Objective To construct Gadd45a expression plasmid and induce its expression in human T cells.Methods Gadd45a was amplified by reverse transcription PCR from human embryonic stem cells,and cloned into the pcDNA3.1 vector.The recombinant plasmid or blank plasmid was transfected into Jurkat cells or normal human CD4+T cells using electroporation,and the expression of Gadd45a was detected by quantitative RT-PCR and Western blot.Results Human Gadd45a expression plasmid was constructed successfully.Gadd45a was overexpressed both in Jurkat cells and normal human CD4+T cells after these cells were transfected with pcDNA3.1-Gadd45a.Conclusion The construction of Gadd45a expression plasmid and induction of Gadd45a overexpression in human T cells lay the foundation for further research on the role of Gadd45a in the epigenetic mechanism.

Objective To explore the effects of UVB on the expression of Gadd45a gene and DNA methylation levels in Jurkat cells. Methods Jurkat cells were irradiated with UVB of 1.0 J/cm2 and 1.5 J/cm2 respectively, and collected at 6, 12, 24 and 48 hours after the irradiation. Real-time RT-PCR was used to detect the mRNA expression of Gadd45a gene and methylation-sensitive genes CD11a and CD70. Global methylation level was also measured by MethylAmp global DNA methylation quantification kit. Results After irradiation with UVB at 1.0 J/cm2, the mRNA level of Gadd45a increased but global methylation level decreased at 6, 12, 24 and 48 hours, and significant changes were observed at 6 and 12 hours for the level of both Gadd45a mRNA expression and global methylation (P ＜ 0.01 or 0.05). Elevated mRNA expressions of CD11 a and CD70 were also noted in Jurkat cells after irradiation with UVB of 1.0 J/cm2, and significant elevation was observed at 12 hours (both P ＜ 0.05 ). After irradiation with UVB of 1.5 J/cm2, there was a statistical increase in the mRNA expressions of Gadd45a, CD11 a, CD70, together with a statistical decrease in global methylation level in Jurkat cells, at 6, 12, 24 and 48 hours (P ＜ 0.01 or 0.05). The mRNA expression of Gadd45a negatively correlated with the global level of DNA methylation in Jurkat cells (r = -0.395, P ＜ 0.05). Conclusion UVB irradiation can upregulate the expression of Gadd45a, but downregulate the global DNA methylation level in Jurkat cells.

Objective To investigate the clinical values of laparoscopic radiofrequency ablation in liver cancer and its impacts on serumal VEGF and MMP-2. Method From Jan, 2012 to Dec, 2013, a series of patients with primary liver cancer were studied, patients were randomly signed into LAFA group or control group. During the study period, LRFA group were treated with laparoscopic radiofrequency ablation combined with FOLFOX4 chemotherapy while patients in control group were treated with PIAF chemotherapy only. The primary outcomes were the Health related quality of life score (HRQL), the degree of solid tumors classification, progression-free survival duration and 2-year mortality. The secondly primary outcomes included the level of serumal VEGF and MMP-2. Result When compared with the control group, patients in LRFA group got a significantly lower rate of disease progression (28.33 % vs 50.00 %, P = 0.015); a longer progression-free survival duration (500 vs 380 d, P = 0.013); a higher HRQL (80.33 ± 5.84 vs 65.87 ± 9.59, P = 0.000); a significantly lower level of VEGF at 7 days, 14 days, 28 days and 6 months after the clinical intervention were started (all P values were 0.000); a significantly lower level of MMP-2 at 14 days, 28 days and 6 months after the clinical intervention were started (the P values were 0.003, 0.001 and 0.000). Conclusion Laparoscopic radiofrequency ablation improved the long-term clinical outcomes and decreased the serumal level of VEGF and MMP-2.

