Red in vivo recombination is a new kind of genetic engineering technique based on homologous recombination. In this work, plasmid pKD46 which expresses Red recombination proteins is transferred into Escherichia coli strain DH5?.The kanamycine resistant gene is generated by PCR by using primers with homology to hisDCB gene of E.coli chromosome. Thus, the hisDCB gene was replaced with kanamycine resistant gene by the plasmid recombination system, then the resistant gene was eliminated by a helper plasmid encoding the FLP recombinase. At last, a E.coli histidine auxotroph which is sensitive to kanamycine was got. The results indicate that Red in vivo recombination is a convenient method to construct auxotrophs.

OBJECTIVETo investigate the role of the key intracellular signaling molecule glycogen synthase kinase-3 beta in the mechanism of liver ischemia reperfusion (IR).

METHODSC57BL/6 mice were subjected to 90 min warm liver cephalad lobe ischemia, followed by various length of reperfusion. Experiment groups included sham control group, liver IRI model group and glycogen synthase kinase-3 beta inhibitor-treated group (SB216763 in DMSO, 25 g/kg, i.p, 2 hour prior to the onset of liver ischemia). The expression of glycogen synthase kinase-3 beta protein was analysed by Western blotting. The serum ALT levels were determined to reflect the function of liver. The affected liver lobes were harvested for histology analysis. The inflammatory gene expression was detected by Quantitative PCR.

RESULTSBy western blot analysis, we found that ischemia itself activated glycogen synthase kinase-3 beta by a significant decrease of its phosphorylation. Glycogen synthase kinase-3 beta inhibitor SB216763-pretreatment ameliorated the liver damages significantly as compared to the controls (sALT: 2046+/-513 U/L vs 5809+/-1689 U/L, P = 0.0153), and suppressed the gene expressions of IL-12, TNFa, IL-1b and IL-6.

CONCLUSIONSThis study demonstrated that the ischemia process modulated liver innate immune activation via a GSK-3-dependent mechanism which favored the development of a pro-inflammation response and lead to liver tissue damages. GSK-3b may be a new therapeutic target to ameliorate liver IRI in transplant patients.

Animals ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Inflammation ; metabolism ; Liver ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury ; metabolism ; pathology

OBJECTIVETo determine the mechanism underlying the therapeutic activities of glycogen synthase kinase 3b (GSK3b) against hepatic ischemia-reperfusion (H-IR) injury by investigating the inhibitive effects of GSK3b on inflammation mediated by Toll-like receptor 4 (TLR4).

METHODSC57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups: the H-IR untreated model (control group), and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide (LPS) endotoxin alone (inflammation group) or with pretreatment of the SB216763 GSK3b-specific inhibitor (intervention group). To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The cells were then divided into the same three groups as the whole mouse system: control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively.

RESULTSThe phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-stimulated ERK, JNK and p38 phosphorylation in macrophages. In the mouse model, GSK3b activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 (control: 0.21 ± 0.08, inflammation: 0.83 ± 0.21, intervention: 1.76 ± 0.67; F = 3.16, P = 0.027) but to significantly inhibit the gene expression of pro-inflammatory cytokines IL-12 (control: 0.11 ± 0.05, inflammation: 0.85 ± 0.11, intervention: 0.43 ± 0.10; F = 2.67, P = 0.038), TNF-a, (control: 0.052 ± 0.012, inflammation: 8.11 ± 0.98, intervention: 3.9 ± 0.82; F = 4.13, P = 0.016), IL-6 (control: 0.22 ± 0.08, inflammation: 6.37 ± 0.81, intervention: 2.11 ± 0.63; F = 3.21, P = 0.024), and IL-1b (control: 0.12 ± 0.07, inflammation: 2.51 ± 0.62, and intervention: 1.28 ± 0.33; F = 2.22, P = 0.030).

CONCLUSIONInhibition of GSK3b selectively regulates the expression of anti-inflammatory and pro-inflammatory cytokines in liver Kupffer cells (liver macrophages). This process may be one of the mechanisms underlying the ability of GSK3b to ameliorate hepatic ischemia-reperfusion injury, possibly because inhibition of pro-inflammatory cytokines may indirectly mediate liver cell apoptosis.

Animals ; Cells, Cultured ; Cytokines ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Inflammation ; metabolism ; pathology ; Lipopolysaccharides ; adverse effects ; Liver ; pathology ; Macrophages ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo study the relationship between pulmonary surfactant-associated protein B (SP-B) gene polymorphisms and their susceptibility to neonatal respiratory distress syndrome (RDS).

