Aim To express and purify a new sub-type of interferon α , α 1c in E.coli. Methods To establish a brief protocol for expression and purification of IFN-α 1c. In vitro assay for the IFN activity is required for the measurement of antiviral, antiproliferative and immunoregulatory properties. Results Expression of IFN-α 1c was verified by its reactivity to IFN-α 1-specific neutralizing antibodies by ELISA. Western blot also detected a band with relative molecular mass(Mr) of 19 000. IFN-α 1c possessed antiviral biological activity(3.2× 107) higher than IFN-α 1b(1.0× 107～ 1.8× 107) did on VSV challenged WISH cells. IFN-α 1c also inhibited growth rate of A549 comparable to IFN-α 1b. IFN-α 1c as an immunologic effector also augmented the cytotoxic activity of NK cells. Conclusion These results demonstrate that IFN-α 1c can be successfully expressed in E.coli. This interferon with high bioactivity may be a good candidate for clinical use.

Objective To explore the association of lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1),CX3C chemokine receptor 1 (CX3CR1) with coronary artery stenosis disease and its outcomes.Methods A case-control study was conducted.A total of 176 cases of coronary artery stenosis which were confirmed coronary artery stenosis ≥ 50％ by coronary angiography(CAG) were served as case group from department of cardiology of TEDA International Cardiovascular Hospital of Tianjin from May 2011 to April 2013.A total of 129 patients without coronary artery lesion by CAG from this hospital in the same period were served as control group,which has no history of heart disease,liver and kidney dysfuction,brain disease,hematological disease,other disorders that could bring out atherosclerosis and thrombosis.General information and laboratory parameters,LOX-1,CX3CR1,uric acid (UA) and creatinine (CREA) were measured in 2 groups.These parameters of each group were compared,the levels of LOX-1 and CX3CR1 in one-vessel stenosis were compared than that in multi-vessels stenosis in case group,the correlations between LOX-1,CX3CR1 and Gensini score and other variables were analyzed.Comparison of the levels of LOX-1 and CX3CR1 between major adverse cardiovascular events (MACEs) group and nonmajor adverse cardiovascular event (MACE) group was made during follow up 1.5 years.MACEs in patients with different levels of LOX-1 and CX3CR1 were compared during 1.5-year follow up.All of the data were analyzed by SPSS 16.0 software.The independent-samples T test,Mann-Whitney U test,Chi-square test,Spearman correlation,Binary Logistic Regression and Kaplan-Meier probability were adopted for data analysis.Results Comparison between case group and control group,LOX-1:3.72 (1.44,8.15) μg/L vs 0.75(0.50,1.19) μg/L,z =11.072,P ＜0.001 ;CX3CR1:(2.82 ± 1.85) μg/L vs (2.32 ±0.79) μg/L,t =2.021,P ＜ 0.05 ; UA:(351.34 ± 94.82) μmol/L vs (326.74 ± 79.51) μmol/L,t =2.094,P ＜ 0.05 ;CREA:(70.86 ± 20.94) μmol/L vs (65.55 ± 12.96) μmol/L,t =2.077,P ＜ 0.05.CX3CR1 level was significantly higher in patients with multi-vessels stenosis (2.84 ± 1.78) μg/L than that in one-vessel stenosis(2.48 ± 1.64) μg/L,there was significance in difference (t =2.207,P ＜ 0.05).There were no statistically significant correlation between LOX-1,CX3CR1 and Gensini score (R was 0.032,0.079 respectively,P＞ 0.05).LOX-1 was negatively related to left ventricular ejection fraction(LVEF) (R =-0.272,P ＜ 0.01),but positively related to left ventricular end-diastolic diameter (LVDD)(R =0.190,P＜0.05),positively related to UA (R =0.121,P ＜ 0.05).Comparison between MACE group and nonMACE group,LOX-1:7.38(4.97,11.88)μg/L vs 3.52(1.45,7.75) μg/L,z =2.762,P ＜0.01;CX3CRl:(4.02 ±2.90) μg/L vs (2.67 ± 1.48) μg/L,t =3.086,P ＜0.01.LOX-1 and TG were independent risk effects of coronary artery stenosis disease.MACEs were increased in patients with high levels of LOX-1 after PCI during following up 1.5 years (comparison between high-LOX-1 group and lowLOX-1 group,the probability of non-MACE was 87.1％ (115/132) vs 97.7％ (43/44),Log-ranK test,x2 =6.957,P ＜ 0.01).Conclusions LOX-1 and CX3CR1 may be involved in the process of coronary artery stenosis,and a high level of LOX-1 may be associated with left ventricular systolic dysfunction in patients with coronary artery stenosis.Elevated LOX-1 level are closely related to afterwards MACE incidence after PCI in patients with coronary artery stenosis.

OBJECTIVE: Interventional occlusion of intracristal ventricular septal defects (IVSD) is a node point. Reports concerning angiography features and successful occlusion of IVSD using domestic occluder are rare. The aim of this paper is to investigate the angiography features of IVSD and the experience of interventional occlusion with domestic occluder. METHODS: Totally 46 cases of IVSD were diagnosed by color Doppler echocardiography that they had interventional indications.According to the short axis view of aortic, they were divided into 4 groups: A (11:30-12:00), B (12:00-12:30), C (12:30-13:00) and D (13:00-13:30). Different groups were performed with adequate angle angiography to show ventricular septal defect, as well as to decide strategy, which to choose occluder and occluded methods.RESULTS: All 46 patients were occluded successful. Technical success rate was 100%, without related complications. A group included 10 cases, B group 13 cases, C group 16 cases and D group 7 cases. The left anterior oblique angle of IVSD left ventricular angiography was greater than other type of VSD, and increased gradually with the changes in the defect location (Group A to D). According to the defect size and different groups, the defects were successfully occluded with symmetric,decentered, zere-decentered type occluder. Modified pigtail catheter was good value in practice occlusion of IVSD.CONCLUSIONS: Left anterior oblique angle in IVSD left ventricular angiography should be bigger. Domestic occluder is safe,effective, with less complication and cost, which can be the first choose for patients with LVSD.

To understand the sero-epidemiological features of rickettesiosis of humans and domestic animals in Yunnan province, blood samples from 237 adults in different geographic area, including Xundian country,Yulong country and Simao country and 81 children aged from 4 to 6 years old were collected for serological testing. In addition, 90 blood samples from dogs, goats and ox in each investigated area were also collected. Antibodies against 8 rickettsiae, including R.typhi, R.heilongjiangii or R.sibirica, Orientia tsutsugamushi Karp or Kato, Ehrlichia chaffeensis, Anaplasma phagocytosis, Bartonella henselae, Coxiella burnetii and Hainan spotted fever group of rickettsia were examined by using immunofluorescence assays(IFA).It was found that the sero-epidemiologic rates of R.typhi, B.henselae and C.burnetii (16.46%、6.33% and 9.28%) of adults were higher than those of other rickerrsiae investigated. The positive rates of IgG antibody against R.typhi for children also shared the higher rate (12.35%). Similar sero-epidemiologic features were found for domestic animals. Among the 8 rickettsia tested in this study, the positive rate of IgG antibody against R.typhi appeared to be the highest(61.48%) without significant difference among these investigated sites. From this investigation, it is evident that the rickettsial infection of farm population and domestic animals are common in Yunnan province, and the active surveillance of rickettsiosis and differential diagnosis of unknown febrile patients in clinical practice should be enforced.

BACKGROUND:Silver nanoparticles (AgNPs) show strong antibacterial effect and are not easy to have drug resistance. But the antibacterial mechanisms of AgNPs have not been wel developed.
OBJECTIVE:To explain the antibacterial mechanisms of AgNPs.
METHODS:We investigated the influence of Ti, TiO2 and TiO2 containing AgNPs onEscherichia coliand Staphylococcus aureus by bacterial inhibition ring test. Escherichia coli was cultured in LB liquid medium with 0, 5, 10 mg/L AgNPs. We measured the absorbance value of bacterial culture. DNA gel electrophoresis was used to study the effect of AgNPs onEscherichia coliDNA. Then we researched the character of apoptosis on Escherichia coli by Annexin V and PI staining, using flow cytometry.
RESULTS AND CONCLUSION:The inhibiting effect of Ti and TiO2 onEscherichia coliandStaphylococcus aureus was not obvious. But the inhibition rings of TiO2 containing AgNPs to bacteria appeared. The absorbance value of Escherichia coliculture was reduced whenEscherichia coliwas co-cultured with AgNPs. And this decrease tendency was in direct proportion with AgNPs concentration. AgNPs reduced the amount of DNA of Escherichia coli and this tendency was directly proportional with AgNPs concentration. TheEscherichia coli apoptosis rate induced by AgNPs was increased and this tendency was positively correlated to the AgNPs concentration. These results indicate that AgNPs can induce bacterial apoptosis to influence the growth of bacteria.

ObjectiveTo test the the association between PRODH gene variant rs385440 and the susceptibility to schizophrenia and the severity of schizophrenia in Guangxi Zhuang and Han population,further exploring the genetic mechanisms of schizophrenia in Guangxi Zhuang and Han population.MethodsThe schizophrenia patients were diagnosed according to ICD-10 criteria in this study.The subjects in the association analysis were 282 unrelated schizophrenia patients(94 Zhuang and 188 Han) and 282 healthy controls (94 Zhuang and 188 Han).A quantitative real-time PCR TaqMan MGB experimental method was carried out to analysis rs385440.The clinical psychotic symptoms of 246 schizophrenia patients (83 Zhuang and 163 Han) were assessed by PANSS.Statistical analyses were carried out with SPSS13.0 for windows.ResultsThere was no statistically significant difference in different allele and genotype frequencies of rs385440 between schizophrenia cases and controls in Zhuang samples,Han samples and combined samples respectively (P＞ 0.05 ).In Zhuang schizophrenia patients the score of N4 (passive/apathetic social withdrawal) item in A allele carriers (3.28 ± 1.34) was higher than that of G allele carriers ( 2.40 ± 1.36 ) significantly (P ＜ 0.05 ),and the score of G12 ( lack of judgment and insight) item in A allele carriers(4.92 ± 1.55 ) was higher than that of G allele carriers ( 4.12 ± 1.85 ) significantly (P ＜ 0.05 ).Conclusion There is no association between PRODH gene variant rs385440 and the susceptibility to schizophrenia in Guangxi Zhuang and Han population.Rs385440 associated the severity of passive/apathetic social withdrawal symptom and poor attention symptom of schizophrenia in Zhuang.

BACKGROUND:Bone marrow mesenchymal stem cel s have attracted widespread attention for the capabilities of self-renewal and muti-differentiation, which have been used in treatment of various diseases.
OBJECTIVE:To study the effect of three-dimensional spheroid culture system on the stemness and senescence of bone marrow mesenchymal stem cel s.
METHODS:Mesechyaml stem cel s were isolated from the bone marrow of C57/B6 mice, 3 weeks old, and cultured onto the culture plates coated with or without chitosan. After 5 days of culture, the cel phenotype and expression of stemness related markers CD44 and Sca-1 were analyzed by flow cytometry. PI and Annexin-V staining were used to detect cel apoptosis. Also,β-Gal staining was applied for identification of aging.
RESULTS AND CONCLUSION:The mouse mesenchymal stem cel s began to form spheroids on day 3. The stemness-related markers, including CD44 and Sca-1, expressed higher in spheroid mesenchymal stem cel s than the cel s under normal culturing. Compared with the normal culture group, the apoptosis and senescence of cel s from spheroid culture were lower. The results indicate that the formation of spheroids on chitosan films can increase the stemness, decrease the apoptosis and slow the senescence of mesenchymal stem cel s.

Objective To clarify the role of syk kinase in inflammasome activation in mouse peritoneal macrophages during Listeria monocytogenes (LM) infection. Methods Murine peritoneal macrophages were randomly divided into BAY treatment group, SB treatment group, WO treatment group, no treatment group and negative control group (NI). There were three wells in each group. The syk inhibitor BAY 117082, P38MAPK inhibitor SB203580 and PI3K inhibitor wotamine were used to treat murine peritoneal macrophages for 1h in BAY treatment group, SB treatment group and WO treatment group. Murine peritoneal macrophages were infected with LM for 24 h except NI group. The protein level of interleukin (IL)-18 in the supernatant was detected by ELISA kit. The activation condition of key molecule ASC in the infected-macrophages cyto-plasm was observed under fluorescence microscope. The phosphorylation levels of syk protein kinase at different time points during LM infection were determined by Western blot assay. Results There was no significant difference in IL-18 protein level before and after BAY treatment in NI group (P>0.05). The IL-18 protein level was significantly lower after LM infec-tion in BAY treatment group compared with that in no treatment group (P<0.05). In contrast, there was no significant differ-ence in IL-18 production between SB treatment group, WO treatment and no treatment group (P>0.05). Meanwhile, the per-centage of ASC-speck positive cells was obviously diminished in BAY treatment group compared with that in no treatment group (P<0.01). The phosphorylation levels of syk were significantly increased in 5 min, 15 min and 30 min post-infection. Conclusion Syk kinase signaling is involved in the inflammasomes activation upon Listeria monocytogenes infection in mu-rine macrophages.