Objective To explore the anti-HBV activity of anodonta polysaccharides (AP) and dose-effect relationship in vitro.Methods HepG2.2.15 cells were cultured in vitro and incubated at 37℃ for nine days with AP at a dilution ratio of 1∶10.The expression of HBsAg and HBeAg were detected using ELISA and HBV-DNA copies were detected by real-time fluorescent quantitative PCR.Based on Thiele-type continued-fraction interpolation method,the anti-HBV activity of AP was studied,and the IC50 and the maximum inhibition rate were calculated.Results AP had significant inhibitory effect on the expression of HBsAg and HBeAg in HepG2.2.15 cells in vitro,as well as HBV DNA replication.By Thiele-type continued-fraction interpolation the equations of dose-effect relationship were obtained to determine the maximum inhibition rates of AP on HBeAg and HBsAg secretion being 47.7％ and 56.4％,and the IC50 inhibiting the expression of HBeAg being 143.7mg/L.AP was also able to inhibit HBV-DNA replication and the maximum inhibition rate was 17.8％ with the same method above.Conclusion Anodonta polysaccharides have anti-HBV activity.The thiele-type continued-fraction interpolation method is simple and practical and could be used as a new method for the analysis of drug activity.

AIM: To observe anti inflammatory, anti bacterial and immune regulation effects of shuyanling soft capsule. METHODS: The dimethylbenzene induced the degree of ear swelling in mice and turpentine induced proliferation in rats were used to observe the anti inflammation effects of shuyanling soft capsule. The anti bacterial effects were observed both in vitro and in vivo. The immune regulation effects were studied by the splenocyte proliferation response, the activity of antibody producing cells and the phagocytosis of leukocyte in mice. RESULTS: Shuyanling soft capsule inhibited dimethylbenzene induced the degree of ear swelling in mice and turpentine induced proliferation in rats, protected the mice from the infections of staphylcoccus aureus and salmonella typhi, and repressed sixteen kinds of bacterium in vitro. Furthermore, the capsule remarkably enhanced the phagocytosis of neutropil and increased the activity of spleen antibody producing cells and the transformation of the T and B cell in spleen in mice. CONCLUSION: Shuyanling soft capsule has anti inflammatory, anti bacterial and immune regulation effects.

Objective To investigate the relationship between the methylation of CpG island of RUNX3 gene promoter and its expression in a human cutaneous malignant melanoma cell line A375, and to assess the role of RUNX3 gene methylation in the pathogenesis of human cutaneous malignant melanoma. Methods Cultured A375 cells were treated with various concentrations (0, 1, 5, 10, 20 μmol/l) of 5-azacyti-dine for 24 or 72 hours followed by another 5 days of culture. Then, methylation-specific PCR (MSP) was performed to evaluate the methylation status of RUNX3 promoter region, and Western-blot analysis to detect the protein expression of RUNX3 in A375 cells. Results The RUNX3 gene promoter region was hypermethylated in untreated A375 cells, along with the absence of protein expression of RUNX3. However, after the treatment with 5-azacytidine, the promoter region of RUNX3 gene was demethylated partly, and the expression of RUNX3 protein was restored in A375 cells. Further, the expression intensity was directly correlated with the concentration of 5-azacytidine. Conclusions The promoter hypermethylation of RUNX3 gene may be related to the silencing of RUNX3 gene expression in A375 cells, whereas 5-azacytidine can cause the demethylation of RUNX3 gene, reactivate the gene which has been inactivated by the promoter hypermethylation, and finally induce the re-expression of RUNX3 protein.

