A. 1 and THP-1 cells. On the contrast,the majority of infected J774A. 1 and THP-1 cells were stagnated at G1 phase, but the amount of S phase THP-1 cells was elevated while that of S phase J774A. 1 cells was not(P ＜0.05). No remarkable early-apoptosis in both the infected G1 phase J774A. 1 and THP-1 cells was found, whereas the rates of early-apoptosis and late-apoptosis/necrosis in the infected M phase J774A. 1 and THP-1 cells were significantly increased (P ＜0.05 ). Additionally, late-apoptosis / necrosis rate in the infected G1 phase THP-1 cells (P ＜ 0.05 )that not found in the infected G1 phase J774A. 1 cells. Compared to the non-infected cells, the p21 mRNA levels in the infected J774A. 1 and THP-1 cells were significantly elevated(P ＜0.05), and the c-myc and p27 mRNA levels in the infected J774A. 1 cells and the cycA mRNA level in the infected THP-1 cells were also higher than those in both the non-infected cells ( P ＜ 0.05 ). Conclusion Different cell cycles and their regulating genes have a role to affect the apoptosis of human and murine mononuclear-macrophages caused by L. interrogans with a diversity of cell line origins.

BACKGROUND:Valvular interstitial cel s are the main components of the heart valves. Myofibroblasts, as a kind of valvular interstitial cel s, can express alpha-smooth muscle actin and type I col agen fiber, and hold differentiation potential. These cel s cannot only play a support role in the valve structure, but also play a regulatory role in the process of the valve normal physiological and pathological responses.
OBJECTIVE:To obtain a reliable method of separation, primary culture and identification of myofibroblasts laying a foundation for further study on the cardiac valvular calcification.
METHODS:Aortic valve myofibroblas extracted from porcine hearts were primary cultured by trypsin and col agenase combined digestive method, common enzyme-digestion method and tissue-culture method, respectively. The myofibroblast activity and morphology were observed using microscope, and myofibroblasts were identified using light microscope and immunocytochemistrial method.
RESULTS AND CONCLUSION:Myofibroblasts had a higher activity and purity cultured by trypsin combined with col agenase II digestion method. Aortic valve myofibroblasts were positive for alpha-smooth muscle actin and negative for von Wil ebrand factor under fluorescence microscope, suggesting that myofibroblasts were successful y obtained.

Objective To investigate the role and possible mechanism of P120 catenin involving in the hemodynamic changes by inducing vascular endothelial cells injury through an in vitro experiment. Methods The hemodynamic environment under the different hemodynamic conditions at the vascular bifurcations was simulated through a T-shaped flow chamber system designed by ourselves. The human umbilical vein endothelial cells (HUVEC)cultured in vitro were stimulated and used the HUVEC cells of the small interfering RNA (SiRNA)after P120ctn gene fragments being knocked out. After loading flow rate of 250 and 500 ml/ min respectively and acting on for 12 h,the HUVEC morphology,growth pattern,and expression of P120ctn and other proteins were observed. Results (1)Normal HUVEC:500 ml/ min was loaded for 12 h,the cells were fused excessively at the impinging point,the cell gaps became narrowed,the cell density decreased and the morphology was elongated in the high wall shear stress (WSS)and wall
shear stress gradient (WSSG)regions. A part of cells migrated downstreamly,and their arrangement direction was consistent with the direction of impinging flow. Compared with the unloaded impinging flow field,after the 2 kinds of impinging flows being loaded for 12 h,the expression levels of P120ctn,vascular endothelial calcium (VEC),Kaiso,α-catenin,and other proteins were decreased. The expression level of matrix metalloproteinase 2 (MMP-2)was increased. There were significant differences (all P < 0. 05). (2)HUVEC after P120ctn being knocked out:Under the impact of the impinging flow,the cell growth time was reduced to 60 min. 250 ml/ min being loaded for 60 min,the impinging point and its surrounding cells still maintained the polygon,but some cells in the high WSS and high WSSG regions began to move downstreamly and aggregated,the cell arrangement mode partly arranged along with the direction of the flow;500 ml / min being loaded for 60 min,the cell density in the high WSS and high WSSG regions were decreased significantly and the morphology was elongated. A large number of cells migrated downstreamly and aggregated. The arrangement mode was parallel and consistent with the direction of the impinging flow. Compared with the unloaded impinging flow field,after the 2 kinds of velocities being loaded for 60 min,the expression levels of VEC,Kaiso,α-catenin proteins were decreased. The expression level of MMP-2 was increased,There were significant differences (all P < 0. 05) Conclusions The hemodynamic change may induce the changes in vascular endothelial cell morphology,growth pattern,and expression of P120ctn and other related proteins, leading to the decrease of vascular endothelial cell adhesion connection stability and the expression changes of related proinflammatory factors. The knockout of P120ctn may result in a further decrease of the vascular endothelial cell adhesion connection stability.