METHODSEighty-eight preterm infants with RDS (RDS group) and 103 infants without RDS (control group) were enrolled. The genomic DNA was isolated using DNA kits. Polymerase chain reaction with restriction fragment length polymorphism technique was used to detect the genotype and allele frequency of the SP-B -18A/C and SP-B 1580C/T single nucleotide polymorphisms. The association between the polymorphisms and RDS was analyzed.

RESULTSSP-B -18A/C and SP-B 1580C/T were found to be polymorphic in both RDS and control groups. The frequencies of CC genotype (X2=12.26, P<0.01) and C allele (X2=11.97, P<0.01) of SP-B 1580C/T were significantly higher in the RDS group than in the control group. The C allele significantly increased the risk of RDS (OR=2.26, 95%CI: 1.42-3.60). The frequencies of genotype and allele of SP-B -18A/C showed no significant difference between the two groups.

CONCLUSIONSSP-B 1580C/T polymorphism contributes to the etiology of RDS and may serve as the susceptibility gene for RDS. The C allele increases the risk of RDS. SP-B -18A/C shows no association with the etiology of RDS.

Genetic Predisposition to Disease ; Genotype ; Humans ; Infant, Newborn ; Polymorphism, Single Nucleotide ; Pulmonary Surfactant-Associated Protein B ; genetics ; Respiratory Distress Syndrome, Newborn ; etiology ; genetics

OBJECTIVETo identify independent risk factors for myocardial infarction (MI) in Chinese men and to develop a model to predict risk profile of an individual suffering MI.

METHODSStudy sample included 5 137 men aged 45.2 +/- 7.8 years who came from a cohort in Beijing Capital Steel and Iron Company, based on the three surveys on coronary heart disease conducted in 1974, 1979 and 1980, respectively. Demographic data and other risk factors, such as life style, medical history, blood pressure, total serum cholesterol level (TC), etc. were collected according to the same protocol in 1980. All the participants were followed up for MI in an average period of 20.84 years until 2001.

RESULTS(1) There were 122 cases with MI identified during the period of follow-up, with an incidence of MI 117.4 per 100 000 person-years. Age of more than 50, smoking, higher systolic and diastolic blood pressure (SBP and DBP) levels, higher TC all were identified as important risk factors of MI. (2) Incidence of MI increased with TC. An increment of 0.52 mmol/L of TC significantly increased relative risk of MI by approximately 40% after adjusted for age, blood pressure and smoking. (3) An increment of 20 mm Hg in SBP or 10 mm Hg in DBP associated with a 40% increase in incidence of MI, adjusting for age, TC and smoking. (4) Smoking was the most risky factors for MI. Smokers had 2.3 times risk of MI, after as compared to non-smokers (or its incidence increased by 137%), after adjusting for blood pressure, TC and age, etc. (5) Incidence of MI increased by 20% with increment of five-year of age in those aged over 50 (P < 0.05), after adjusting for blood pressure, TC and smoking. And, (6) finally, based on multivariate logistic and Cox regression analyses, a model containing several risk factors, such as age, blood pressure, TC and smoking, was developed to predict individual's risk for afflicting MI.

CONCLUSIONSResults of this prospective study showed several established risk factors for MI, including age, blood pressure, TC and smoking all as independent predictors of MI in Chinese men. It is clear and rational that intervention and modification of those traditional risk factors can lead to a decrease in coronary events in Chinese population.

Adult ; Age Factors ; Blood Pressure ; physiology ; China ; epidemiology ; Cohort Studies ; Follow-Up Studies ; Humans ; Incidence ; Iron ; Male ; Metallurgy ; Middle Aged ; Multivariate Analysis ; Myocardial Infarction ; epidemiology ; etiology ; Proportional Hazards Models ; Prospective Studies ; Risk Factors ; Smoking ; adverse effects ; Steel ; Triglycerides ; blood

Aim To evaluate the effects of different doses of baicalin on rheumatoid arthritis (RA) in SD rats and explore the possible mechanism.Methods Firstly,the model of RA in SD rats was prepared and the hind foot swelling was measured;HE staining was used to observe the pathological changes of the knee joint synovial tissue;RT-qPCR was adopted to determine the mRNA expressions of TLR2 and MyD88 in synovial tissue;Western blot was used to determine the protein expressions of TLR2,MyD88 and NF-κB p65 aher intragastric administration of different doses of baicalin solution.Results Compared with the model,baicalin (60 and 30 mg · kg-1) could inhibit the proliferation of fibroblasts and inflammatory damages in synovial tissue,significantly cut down mRNA expressions of TLR2 and MyD88 (P ＜ 0.05) and markedly reduce protein expressions of TLR2,MyD88 and NF-κB p65 (P ＜ 0.01).Conclusion Baicalin has good effects on RA,which may be realized by inhibiting the activation of TLR2-NF-κB signaling pathway.

