Objective To identify the expression of a fusion gene GCA formed from GM-CSF gene and LMP2A gene of Epstein-Barr virus (EBV) in a recombinant BCG (rBCG) and to study its immunoge-nicity.Methods The rBCG was constructed to express the fusion gene GCA and the expressed products were detected by Western blot assay .ELISA was performed to measure specific antibody titers in serum sam-ples from mice immunized with rBCG .Lactate dehydrogenase assay was used to analyze the cellular immuni-ty of mice.A mouse model of EBV-positive gastric carcinoma was established to evaluate the therapeutic effects of rBCG.Results The target proteins of GM-CSF and LMP2A were successfully expressed in rBCG . The specific antibodies were detected in rBCG immunized mice as indicated by ELISA .The maximum anti-body titer reached 1 ∶27 900 [(326.5±7.8) pg/ml] as injection with rBCG 5×108/mouse.The rBCG in-duced cytotoxicity of cytotoxic lymphocytes (CTLs) to EBV-positive gastric carcinoma cells (GT39) (with a killing rate of 89.6%±6.8%) was significantly higher than that of control group (P<0.05) The sizes of tumor in PBS control group [(1964.0±548.7) mm3] and BCG group [(1268.65±72.4) mm3] were big-ger than those in rBCG group [(168.64±78.80) mm3].Conclusion The rBCG expressing GM-CSF and LMP2A fusion gene was successfully constructed .The rBCG could induce humoral and cellular immune re-sponses in mice and inhibit the growth of tumor .

Objective:To construct GM-CSF and EB virus LMP2A fusion gene recombinant adenovirus and identificate it , using recombinant adenovirus to do immunologic study.Methods:With RT-PCR and Western blot to detect GC 2A gene expression of the recombinant adenovirus , with ELISA to analysis antibodies of vAd-GC2A stimulation in murine body , lactate dehydrogenase assay to detect the mouse cellular immunity effect , thereby the recombinant adenovirus immune effect was initially analysis.T-test method was used to do preliminary analysis and evaluation of the immune effect of recombinant adenovirus .Results:GC2A fragment amplified by PCR, the size was 1 961 bp and it was the expected.The results showed, the recombinant adenovirus vAd-GC2A proteins can be recognized by sera antibody of GM-CSF ( LMP2A); the results of ELISA showed that vAd-GC2A can stimulate the generation of antibodies.EBV-positive tumor cell killing rate (%) by CTL induced recombinant adenovirus was (66.7 ±6.9)%, at the same time the adenovirus type 5 wild strains and PBS were (24.3 ±2.5)%and(32.4 ±3.1)%only(P≤0.05).Conclusion:Fusion gene has been successfully constructed , vAd-GC2A in mice have humoral and cellular immune function.The data suggest that the vAd-GC2A should play a potent role in preventing the positive EB virus tumor.

ObjectiveTo investigate the expression of T helper (Th) 17 cells and the related interleukin 17 (IL-17) in acute renal allograft rejection in mice and its significance.Methods We established a mouse renal allograft model,in which mice were randomly divided into a renal isograft group and an acute renal allograft rejection group.Three and 7 d after the transplantation,the serum interferon (IFN)-γand IL-17 levels in the mice were determined by enzyme-linked immunosorbent assay,the percentage of Th1 and Th17 cells in the total kidney-infiltrating lymphocytes was investigated by flow cytometry,and the transplanted kidney species were given routine pathological examination after fixation with 10％ formalin.ResultsCompared with the isograft group,the allograft mice showed a significantly higher content of IL-17 (P ＜0.05 ) but not IFN-γ in the serum 3 d after transplantation,and showed significantly higher serum IL-17 and IFN-γcontents 7 d after transplantation (P ＜ 0.05 ).Also,compared with the isograft group,the allograft mice exhibited significantly higher percentage of Th1 and Th17 cells on both day 3 and day 7 ( P ＜ 0.05 ).In the allograft group,the contents of serum IFN-γand IL-17 and the percentage of Th1 and Th17 cells were significantly higher on day 7 than on day 3 (P ＜ 0.05 ).Routine pathological examination indicated that,as time passed,the allograft mice showed gradually stronger rejection responses.ConclusionTh17 cells might play an important role in the development of acute renal allograft rejection,and IL-17 can be used as an early indicator of acute rejection.