Objective To screen and identify the predominant T-and B-cell ( T-B) combined an-tigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans ( L.interrogans) stains and to further analyze their immunogenicity.Methods PCR analysis was used to detect loa22 gene in L.interrogans strains belonging to eight different serogroups or serovars prevalent in China.The PCR prod-ucts were sequenced after T-A cloning.A prokaryotic expression system for loa22 gene of L.interrogans sero-group Icterohaemorrhagiae serovar Lai strain Lai was constructed.The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography.The rabbit anti-rLoa22 serum samples and IgG were pre-pared.The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinfor-matic softwares.Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B com-bined antigenic epitopes, respectively.MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γinduced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8%homologies in nucleotide sequences and 93.9%-99.5%homologies in amino acid sequences.The construc-ted prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein.Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis.The proliferation of CD4+T cells and the expres-sion of IL-2 ( Th1 ) and IL-4 ( Th2 ) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05).Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L.interrogans.The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic pep-tide ( MAP) vaccines against Leptospira infection.

Objective To isolate and identify the pathogenic bacteria from peripheral blood of a patient with septicemia of unknown etiology and to analyze their drug resistance genes.Methods Two peripheral blood samples were collected from the patient after having fever.Several assays including culturing bacteria on blood agar plates by using streaking technique,Gram-staining of bacterial colonies and microscopic observation,VITEK 2-compact automatic bacterium detection and analysis system as well as a sequencing analysis of the 16s rRNA gene were performed to identify the bacterial pathogens in blood samples.Microdilution test was performed to detect the drug susceptibilities of isolated bacteria to antibiotics.Confirmatory tests were performed to detect the production of β-lactamase and extended spectrum β-lactamase by the isolated strains and the phenotypes of AmpC enzyme and carbapenemase.PCR was used to identify the β-lactamase-encoding genes in the isolated strains by using the primers of 19 common β-lactamase-,AmpC enzyme-and carbapenemase-encoding genes in Enterobacteriaceae strains and the primers of 21 annotated gene sequences encoding the β-lactamase of a Ralstonia mannitolilytica strain.The PCR products were sequenced and analyzed after T-A cloning.Results Ralstonia mannitolilytica strains were isolated from the two peripheral blood samples.The isolated strains were sensitive to ceftriaxone,cefepime,ciprofloxacin,ofloxacin,tigecycline and compound sulfamethoxazole (SMZ-TMP),but resistant to the other 11 tested antibiotics.Results of PCR amplification by using the primers of common genes encoding β-lactamase of Enterobacteriaceae strains were all negative.Fragments of genes encoding the β-lactamase of the isolated Ralstonia mannitolilytica strain were successfully amplified,which were TK49_09850,TK49_12955,TK49_14470,TK49_14495and TK49_18990.The sequences of the amplified gene fragments were not similar to those of the common β-lactamase-encoding genes in Enterobacteriaceae strains.Conclusion The patient was infected with Ralstonia mannitolilytica.The isolated Ralstonia mannitolilytica strain showed a high-level drug resistance with a noticeable diversity against different β-lactam antibiotics.The genes encoding β-lactamase of the isolated Ralstonia mannitolilytica strain were completely different to those of Enterobacteriaceae strains.

Objective To evaluate the results of rotary self-locking intramedullary nail (RSIN) for treating long tubular bone fracture of extremities, and discuss the current problems. Methods One hundred and twenty-two patients with long tubular bone fracture of extremities,including 59 femoral fractures,57 tibial fractures and 6 humeral fractures,who had been treated by RSIN were retrospectively investigated. Results All of the patients achieved clin-ical healing,with an average time of 24 weeks. Nobody was found to appear maluniun,infection and the break of inter-nal fixture. Internal fixations were removed after the fracture healing,with an average time of 14 months. It was diffi-cult to remove the internal fixations in 5 cases, and one case refractured after removing the internal fixations. Conclu-sion RSIN has the advantages such as easy operation,less trauma,no pendulum effect,early motion after operation and so on, except the characters of general interlocking intramedullary nail such as anti-rotation, anti-crispition and anti-displacement.