AIM:To investigate the effect of recombinant bovine basic fibroblast growth factor (rb-bFGF) eye drops and hydroxyl indican eye drops on tear film stability and dry eye symptoms after age-related cataract surgery.METHODS:Totally 80 patients with 80 affected eyes undergoing age-related cataract surgery in our hospital from January 2015 to October 2016 were selected as study subjects,and they were randomly divided into control group and experimental group with 40 patients (40 affected eyes) in each group.The two groups were treated with hydroxyl indican eye drops and rb-bFGF eye drops,respectively.The clinical curative effect,inflammation related factors [interleukin 6 (IL-6),tumor necrosis factor α (TNF-α)],Schirmer test (S Ⅰ t),break-up time (BUT) of tear film,corneal sodium fluorescein staining (FL) and scores of dry eye symptoms in the two groups were observed.RESULTS:The total treatment effective rate of experimental group after treatment was significantly higher than that of the control group (90.0％ vs 72.5％;x2 =4.021,P＜ 0.05).Before treatment,there was no significant difference in IL-6,TNF-α,S Ⅰ t,BUT,FL score and scores of dry eye symptoms between the two groups (P＞0.05).After treatment,IL-6,TNF-α,S Ⅰ t,FL score and scores of dry eye symptoms in two groups significantly decreased while BUT significantly increased (P＜ 0.05),and changes of the indexes were more significant in the experimental group than the control group (P＜0.05).CONCLUSION:In the treatment of patients after age-related cataract after surgery,rb-bFGF eye drops has more advantages over hydroxyl indican eye drops in regulating the expression of inflammatory factors,improving the tear film stability and relieving dry eye symptoms.

OBJECTIVETo express recombinant human interferon lambda2 in E.coli and to study its antiviral activities.

METHODSAccording to preferred codons used in E.coli, the highly-expressed human interferon lambda2 gene was designed, synthesized and cloned into expression vector pBV220 and transfected into E.coli DH5alpha. The expressed product was purified by using CM FF and size exclusion chromatography. Its antiviral activities were tested on different cells.

RESULTSThe expressed product was calculated about 15% of the total E.coli protein. The purified protein reached about 90% purity. Its specific antiviral activity was about 1.5 x 10(6) IU/mg on WISH/VSV test system. It was shown that the antiviral activity of the product on primates-origin cells seemed to be much higher than that on other non-primates-origin cells, indicating that interferon lambda2 possessed more stringent species specificity as compared with interferon-alpha2b. New interferon lambda2 showed similar anti-HBV activity as interferon-alpha2b.

CONCLUSIONRecombinant human interferon lambda2 could be expressed on E.coli. The purified product showed more stringent species specificity and similar anti-HBV activity as compared with interferon-alpha2b.

Animals ; Antiviral Agents ; pharmacology ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cercopithecus aethiops ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Hepatitis B virus ; drug effects ; Humans ; Interleukins ; biosynthesis ; genetics ; isolation & purification ; pharmacology ; Microbial Sensitivity Tests ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Vero Cells

The expression of human clotting factor VIII gene was observed in transgenic off spring of mice through artificial insemination with sperm as carriers. Female mice were impregnated through artificial insemination by introducing sperm carrying pRC/RSV-hF VIII BD, which contained human F VIII BD (B-domain deleted) cDNA (hF VIII BD c DNA), into the uteri. During the fourth week after the birth of new-born mice, PCR was used to screen hF VIII BD cDNA positive transgenic mice, then blood of which was collected for detecting the antigen and Anti-hF VIII inhibitors, simultaneously, the transcription and expression of hF VIII BD cDNA were investigated by Northern blot and Western blot. The results showed that 7 became pregnant of 20 inseminated mice, and 11 new-born mice came into the world, out of which 9 survived at last. Three hF VIII BD cDNA-positive-transgenic mice had been screened out by PC R, in which the antigen of human F VIII in plasma was 8.65 ng/ml, 7.84 ng/ml and 8.44 ng/ml, respectively, the Anti-hF VIII inhibitors were all negative. Northern blot and Western blot showed that the transcription and expression of hF VIII BD cDNA existed in tissues such as spleen, liver, lung and kidney of 3 transgenic mice. It was concluded that transgenic mice carrying human F VIII gene can be generated by sperm-carrier techniques and express human F VIII protein. This experiment provides important data for manufacturing transgenic animal carrying human F VIII gene, which can work as a biological reactor to produce human F VIII protein, through sperm-carrier techniques.
Animals ; Blotting, Northern ; Blotting, Western ; Factor VIII ; genetics ; Female ; Gene Expression ; Humans ; Insemination, Artificial ; Male ; Mice ; Mice, Transgenic ; Pregnancy ; Spermatozoa ; metabolism