Background Ultraviolet irradiation promotes cellular apoptosis by affecting the mitochondrial transmembrane potential,including human lens epithelial cells (LECs).Gene associated with retinoid-interferoninduced mortality-19 (GRIM-19),a cell death regulatory protein,is essential for the assembly and function of mitochondrial complex Ⅰ.However,whether LECs apoptosis induced by ultraviolet irradiation is related to GRIM-19 is still unclear.Objective The purpose of this study was to investigate the relationship between the apoptosis of human LECs caused by ultraviolet with GRIM-19 expression in vitro.Methods Human LEC line(SRA01/04)was cultured in α-MEM containing 10％ fetal bovine serum.The cells were exposed to ultraviolet ray at doses of 0,30,60,90,120 or 150 mJ/cm2 when cell growth reached the logarithmic phase and 80％ confluency.The rate of apoptosis of the cells was assayed using flow cytometry,and the level of expression and relative amount of GRIM-19 protein (GRIM-19/β-actin) were detected by Western blot.The relationship between apoptosis and the GRIM-19/β-actin value among the different treatment groups was compared using One-way ANOVA,and the correlation of LECs apoptosis rate and GRIM-19 expression level was assessed by Pearson linear analysis.Results A significant difference was found in the apoptosis rate among the different treatment groups(F=149.32,P＜0.01).Compared with the 0 mJ/cm2 ultraviolet irradiation group,the apoptosis rate of LECs was significantly increased in the 60,90,120 and 150 mJ/cm2 ultraviolet irradiation groups (q =17.02,-25.20,-29.41,-8.61,P ＜ 0.01).The expression of the GRIM-19 protein in the LECs suspension was enhanced by ultraviolet irradiation at 60,90,120 and 150 mJ/cm2.The relative expression of the GRIM-19 protein (GRIM-19/β-actin) was significantly different among the various groups (F=6.87,P＜0.05),and the GRIM-19/β-actin values in the 60,90,120,150 mJ/cm2 ultraviolet irradiation groups were elevated in comparison with the un-irradiated group(2.01±0.76,2.98± 1.80,3.97± 1.61,2.42± 1.28 vs.0.56±0.23),which showed statistically significant differences (q =4.12,-5.04,-7.09,-3.85,P ＜ 0.01).In addition,a positive correlation was seen between the rate of apoptosis and the expression of the GRIM-19 protein(r=0.71,P＜0.01).Conclusions GRIM-19 is expressed in normal human LECs.The apoptosis of human LECs accompanies the up-regulation of GRIM-19.The expression of GRIM-19 in LECs increases with ultraviolet irradiation in a doseindependent manner.