Objective To evaluate the predictive value of neutrophil-to-lymphocyte ratio (NLR) and hemoglobinto-white blood cell ratio (HWR) prior to treatment in prognosis of non-operative esophageal squamous cell carcinoma (ESCC) patients.Methods From October 2009 to November 2014,a total of 362 non-operative ESCC patients were enrolled in this prospective study.x2 test was used to analyze the relationship between NLR,HWR and general clinical features;Univariate and multivariate Cox regression analysis was performed to evaluate the association between variables and prognosis of non-operative ESCC patients.Results The optimal cutoff level of 1.23 for NLR and 24.92 for HWR were identified by X-title software.x2 test indicated NLR was significantly associated with gender,tumor length,T stage,TNM stage and fasting blood glucose level.Moreover,univariate and multivariate analyses showed that high NLR and high HWR were all associated with a poor prognosis in non-operative ESCC.However,when patients were specifically stratified by treatment modus,subset analysis showed that NLR was only predictive of prognosis in the concurrent chemoradiotherapy group (HR =4.080,95％ CI:1.074-15.497,P =0.039),whereas the influence of HWR on prognosis was just existed in the sequential chemoradiotherapy group (HR =2.625,95％ CI:1.311-5.254,P =0.006).Conclusions In this investigation,we found that high NLR and high HWR were associated with poor prognosis in non-operative ESCC.

Objective To analyze the platelet activating factor acetylhydrolase ( PAF-AH) activity of a gene product encoded by LA2144 gene of Leptospira interrogans ( L. interrogans) , to investigate the ex-pression and secretion of LA2144 protein in various cell cultures and to further understand its function in in-ducing internal hemorrhage in an animal model. Methods The DNA sample containing LA2144 gene was extracted from L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai and used as the template for gene cloning by PCR. The LA2144 gene without the signal sequence coding region was amplified by PCR and inserted into a prokaryotic expression construct for the protein expression. The expressed recombinant protein, rLep-PAF-AH, was purified by Ni-NTA affinity chromatography. Spectrophotometry was used to measure the hydrolytic activity, hydrolytic efficiency, Km and Kcat values of the rLep-PAF-AH protein in hydrolyzing PAF substrate. Real-time fluorescent quantitative RT-PCR ( qRT-PCR) and Western blot assay were performed to measure the expression of LA2144 gene at mRNA and protein levels in human umbilical vein endothelial cells (HUVEC), human monocytes (THP-1) and murine macrophages (J774A. 1) with L. interrogans strain Lai infection, respectively. Each syrian hamster was intravenously injected with 100 μg of LPS-free rLep-PAF-AH for two times. Hemorrhage in the lungs, livers and kidneys were observed in three days after the injection. Results The constructed prokaryotic expression system for LA2144 gene of L. inter-rogans strain Lai could highly express the rLep-PAF-AH upon the induction of IPTG. The purified rLep-PAF-AH showed high purity with a single protein band in gel as indicated by SDS-PAGE. The efficiency of 5 μg of rLep-PAF-AH in hydrolyzing PAF substrate was 26. 6 U/L with a Km value of 82. 79 μmol/L and a Kcat value of 0. 24 S-1 . The expression of Lep-PAF-AH at mRNA level in HUVEC, THP-1 and J774A. 1 cells were significantly elevated after co-culture with L. interrogans strain Lai for 1 or 2 hours (P<0. 05). A large amount of Lep-PAF-AH were detected in the supernatants from co-cultures of L. interrogans strain Lai with the three cell lines, but not from the culture of the spirochete in EMJH medium. The signs of hemor-rhage were observed in the lung of hamsters injected with rLep-PAF-AH, but not in tissue samples from liver and kidney. Conclusion The LA2144 gene product was characterized by a stronger PAF-AH activity. The expression of LA2144 gene at mRNA and protein levels in various cell lines were enhanced during L. interro-gans infection. Moreover, the rLep-PAF-AH could induce the pulmonary hemorrhage in hamsters. This stud-y indicated that the protein encoded by LA2144 gene was an important virulence factor causing hemorrhage in hosts during L. interrogans infection.