A 880 bp cDNA localized to the putative NS5 region of HGV genome was expressed in E.coli BL21(DE3). The cDNA fragment was inserted into a plasmid pGEX-5X-1,at the downstream of the DNA sequence encoding Schistosoma japonicum glutathione S- transferase(GST),in the same reading frame with the gene of GST. A 60KD GST-NS53 fusion protein was expressed at 37℃ in a form of inclusion bodies amounting to 30 percent of total host protein whereas at 20℃ mainly in a form of soluble protein . The fusion protein was extracted and purified to homologue. The purified GST-NS53 fusion protein could be specifically recognized with either the sera from the patient infected by HGV or the antisera directed against GST.

Objective To investigate the effects of Helicobacter priori (H.prlori) strains from the patient with gastric cancer on the expression of DNA mismatch repair (MMR) genes in AGS cells in vitro. Methods AGS cells were co-cultured with ten strains of H.prlori from five patients with gastric cancer and five patients with gastritis respectively.The expressions of DNA MMR genes (hMSH2 and hMLH1) in mRNA and protein levels were determined by RT-PCR and Western blot.The enteropathogenic E.coli served as a bacterial control.Results E.coli and H.priori strains from gastritis have no effects on the expression of hMSH2 and hMLH1 in both protein and mRNA levels on the whole,while all the H.prlori strains from gastric cancer reduced expression levels of hMSH2 and hMLH1.Conclusions There are different effects between H. prlori strains from gastric cancer and those from gastritis on DNA MMR in AGS cell line,indicating that infec- tion of some H.prlori strains might lead to the inhibition of DNA MMR,and that in turns increases the risk of gastric carcinogenesis during chronic H.prlori infection.

Objective To evaluate the early diagnostic value of circulating microRNA-1 (miR-1) on acute myocardial infarction (AMI). Methods A prospective cohort study was conducted. The patients with chest pain admitted to the Second People's Hospital of Wuxi from November 2012 to June 2015 were enrolled. According to AMI diagnostic criteria, the patients were divided into AMI group and non-AMI group, and healthy individuals during the same period were served as heath controls. The venous samples of the onset patients were collected within 3 hours after admission. The plasma miR-1 was determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the levels of plasma cardiac troponin I (cTnI) and MB isoenzyme of creatine kinase (CK-MB) were measured by electrochemiluminescence. The correlation between plasma miR-1 and cTnI as well as CK-MB was performed by Spearman analysis. The early diagnostic performance of plasma miR-1, cTnI, and CK-MB for AMI was estimated by receiver operating characteristic (ROC) curve analysis. Results There were 127 patients in AMI group, and 107 in non-AMI group, including 82 patients with angina pectoris, 2 with pulmonary embolism, 3 with aortic dissection, 2 with acute pericarditis, 3 with myocarditis, 13 with acute heart failure, and 2 with peptic ulcer. Ninety volunteers were served as healthy controls. There was no difference in clinical characteristics including gender and hyperlipidemia between AMI group and non-AMI group. The expressions of plasma miR-1, cTnI and CK-MB were significantly increased in AMI patients as compared with those of the healthy controls [miR-1 (2-ΔΔCt): 4.32±2.60 vs. 1.44±0.75 and 0.98±0.18, cTnI (μg/L): 3.23 (0.63, 10.70) vs. 0.02 (0.00, 0.17) and 0.00 (0.00, 0.00), CK-MB (U/L): 32.40 (14.20, 95.40) vs. 14.40 (11.20, 17.10) and 8.90 (8.28, 9.50), all P < 0.01]. The expression of plasma miR-1 had a significantly positive correlation with cTnI and CK-MB in AMI patients (r1 = 0.395, r2 = 0.490, both P < 0.000). It was demonstrated by ROC curve analysis that the area under ROC curve (AUC) for the diagnostic value of miR-1 on AMI was 0.905 [95% confidence interval (95%CI) = 0.860-0.950, P = 0.000], the sensitivity was 86.6%, and the specificity was 95.4%; the AUC for cTnI was 0.908 (95%CI = 0.870-0.946, P = 0.000), the sensitivity was 81.9%, and the specificity was 95.9%; the AUC for CK-MB was 0.795 (95%CI = 0.736-0.854, P = 0.000), the sensitivity was 63.0%, and the specificity was 92.9%. Conclusions Plasma miR-1 has the capacity in early diagnosis of AMI, superior to CK-MB, and equal to cTnI. It can provide additional diagnostic information beyond cTnI. The diagnostic accuracy for early AMI can be improved with the combination of plasma miR-1 and cTnI.