Background Epithelial-myofibroblast transition (EMT) of human lens epithelial cells (LECs) induced by transforming growth factor-β2 (TGF-β2) is the main mechanism in the pathogenesis of posterior capsular opacification(PCO).Seeking an effective drug capable of inhibiting this process is important for the prevention and treatment of PCO.Objective The purpose of this study was to investigate the inhibitory effect of rapamycin (RAPA)on the proliferation of human LECs and TGF-β2-induced EMT.Methods Human LEC strain(SRA01/04)was cultured in DMEM with high glucose and 10％ fetal bovine serum.The cells were consequently cultured in serumfree DMEM with 5 mg/L TGF-β2,TGF-β2+10 mg/L RAPA,TGF-β2 + 100 mg/L RAPA,TGF-β2 + 1000 mg/L RAPA or TGF-β2 +10 000 mg/L RAPA for 72 hours,and SRA01/04 cultured in serum-free DMEM were used as control.The proliferation rate(A490)of SRA01/04 in the different groups was detected using the MTT assay and the rate of inhibition of RAPA was calculated.The expressions of the α-smooth muscle actin(α-SMA) and E-cadherin(E-cad)mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The changes in the expression of α-SMA and E-cad in SRA01/04 were evaluated by Western blot 24,48 and 72 hours after TGF-β2 +400 mg/L RAPA treatment.Results The A490 value of SRA01/04 was 0.680±0.020,0.550±0.013,0.480±0.014,0.400±0.011 and 0.200±0.019 in the control group,TGF-β2 group,TGF-β2 + 10 mg/LRAPA group,TGF-β2 + 100 mg/L RAPA group,TGF-β2 + 1000 mg/L RAPA and TGF-β2 + 10 000 mg/L RAPA group,respectively,showing a gradually declining trend in SRA01/04 rate of proliferation with increasing RAPA concentrations (F =101.920,P =0.000).RT-PCR and Western blot assay showed that the relative expression levels of α-SMA mRNA (α-SMA mRNA/β-actin mRNA)and protein (α-SMA/β-actin)in the cells were significantly increased in the TGF-β2 treatment group.However,with exposure to RAPA,the relative expression levels of α-SMA mRNA and protein were significantly lowered with increasing RAPA concentrations,but the expression levels of E-cad mRNA and protein were raised (α-SMA mRNA:F =294.660,P =0.000 ; α-SMA protein:F =346.950,P =0.000 ; E-cad mRNA:F =264.250,P =0.000 ; E-cad protein:F =317.327,P =0.000).In addition,after exposure to 400 mg/L RAPA,the expression levels of α-SMA protein gradually reduced and those of E-cad protein gradually increased with increasing treatment durations,showing significant differences among the different time points (α-SMA:F =693.864,P =0.000 ;E-cad:F=369.286,P =0.000).Conclusions RAPA can inhibit the proliferation of SRA01/04 in vitro and arrest EMT of SRA01/04 induced by TGF-β2 in a dose-and time-dependent manner.

The effect of phenol concentration on the structure and function of microbial communities,which were cultured in different conditions using coking wastewater biofilm as seeding,was investigated by Biolog and denaturing gradient gel electrophoresis(DGGE)methods.The less number of bands of cultivated sam-ples on the denaturing gradient gel electrophoresis fingerprint of 16S rRNA gene indicated reduction of di-versity after enrichment and cultivation.Some bands on the DGGE gel were significantly influenced by the phenol concentration in medium.The results of Biolog showed that the original biofilm sample had the highest substrate utility capacity as measured by average well color development(AWCD).But low concen-tration of phenol enriched sample S7 showed more diverse activity on the utility of Carboxylic acids.The principal component analysis(PCA)of Biolog data revealed that the metabolic patterns were similar when using the same phenol concentration,although the sample S7 much less similar to other cultivated samples.These results suggested that the enrichment and cultivation with phenol supplemented decreased the diver-sity and also changed the metabolic function of the microbial community.Lower phenol concentration in-creased the microbial community metabolic activity.The phenol degrading capacity of isolates from each samples indicated that the enrichment and cultivation condition had changed the type and property of cul-truable bacteria.Based on these results,we concluded that the different microorganisms will be isolated un-der different cultivation condition.

