Objective To explore the mechanism of immune tolerance in chronic B hepatitis, the effects of HBV infection on the functions of macrophages and related mechanism of signal transduction by transfection in vitro were studied. Methods Peritoneal macrophages of mice were isolated regularly and cultured, transfected transiently with pcDNA3-HBV or pcDNA3 plasmid DNA and cultured under the stimulation of LPS. After 72 h, RT-PCR was performed to detect the expression of PreS1, TNF-? or IL-1? mRNA. To detect the expression of NF-?B RelA protein by FCM, and the level of nitric oxide in cultural supernatant was measured with Griess reaction. Results After being transfected with pcDNA3-HBV,peritoneal macrophages had the expression of PreS1 mRNA, but have lower level of TNF-?、IL-1? mRNA and transcriptional factor NF-?B, compared with pcDNA3-transfected control group; the level of nitric oxide in pcDNA3-HBV group was also decreased. Conclusions Transient transfection of pcDNA3-HBV could decrease the function of macrophages directly by inhibiting NF-?B activity and effector molecules production, which may be one of the mechanisms of immune tolerance in chronic B hepatitis.

Objective　To study　how　ConA actives macrophages in vivo to produce cytotoxic effectors and its phagocytic functions,and　 cytotoxicity. Methods　 ConA was intraperitoneally injected(ip). Cock red blood cells(cRBC) were used to evaluate MΦ phagocytic activity,and S180 cells as target cells to analyze MΦ dependent cytotoxicity(MTC).Nitric oxide(NO),TNF-α　and IL-1 levels of MΦ cultural supernatant were measured using griess reagent,L929 cells MTT method and thymocytes proliferation test respectively. Results　 ConA could promote MΦ　to phagocytize cRBC and kill S180 cells,enhance the production of such factors as NO,TNF-α　and IL-1 by MΦ. There was significant difference compared with PBS　control group(P＜0.01). Conclusions　 ConA could stimulate MΦ to produce effectors, which mediate immune regulation of ConA to MΦ.

Objective:To explore effect of early rehabilitation therapy on improving prognosis and quality of life (QOL) in patients with acute myocardial infarction (AMI) .Methods :A total of 120 AMI patients treated in our hospital were selected .According to random number table ,they were randomly and equally divided into routine treatment group (n=60 ,received routine treatment) and rehabilitation group (n=60 ,received routine treatment combined rehabilitation therapy ) .Clinical symptoms ,myocardial enzymes ,cardiac function indexes ,complications , QOL ,length of hospital stay and hospitalization cost were statistically analyzed and compared between two groups . Results :Compared with routine treatment group after treatment ,there were significant reductions in incidence rates of dizziness ,low back pain ,leg weakness ,constipation ,palpitations (P<0.05~ <0.01) ,abdominal distension and complications (36.7% vs .5.0% );significant rise in Barthel index [ (61.9 ± 8.7) scores vs .(86.4 ± 6.9) scores] , significant reductions in length of hospital stay [(13.8 ± 3.2) d vs .(5.9 ± 2.6) d] and hospitalization cost [(13600 ± 1450) RMB vs .(8600 ± 1240) RMB] in rehabilitation group ,P<0.05~ <0.01 .Conclusion:Early rehabilitation therapy can significantly improve prognosis and quality of life ,shorten length of hospital stay , lower hospitalization cost in patients with acute myocardial infarction ,which is worth clinical extending .

Objective To detect the integration of hepatitis B virus (HBV) DNA in HBVrelated human hepatocellular carcinomas (HCC). Methods Extracted DNA from the liver tissue samples and amplified by nested polymerase chain reaction (PCR) with specially designed U-base primers. According to the known genes and human Alu repeat sequences (Alu repeat) , primers were designed respectively. Integrated clones combined target HBV DNA and the adjacent cell gene sequences were established by PCR and products were sequenced by biotechnology companies.Accurate locations of HBV genes integrated in the human genomes were analyzed by national center for biotechnology information (NCBI) basic local alignment search tool (BLAST) and Map Viewer search. Results In 24 HBsAg positive HCC samples, 15 cases showed the integrations of HBV fragment. And the other 8 samples didn't show any evidence of integration. Among 14 samples with integration, forward insertions of HBV DNA into the host chromosomal DNA were found in 10 samples and reverse insertions were found in 8 samples while both forward and reverse insertions were found in 5 samples. Analysis from viral-cellular junctions suggested that the integrations were all happened with truncated viral DNA and could be in any locus of X gene. Conclusion HBV DNA integration is not distributed evenly throughout the host genome.