Aging is associated with declines of the muscular strength and cardiorespiratory functions, resulting in an impaired capacity to perform daily activities. Frailty is a clinical geriatric syndrome characterized by decreases in the biological functional reserve and resis-tance to stressors, which puts older individuals at special risk of disability. To counteract the muscular strength and cardiorespiratory func-tions declines associated with aging, as well as to prevent and treat the frailty syndrome, the strength and endurance training seems to be an effective strategy to improve muscular strength and endurance performance. This article reviewed the exercise prescription in health and frail elderly subjects according to the current situation of the researches about muscular strength and cardiorespiratory function training. Based on the results found, for the healthy older adults, strength training should be performed at moderate-to high-intensity (60%-80%of 1 RM ), and moderate volume (2-3 sets per exercise, 2-3 times/week ). Also, endurance training should be performed at moderate-to high-in-tensity (60%-85% of VO2max), and moderat volume (25-40 min). The frail older adults should exercise 2-3 times a week, for 45-60 min. Strength, endurance, flexibility, and balance training components should be incorporated, and strength and balance training should be empha-sized.

Objective To explore the effects of strength training on aging muscular atrophy and inflammatory factors levels in old men.Methods 11 old men participated in 12 weeks of strength training. Muscle strength/muscle thickness and serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, high sensitivity C-reactive protein (hsCRP) and adiponectin were measured before and after training. Results After training, the muscle thickness increased, but there was no significantly difference (P>0.05); the levels of hsCRP decreased (P<0.001) and adiponectin increased (P<0.05). Conclusion Strength training may assist in maintaining or improving muscle strength/muscle mass and reducing inflammatory factors levels.

Objective To summarize the experience in surgical treatment of Ebstein anomaly,and evaluate the therapeutic effect.Methods Thirty-five patients of Ebstein anomaly were treated by operation.New York heart association(NYHA)heart function classⅠ?was in 11 cases,classⅡ?was in 14 cases,class III-Ⅳ?was in 10 cases.The cue of echocardiogram about tricuspid valve regurgitation in 24 cases were severe,5 cases were moderate and 6 cases were mild.Two patients accepted tricuspid valve replacement.Thirty-three cases were accepted tricuspid valve repair and right ventricle folded,in which 2 cases accepted Danielson method,and 31 cases accepted Carpentier method,among them,9 cases accepted one and a half ventricular repair.Associated heart anomaly was corrected at the same time.Results One patient died.There were 2 cases with third degree atrioventricular block after tricuspid valve replacement,2 cases with low cardiac output syndrome and 2 cases with atrial fibrillation combined premature ventricular contraction.The heart was lower by chest X-ray and echocardiogram examination,tricuspid valve regurgitation in 22 cases were disappeared,9 cases were mild,3 cases were moderate.All patients were followed up from 1 month to 7years,tricuspid valve regurgitation in 21 cases were disappeared,12 cases were mild,heart function were class Ⅰ-Ⅱ.Because of heart function aggravation,1 patient was operated again 3 years after operation.Condusions Ebstein anomaly is a rare congenital heart disease,Carpentier method tricuspid valve repair can decrease regurgitation obviously and protect right ventricular function.One and a half ventricular repair should be adopted according to the function of tricuspid valve and right ventricle.

Objective To observe hypoxia-induced pulmonary arterial endothelial-to-mesenchymal transition and investigate the role of transforming growth factor β1 (TGF-β1) in the process. Methods Pulmonary arterial cells improved by adherence method were cultured in normoxia (containing 21%O2,5%CO2 and 74%N2) or hypoxia (containing 1%O2,5%CO2 and 94%N2) for 1,4,or 7 days,respectively. Endothelial-to-mesenchymal transition was confirmed with morphological observation and expression of α-smooth muscle actin (α-SMA) by immunocytochemistry. Expression of TGF-β1 was evaluated by RT-PCR and Western blot,and α-SMA by Western blot. Results Hypoxia-induced paving-stone-like pulmonary arterial endothelial cells transdifferentiating to polygonal cells with high-expression of α-SMA. TGF-β1 expression was increased significantly after 7 days of hypoxia. TGF-β1 stimulating alone increasedα-SMA expression of pulmonary arterial en-dothelial cells;while,SD-208,inhibitor of TGF-β1,abolished the above effect. Conclusion Hypoxia can induce endothelial-to-mesenchymal transition. And TGF-β1 plays an important role in the process.