In the previous study, a high-throughput screening method was established to find the antagonists of CD36. In the present study, a new compound named IMB-1680 was found using this method. The anti-atherosclerotic activities of IMB-1680 were then evaluated. Dose-dependent activities of IMB-1680 were detected by using Sf9 [hCD36] and CHO [hCD36] models. Fluorescence microscopic photography and flow cytometry were used to analyze uptake of mLDL. Foam cell test with RAW264.7 macrophages was used to examine lipid accumulation. The results showed that IMB-1680 inhibited CD36 activity with IC50 of 2.80 and 8.79 micromol x L(-1) in Sf9[hCD36] and CHO [hCD36] cells, respectively. Fluorescence microscopic photography and flow cytometry revealed that IMB-1680 could significantly reduce DiI-AcLDL uptake. Meanwhile, IMB-1680 also could reduce lipids accumulation in RAW264.7 macrophages. In all, the data indicated that IMB-1680 might be a potent effective anti-atherosclerotic leading compound.
Animals ; CD36 Antigens ; antagonists & inhibitors ; genetics ; metabolism ; CHO Cells ; Cells, Cultured ; Cricetulus ; Dose-Response Relationship, Drug ; Foam Cells ; cytology ; High-Throughput Screening Assays ; Humans ; Lipoproteins, LDL ; metabolism ; Macrophages ; cytology ; metabolism ; Mice ; Molecular Structure ; Plasmids ; Receptors, Scavenger ; antagonists & inhibitors ; Sf9 Cells ; Spodoptera ; Transfection

0.05).Conclusion GH improves FAH of children with GHD.Height at the initiation of puberty is the most significant determining factor for the long-term efficacy.Hence,it is important that the diagnosis should be made and treatment be initiated as early as possible to afford children with GHD the opportunity to make up much of their height deficit before puberty.Adequate dosage of GH should be used for the children taking initial treatment at puberty to attain satisfactory FAH.

Objective To explore the correlation of bone metabolism levels and risk of coronary heart disease in elder women patients .Methods A total of 163 elder women patients were divided into three group:CON group ,CAD group ,and CHD group .We explored related atherosclerosis risk factors and factors related to bone metabolism .Results Compared with CON group ,there was no statistical significance in CAD group in factors related to bone metabolism(P>0 .05) .In CHD group ,serum 25‐OH‐Vitamin D significantly decreased and β‐C‐terminal telopeptide of type Ⅰ collagen significantly increased compared with CON group(P<0 .05) .Compared with CAD group ,the serum 25‐OH‐Vitamin C also significantly decreased andβ‐C‐terminal telopeptide of type Ⅰcollagen significantly increased(P< 0 .05) .Spearman correlation analysis showed that BMI ,HDL‐C ,triglycerides ,LDL‐C ,blood glucose and 25‐OH‐Vitamin D were correlated with coronary heart disease .With coronary heart disease as the dependent variable , the results showed lower LDL‐C ,25‐OH‐Vitamin D had independent predictive value for the risk coronary heart disease .Conclusion Lower 25‐OH‐Vitamin D levels in elder patients were positively correlated with coronary heart disease ,and it might also be an in‐dependent predictor .

This paper was aimed to study the human plasma protein binding rate of diterpene ginkgolides meglumine injection.The equilibrium dialysis was used to determine the human plasma protein binding rate of ginkgolide A (GA),ginkgolide B (GB) and ginkgolide K (GK) in diterpene ginkgolides meglumine injection.The LC-MS/MS method was used for the content determination of ginkgolides.And then,the plasma protein binding rate was calculated.The results showed that there was no interference from other ingredients for the determination of ginkgolides.The calibration curve of the analytes was in good linearity in certain range of contents.The precision and stability of the analytes met the methodology requirements.After 8 h incubation,the human plasma protein binding rate of GA,GB and GK achieved balance.The human plasma protein binding rate of GA (0.34,1.70 and 8.51μg·mL-1) was 84.03%-88.11%; the human plasma protein binding rate of GB (0.62,3.09 and 15.5μg·mL-1) was 41.21%-53.56%; the human plasma protein binding rate of GK (0.04,0.20 and 1.01μg·mL-1) was 45.24%-59.59%.It was concluded that the method was simple,rapid and sensitive,which met the analysis requirement for biological samples.GA had a high plasma protein binding rate; GB and GK had medium plasma protein binding rate.