Objective To investigate the nutritional risks in hospitalized patients with liver diseases and work out nursing countermeasures.Methods Forty patients with liver cirrhosis, 40 patients with primary liver cancer and another 40 revisiting after liver transplantation involved in the investigation with a self-designed general information questionnaire and the nutritional risk screening 2002. The three groups were compared in terms of nutritional risks.Results Among the total 120 patients,38.3%(46/120)of them took the nutritional risk and even 12.5%(15/120)had the risk of undernourishment.The risk in the patients with liver cirrhosis was higher than that in the other two groups(χ2=9.899 and 11.4299 and P=0.002 and 0.001,respectively).Conclusions The nursing staffs should pay attention to the nutritional status of patients with liver diseases, especially the patients with liver cirrhosis.It is necessary to take effective measures in order to improve the nutritional status of the patients to reduce the nutritional risks.

Objective To investigate the advantage of detaining peripherally inserted central catheter through the veins of lower extremity in neonatal intensive care unit.Methods 165 cases of infants were randomized into veins of lower extremity group (first saphenous veins,then popliteal vein) and veins of upper extremity group (first basilic vein,then median cubital vein,cephalic veins) from January to December 2012.We observed the success rate of detaining,catheter tip location,and complications such as phlebitis in the two groups.Results The success rate of detaining of lower extremity group was higher than that of upper extremity group,and ectopic rate of lower extremity group was lower than that of upper extremity group.There was no significant difference in complications such as phlebitis between two groups.Conclusions Peripherally inserted central catheter through the veins of lower extremity has more advantage over through the veins of upper extremity group.

AIM: To explore the relationship between caspase activation and the evasion of Legionella from macrophage elimination through a Legionella-infected macrophage model. METHODS: After infected by Legionella, the activity of caspase 3 in macrophages was analyzed by confocal microscopy as well as fluorescence reader. Growth and replication of Legionella in macrophage was assayed. Replication of Legionella was analyzed again to see the effect of caspase 3 inhibition on the growth of Legionella after use of caspase 3 inhibitor. RESULTS: Both confocal microscopy and caspase 3 fluorescent substrate analysis showed that Legionella virulent strain had powerful capability of activating caspase 3 while the mutant non-virulent strain did not have this capability. The virulent strain highly replicated in macrophages and the replication was significantly inhibited by caspase 3 inhibitor. CONCLUSION: Our results indicate that the intracellular caspase 3 is activated shortly after infection by Legionella virulent strain. The evasion of Legionella from the elimination of macrophages may be mediated by caspase 3 activation to a great degree.

Polymerase chain reaction (PCR) is a simple, quick and highly sensitive method. However the accuracy of the conventional PCR assay was often affected by false positives and false negatives. In this study, a protocol competitive PCR was used to reduce the false results in screening for selectable marker-free (SMF) Xa2l transgenic rice plants. The competitive template of Xa21 was the endogenous Xa2l homologous sequence located on chromosome 11. The competitive template of the selectable marker gene, hygromycin phosphotransferase (hpt), was an additive DNA extracted from hpt transgenic Nipponbare (Oryza sativa L). Through competitive PCR analysis of transgenic T1 plants produced by double right border binary vector, false positive or false negative samples were effectively diminished, and genuine SMF Xa21 transgenic plants were obviously obtained. Comparing with the conventional non-competitive PCR, competitive PCR increased the accuracy for selecting SMF Xa21 transgenic plants. The results of bacterial blight (BB) resistance tests and hygromycin B resistance assay of SMF Xa21 transgenic plants testified the reliability of this method.
Genetic Vectors ; Oryza ; genetics ; metabolism ; Plant Diseases ; genetics ; prevention & control ; Plant Proteins ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Transformation, Genetic

AIM: To explore the effect of TNF-related apoptosis inducing ligand (TRAIL), a new apoptotic inducing molecule on the biological activity of hepatocarcinoma cell line. METHODS: The expression of membrane binding TRAIL on HepG2 cells was detected by immuno-cytochemistry. Quantity of secretory TRAIL was assayed by ELISA method. The cytotoxicity and apoptosis induced by TRAIL was detected by MTT and TUNEL method, respectively. The telomerase activity of HepG2 cells was detected by TRAP-PCR assay kit. The expression of hTERT, the catalytic subunit of telomerase, was detected by FCM. RESULTS: TRAIL was constitutively expressed on the membrane of HepG2 cell line. Soluble TRAIL was also expressed to a certain degree. Cytotoxicity assay showed that TRAIL significantly inhibited the growth of hepatocarcinoma cells. TUNEL assay indicated that TRAIL induced apoptosis in hepatocarcinoma cells. Detection of telomerase activity showed that TRAIL inhibited telomerase activity and the expression of telomerase catalytic subunit. CONCLUSION: TRAIL is an effective molecule to inhibit the growth of hepatocarcinoma through multiple pathways, such as inducing apoptosis and inhibiting the activity of telomerase.