In order to study the feasibility of E2 gene fragment of hepatitis virus G(HGV) as a component of DNA vaccine against the hepatitis virus G infection, a 559bp DNA fragment encoding HGV E2 was cloned into plasmid pCMV-S from pThioHis-E2 in the same reading frame with HBsAg gene to form a recombinant plasmid named pCMV-S-E2. BALB/c mice of Kunming strain were immunized with purified plasmid DNA of pCMV-S-E2 by intra-muscularly inoculation. The immunizations were boosted twice at an interval of 14 days. The whole blood was collected from mice orbit on the day-8 after the last boost. Mice sera were screened by ELISA to determine the humoral immune response using E2-GST fusion protein as the immobilized antigen and the sera from mice immunized with pCMV-S as control. The result indicated that the immunization with plasmid DNA of pCMV-S-E2 could induce quite strong humoral immune response.
Animals ; Female ; GB virus C ; immunology ; Hepatitis Antibodies ; blood ; Mice ; Mice, Inbred BALB C ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Hepatitis Vaccines ; immunology

3D Scaffolds with controlled porous structure were designed and fabricated by utilizing CAD and rapid prototyping techniques. A flow perfusion bioreactor, which allowed exposure of the culture cells to low levels of mechanical stimulation by fluid flow-induced shear stress, was developed in our lab. The scaffolds were pre-designed and the negative images of the designs were used to build the molds on a stereolithography (SL) apparatus with epoxy resins. Calcium phosphate cement paste was cast into the molds. And after pyrolysis, the 3D scaffolds with controlled internal pore architectures were obtained. Rabbit osteoblasts were seeded in 3D porous scaffolds, cultured in the flow perfusion bioreactor with media flow rate set at 2 mL/min and 6-well plates. At 3, 7, and 14 days, scanning microscopic evaluation showed excellent growth on the surface of scaffolds and poor viability of cells within microchannels in static cultures. In flow perfusion bioreactor, there was greater cellularity throughout the scaffolds and abundant deposition of extracellular matrix. Cells were also seen throughout the internal microchannels of scaffolds. These results represent that better mass transport of oxygen and nutrient occurred in the flow perfusion bioreactor and cells distribution in 3D porous scaffolds was improved.
Animals ; Bioreactors ; Cell Culture Techniques ; instrumentation ; methods ; Cell Division ; Cells, Cultured ; Osteoblasts ; cytology ; Porosity ; Rabbits ; Skull ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVETo investigate the expression of nuclear factor kappa B (NF-kgr;B) and tumor necrosis factor α (TNF-α) in lung tissue of acute paraquat poisoned rats.

METHODS68 male Wistar rats were randomly divided into 2 groups: the control group (n = 8), the intoxication group (n = 60). On the 1st, the 3rd, the 7th, the 14th and the 28th day after intoxication, the expression of NF-κB p65 and TNF-α in lung tissue were detected by LSAB immunohistochemistry (IH) staining. Meanwhile, the level of malondialdehyde (MDA) in plasma, and lung homogenate, the content of malondialdehyde (HPY) in lung homogenate were detected.