Objective To investigate the interventional treatment strategy for occluding the intercristal ventricular septal defect (VSD) in order to improve the surgical safety and success rate. Methods During the period from January 2012 to December 2013, a total of 31 patients with intercristal VSD were admitted to authors’ hospital to receive interventional catheter occlusion therapy. Preoperative color Doppler ultrasound echocardiography showed that on the short axis view of the aorta the VSD interrupted port was situated at 12:00 - 1:00 o’clock region. Left ventricular and above aortic valve angiography indicated that the VSD location, shape and size, the split vent size on the left ventricle side and its distance from the aortic valve could be correctly measured when the VSD shunt was visualized , which were very helpful in guiding the operator to select the suitable occluder as well as to adjust the release pattern of the occluder. Postoperative imaging findings of the left ventricular and above aortic valve angiography were compared with the preoperative ones. Results Successful occlusion of VSD was obtained in 22 patients , in 13 among them the left ventricular angiography showed that the direction of blood flow beam at the defect hole was from the left ventricle to the right ventricle in an obliquely upward direction. The basal width of the defect on the left ventricle side was (5.12 ± 1.38) mm, and(6 - 10) mm occluder was employed. In the remaining 9 patients the left ventricular angiography showed that the direction of blood flow beam at the defect hole was from the left ventricle to the right ventricle in a direction almost parallel to the aortic valve , and the basal width of the defect on the left ventricle side was (7.18 ± 1.26) mm, and (9 - 12) mm zero-bias occluder was adopted. Interventional occlusion of VSD was unsuccessful in 9 cases as the intercristal hole was rather larger, and two of them had coexisting aortic sinus aneurysm complicated by mid-to-severe degree aortic valve regurgitation. Conclusion Based on the precise analysis of angiographic images by experienced radiologists optimal treatment scheme can be worked out. If conditions permit, symmetrical occluder should be employed so far as possible in order to reduce the degree of operation difficulty and improve the surgical safety and the success rate as well.

BACKGROUND: Human keratinocyte growth factor-2 (hKGF-2) has extensive physiological functions, which plays an important role in embryonic development, tissue-repairing, nervous regeneration, vascularization and development of tumor.OBJECTIVE: To clone hKGF-2 gene, obtain the expression of hKGF-2 in Escherichia coli(E.coli) and determine its bioactivity, so as to provide experimental basis for further investigation.DESIGN: Open experiment.SETTING: Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.MATERIALS: The experiment was conducted in State Key Laboratory of Viral Genetic Engineering, Institute for Viral Disease Control and Prevention of Chinese Center for Disease Control and Prevention. The temperature control expression vector pBV220 was constructed by State Key Laboratory of Viral Genetic Engineering; EcoR Ⅰ , BamH Ⅰ , T4 DNA ligase (Promega Co., Ltd.); The specific polymerase chain reaction (PCR) of hKGF-2 (Manufactured by Shanghai Boya Biotechnology Co., Ltd.); Heparin-Sepharose CL-6B (Pharmacia Company); PCR rapid purification kit,Trizol kits for total RNA extract, Kits for RT-PCR (GIBCO Co., Ltd.); Kits for rapid extraction of plasmid DNA (Boda Company); BL-21-codon plus compent cells (Stratagene Co., Ltd.).METHODS: High-expression strain BL-21 codon plus competent cells was used to express and purify initially recombinant hKGF-2 protein, and its activity was detected. RT-PCR was adopted to obtain hKGF-2 cDNA from lung tissues of naturally aborted fetus and clone it into pBV220 carri er plasmid. The hKGF-2 protein expressed in BL-21 codon plus competent cells of E.coli. Affinity chromatography and ion exchange chromatography were applied in isolation and purification, and the bioactivity of expression protein was determined in cell proliferation test.MAIN OUTCOME MEASURES: The length and sequence of cDNA segment in hKGF-2, the expression of hKGF-2 gene inE.coli and the purification of hKGF-2 activity.RESULTS: The segment of hKGF-2 cNDA was about 500 bp, and hKGF-2 protein highly expressed in BL-21, which had soluble expression in the supernatant. SDS-PAGE showed that the relative molecular mass was about 20000, and hKGF-2 protein could significantly promote the mitotic activity of NIH3T3 cells. The A value (490 nm) of hKGF-2 in the 1 μg/L, 5 μg/L and 10 μg/L groupswere higher than that in the blank control group, and the differences were significant (which were 0.174±0.022,0.220±0.029,0.306±0.050,0.066±0.004 respectively,P ＜ 0.001).CONCLUSION: hKGF-2 gene is successfully cloned, which highly expresses in BL-21 of the E.coli. Purified hKGF-2 protein can stimulate the proliferation of NIH3T3 cells and significantly promote its mitotic activity.