Objective:To investigate the mechanisms of microRNA (miR)-103a-3p/chitinase-3-like protein 1 (CHI3L1) in the proliferation and vascular mimicry of ovarian cancer cells and its effect on transforming growth factor-β (TGF-β) pathway.Methods:The relationship between the expression level of miR-103a-3p and the overall survival rate of ovarian cancer patients was analyzed by bioinformatics. The human ovarian adenocarcinoma SKOV3 cells were divided into 4 groups: control group, miR-103a-3p mimic group, miR-103a-3p mimic+ CHI3L1 group and CHI3L1 group. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the expression levels of miR-103a-3p, CHI3L1 mRNA and CHI3L1 protein respectively. The expression level of YKL-40 in cell culture fluid was detected by enzyme-linked immunosorbent assay. The cell viability, proliferation ability and angiogenesis ability of the 4 groups were detected by CCK-8 method, clone formation experiment and angiogenesis experiment. The dual luciferase report verified that miR-130a-3p targeted CHI3L1.Results:The overall survival rate of ovarian cancer patients with high expression of miR-103a-3p was higher than that of patients with low expression of miR-103a-3p ( χ2=6.187, P=0.048). The differences in miR-103a-3p and CHI3L1 mRNA levels among the control group, miR-103a-3p mimic group, miR-103a-3p mimic+ CHI3L1 group and CHI3L1 group were statistically significant ( F=198.254, P<0.001; F=60.214, P<0.001), miR-103a-3p mimic group and miR-103a-3p mimic+ CHI3L1 group had higher miR-103a-3p levels than the control group (all P<0.001), CHI3L1 group had higher CHI3L1 mRNA level than the control group ( P<0.001). The expression levels of CHI3L1 protein in the 4 groups were 2.25±0.23, 1.19±0.12, 2.29±0.28 and 4.31±0.37, and the difference was statistically significant ( F=18.675, P<0.001). The expression levels of YKL-40 in the cell culture fluids of the 4 groups were (1.84±0.20) ng/ml, (0.95±0.08) ng/ml, (2.64±0.25) ng/ml, (6.27±0.79) ng/ml, and the difference was statistically significant ( F=35.297, P<0.001). The YKL-40 level of the CHI3L1 group was significantly higher than that of the control group ( P<0.001), the miR-103a-3p mimic group was lower than the control group ( P<0.001), and the miR-103a-3p mimic+ CHI3L1 group was higher than the miR-103a-3p mimic group ( P<0.001). The cell viabilities of the 4 groups were 100%±2.54%, 76.23%±2.13%, 104.89%±3.56% and 137.42%±2.80%, and the difference was statistically significant ( F=23.584, P<0.001). The cell viability of the miR-103a-3p mimic group was significantly lower than that of the control group ( P<0.001), the CHI3L1 group was higher than the control group ( P<0.001), and the miR-103a-3p mimic+ CHI3L1 group was higher than the miR-103a-3p mimic group ( P<0.001). The number of clones formed in the 4 groups were 76.85±4.67, 21.56±2.85, 72.06±5.07 and 169.63±9.21, and the difference was statistically significant ( F=31.541, P<0.001). The proliferation capacity of the miR-103a-3p mimic group was significantly lower than that of the control group ( P<0.001), the CHI3L1 group was higher than the control group ( P<0.001), and the miR-103a-3p mimic+ CHI3L1 group was significantly higher than the miR-103a-3p mimic group ( P<0.001). The differences in the relative tube lengths and the tube bramches of the 4 groups were both statistically significant ( F=24.254, P<0.001; F=27.564, P<0.001). The differences in TGF-β and Smad levels of the 4 groups were both statistically significant ( F=30.254, P<0.001; F=34.187, P<0.001). The results of dual luciferase experiments showed that compared with the NC group, the luciferase activity in cells co-transfected of miR-103a-3p and CHI3L1-wt was significantly reduced. The difference of luciferase activity between the cells transfected with NC and co-transfected with miR-103a-3p and CHI3L1-mut was not significant. Conclusion:MiR-103a-3p can directly inhibit the expression of CHI3L1 and inhibit the proliferation and angiogenesis of ovarian cancer SKOV3 cells to inhibit ovarian lymphatic metastasis and distant metastasis, which may be related to the TGF-β pathway.