RESULTSThe levels of MDA in plasma on the 1st, the 3rd, the 7th day and in lung homogenate on the 1st, the 3rd day of the intoxication group [in plasma: (10.15 ± 3.15), (6.97 ± 1.65) and (5.44 ± 0.66) nmol/ml; in lung homogenate: (10.20 ± 2.43), (10.71 ± 171) nmol/ml] were significantly higher than that of the control group [in plasma: (3.84 ± 1.04) nmol/ml, in lung homogenate: (7.66 ± 0.66) nmol/ml]. The content of HPY in lung homogenate on the 14th and the 28th day after intoxication [(19.98 ± 2.86), (26.06 ± 4.06) µg/0.1 g lung homogenate] were higher than that of the control group [(8.80 ± 1.26) µg/0.1 g lung homogenate] significantly. The expression of NF-κB p65 and TNF-α in lung tissue were both significantly increased on the first day and the 3rd day of the intoxication group compared with the control group and weakened obviously after the 7th day.

CONCLUSIONAcute paraquat poisoning can induce increased expression of both NF-κB p65 and TNF-α in lung tissue; the enhanced activity of NF-κB may take part in the process of pulmonary injury in PQ poisoning.

Animals ; Lung ; metabolism ; pathology ; Male ; Paraquat ; poisoning ; Rats ; Rats, Wistar ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

A cDNA fragment locating at the putative envelop protein 2(E2) region of GBV-C/HGV fused with Schistosoma japonicum, glutathione S-transferase(GST) was amplified with PCR from plasmid pGEX-E2. The amplified DNA fragment was inserted into plasmid pGEX-5X-1, at the downstream of the coding sequences of GST, in the same reading frame with the gene of GST. The fusion gene fragment of GST-E2 was amplified with PCR, using the recombinant plasmid pGEX-5X-1-E2 as the template. The amplified 1324 bp DNA fragment of GST-E2 was inserted into Pichia pastoris expression vector pPIC9K in reading frame with alpha-factor secreting signal peptide. The plasmid pPIC9K-GST-E2 was transformed into Pichia pastoris GS115 with electroporation. The transformants (His+ Muts) were selected and induced to express the 54kD GST-E2 fusion protein, which could be specially recognized by both the antisera directed against E2 and against GST. The GST-E2 fusion protein was purified with Sepharose 4B glutathione affinity chromatography to a purity of 95%. The expression was optimized to achieve the highest expression level of GST-E2 fusion protein which was accumulated up to 50% of total proteins in the culture supernatant. The GST-E2 protein derived from the recombinant Pichia pastoris was proved possessing antigenicity and high specificity by ELISA, probed with sera from the patients infected by GBV-C/HGV.
Animals ; Antigens, Viral ; genetics ; immunology ; isolation & purification ; GB virus C ; genetics ; immunology ; Gene Expression ; Genetic Engineering ; Glutathione Transferase ; genetics ; Hepatitis Antibodies ; blood ; immunology ; Humans ; Pichia ; Recombinant Fusion Proteins ; genetics ; immunology ; isolation & purification ; Schistosoma japonicum ; enzymology ; Viral Envelope Proteins ; genetics ; immunology ; isolation & purification

Activation of the innate immune system requires recognition of pathogen-associated molecular patterns, such as NOD-like receptors. The NOD-like receptor protein 3 (NLRP3) inflammasome is involved in induction of the pro-inflammatory cytokine, IL-1beta, and subsequent inflammatory responses. NLRP3 inflammasome plays important roles in the inflammatory and innate immune responses associated with autoimmune/inflammatory syndrome. However, analysis of the tissue distribution and expression profiles in BALB/c mice is still incomplete. In this study, we investigated the tissue distribution and expression pattern of NLRP3 in BALB/c mice to further elucidate its function in innate immunity in this commonly used laboratory animal model. NLRP3 mRNA expression levels and tissue distribution of the protein were investigated by real-time quantitative PCR and immunohistochemical analyses, respectively. NLRP3 mRNA expression was higher in the kidney and inguinal lymph nodes than in other tissues. Cytoplasmic expression of NLRP3 was detected in the epithelial reticular cells of the spleen and thymus, lymphocytes in the inguinal lymph nodes, cardiac muscle cells, cerebral cortex neurons, alveolar macrophages, renal tubule cells and liver sinusoidal endothelial cells. The results of this study will assist investigators in interpreting site-specific functions and roles of NLRP3 in inflammatory responses.
Animals ; Carrier Proteins/*genetics/metabolism ; *Gene Expression Regulation ; *Immunity, Innate ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Organ Specificity ; RNA, Messenger/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA

Objective To detect the expression levels of high mobility group box chromosomal protein 1 (HMGB1) and Th17 cells transcription factors, related cytokines in peripheral blood of rheumatoid arthritis (RA) patients and analyze the relations between HMGB1 and CRP, ESR, RF in RA patients. The other aim of this study is to identify the expression level of HMGBI and the relationship between HMGB1 and Th17 in RA patients. Methods The mRNA levels of HMGB1, RORyt, interleukin (IL)-17 in the peripheral blood mononuclear cells (PBMC) were determined by quantitative real-time PCR (QRT-PCR) from 80 patients with rheumatoid arthritis,including 32 RA patients in stable phase and 48 patients in active phase, and 50 healthy volunteers. The concentration of HMGB1, IL-23, IL-17 in plasma were detected by enzyme linked immunosorbent assay (ELISA), one-way ANOVA and Spearman's correleation were adopted for statistical analysis.Results The mRNAs of HMGBI, RORyt and IL-17 in RA patients were higher than that in healthy control group (P<0.05), especially in active RA patients [ HMGB 1 (0.424±0.262) pg/ml, RORγt (0.34±0.25) pg/ml,IL-17 (1.42±0.38) pg/ml,P<0.01 ] when compared with patients with stable disease. The concentration of HMGB1, IL-23 and IL-17 in the plasma of RA patients was higher than that of the healthy control group (P< 0.05), and was positively correlated with the expression levels of HMGB1, Th 17-associated factors and the level of CRP, ESR, RF in RA patients' plasma(P<0.05). Conclusion The HMGB1 and Thl7 cells levels are higher in active RA patients than those in patients with stable disease, arid there is significant positive correlation between them. Detection of peripheral HMGB1 and Thl7 cell-specific transcription factors or related cytokines can help to understand the development and progress of rheumatoid arthritis and provide clues for new treatment targets for RA.

OBJECTIVETo characterize the genetic aberrations in pediatric acute lymphoblastic leukemia (ALL).

METHODSNinety ALL cases were enrolled in the study from January 2009 to November 2011. Chromosome banding analysis and fluorescence in situ hybridization (FISH) were used to detect genetic aberrations.

RESULTS(1) Chromosome analysis: 35 (53.0%) of 66 cases who had metaphase were abnormal, and 24 cases had no metaphase. (2) FISH analysis: among the 31 cases who had normal karyotypes and 24 who had no metaphase detected by chromosome banding technique, 7 (22.6%) and 14 (58.3%) cases were abnormal detected by FISH, respectively. There were no statistically significant differences compared with chromosome analysis (P = 0.655). Among these 55 ALL cases TEL/AML1, bcr-abl and MLL fusion genes were observed in 16 (29.1%), 3(5.5%) and 2(3.6%) cases, respectively. (3) Cytogenetic aberration was observed in 56 of total 90 ALL cases (62.2%).

CONCLUSIONSCytogenetic changes are common in childhood ALL. Conventional cytogenetic study could reliably detected chromosomal abnormalities for ALL with assessable metaphase. FISH should be used as a complementary method for ALL patients who have poor chromosomal morphology or no metaphase cells, and combination of both methods can improve the detection rate of genetic abnormalities in childhood leukemia.

Adolescent ; Child ; Child, Preschool ; Chromosome Aberrations ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Infant ; Karyotyping ; Male ; Myeloid-Lymphoid Leukemia Protein ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics