Objective: To develop an HPLC method for the determination of rutin, hyperin and quercetin in Vicia sepium L. . Methods:The samples were separated on an Agilent ZORBAX Eclipse XDB-C18 column(250 mm × 4. 6 mm, 5 μm) with the mobile phase consisting of acetonitrile-1‰ phosphoric acid solution with gradient elution at the flow rate of 0. 8 ml·min-1 . The column tem-perature was 30℃, and the detection wavelength was set at 370 nm. Results: The linear range of the three components was 4. 090-130.940 μg ·ml-1(r=0.999 9), 4.600-147.200 μg ·ml-1(r=0.999 9) and 0.810-25.780 μg·ml-1(r=0.999 8), and the average recovery was 103. 45% (RSD=1. 25%), 98. 96% (RSD=1. 77%) and 102. 88% (RSD=0. 84%)(n=6), respectively. Conclusion:The method is stable, reproducible and simple, which can be used in the quality control of Vicia sepium L. .

Objective To investigate the effect of mixed application of lecture-based learning and flipped classroom and problem-based learning in microbiology teaching. Methods The five-year program students of Grade 2013 and Grade 2014 were randomly divided into two groups, and the experimental group (48 students in the teaching reform class) carried out the blended teaching, while the contrast group (48 students of parallel class) carried on the traditional teaching. After the lecture, the teaching effect of the hybrid teaching method and traditional teaching method was analyzed after the theoretical and the experi-mental operation test were adopted, and the four part questionnaire surveys including the integrated use of knowledge, active classroom atmosphere, innovation ability and teaching satisfaction were proceeded in each group. The data of each group was analyzed by t test analysis with SPSS 19.0 respectively. Results The theory test scores of experimental group was (90.16±3.14), which was higher than the control group (82.33± 4.21). The difference between them was very significant (P=0.000). Survey results showed that the integrated use of knowledge, active classroom atmosphere, innovation ability and teaching satisfaction were higher than traditional group. The difference was statistically considered significant (P<0.01). Conclusion Hybrid teaching method is very good for training and development of students' comprehensive quality and ability. It has important significance in improving the students' score.

Objective To explore the changes of human mitochondrial COXI,ND1 and ND6 genes expression induced by ionizing irradiation.Methods Changes of human COXI,ND1 and ND6 gene expression were detected by RT-PCR and Real-time PCR 8 h after the irradiation in human lymphoblastoid cell lines,which were exposed to 1-10 Gy 60Co γ-rays.And the dose-effect relationships between expression changes of the genes and the doses were analyzed.The changes of these three genes expression were also analyzed at different post-radiation time-points between 0.5 h and 72 h after irradiation of 5 Gy in order to explore the time-effect.Results The expression of three genes COXI,ND1 and ND6,showed either the dose-effect or the time-effect after irradiation.The gene expression levels of three genes up-regulated generally and the peak change time-point was 4 h after irradiation.Conclusion Ionizing radiation,msht induce the changes of mitochondrial gene expression,and the gene expression level is up-regulated.

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.

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 establish the analysis criteria of nucleoplasmic bridges (NPB) and a dose-response curve of NPB frequencies in human peripheral blood lymphocytes irradiated by 60Co γ-rays.Methods Human peripheral blood samples were collected from three healthy males,and were irradiated with 0,1,2,3,4,5 and 6 Gy of 60Co γ-rays.A cytokinesis-block micronucleus assay was carried out to analyze NPBs and micronuclei (MN) in binucleated cells.Results NPBs in binucleated cells at each dose level of γ-ray was conformed to the Poisson distribution.The dose-response curve of the γ-ray-induced NPB frequencies in human peripheral lymphocytes followed the linear-quadratic model y =(1.39 × 10-3)x2 + (4.94 × 10-3)x (R2 =0.981,P ＜ 0.05).Conclusions The dose-response curve of NPB frequencies in human peripheral blood lymphocytes induced by 0-6 Gy 60Co γ-rays was established.

BACKGROUND:Recombinant adeno-associated virus serotype 9 has a high affinity in myocardial tissue, and the expression of recombinant adeno-associated virus serotype 9-enhanced green fluorescent protein (rAAV9-eGFP) in the aorta of atherosclerosis mice is not clear. OBJECTIVE:To explore the optimal time point of rAAV9-eGFP expression in the aorta of atherosclerosis mice. METHODS:Atherosclerosis model was established with high-fat diet in 30 ApoE-/-mice for 16 weeks. Among them, 25 mice were injected with 5.0×1011 vg (virus genomes) rAAV9-eGFP through the tail vein, while the remaining 5 mice were injected with saline, serving as the control group. The virus-transfected mice were kil ed at 14, 21, 28, 35 and 60 days after transfection, and aortic tissue was harvested. The expression of enhanced green fluorescent protein was detected with laser scanning confocal microscope. Western blot assays were used to detect the expression of enhanced green fluorescent protein in aorta. The expression of enhanced green fluorescent protein in vivo was observed and the optimal expression time point was determined. RESULTS AND CONCLUSION:rAAV9-eGFP effectively transfected the aorta of atherosclerosis mice, enhanced green fluorescent protein was expressed in aortic tissue, and the expression intensity increased gradual y with the increasing transfection time. The highest expression level was found at 35 days after transfection and then maintained stable at 60 days. There were significant differences at different time points after transfection (P<0.001). These data indicate that rAAV9-eGFP can be effectively expressed in the aorta of atherosclerosis ApoE-/-mice and rAAV9-eGFP can be regarded as the optimal vector in the treatment of atherosclerosis.

Objective To establish the immortalized cell lines of peripheral blood lymphocytes for old male residents in high background radiation area(HBRA)in Guangdong,China,in order to preserve the specific genomic resources of residents in HBRA for the further genetic and molecular biological study on HBRA.Methods The peripheral blood samples of 20 old male residents in HBRA were collected after informed consent.The immortalized B lymphoblastoid cell lines,2 for each resident,were established with Epstein-Barr virus.After being frozen and recovered,the cell viability,the contamination of bacterium and mycoplaama were analyzed.The stabilization of cell lines was decided by comparing the karyotypes of the peripheral blood lymphocytes and the cell lines.Results 40 cell lines for 20 residents in HBRA were successfully established.The recovery rate of cell lines after being frozen was 100%.All the cell viablity after recovery was higher than 90%.and no contamination of bacteria and mycoplasma occurred.The karyotypes of the 20th generation cell lines were not change.Conclusion The immortalized cell lines established in this study could provide biological resources for further study on genetics and molecular biology in HBRA.

Objective To investigate the protective effect of standard dose prednisone(starting dose about 0.4 mg/kg bw) combined with glutamine(2 g/d) for Graves' ophthalmopathy(GO) after 131I treatment.Methods The prospective randomized controlled trial was performed and included 116 consecutive patients with mild-to-moderate GO or no active GO after 131I treatment,but with high risk factors.The experimental group(59 cases) started to take oral prednisone(0.44 ± 0.13)mg/kg at 2 d atter theatment,meanwhile replenishing glutamine,and prednisone was gradually decreased by 5mg per 2 weeks and stopped until 2 months;the control group(57 cases) received the initial dose prednisone(0.43± 0.14)mg/kg without replenishing glutamine,and the rest was same as the experimental group.The GO change and prednisone adverse reactions in 2,4,6 months after treatment were evalua ted in the two groups.Results The baseline characteristics had no significant difference between the two groups.In comparison of the experimental group and control group after 6-month treatment,the CAS score was (2.8 ± 0.8 vs.3.5 ± 0.9),exophthalmos degree was (19.6±8.1)mm vs.(21.7±3.0)mm,eyelid width was (11.3±2.9)mm vs.(13.8±3.1)mm,the improvement degree in the experimental group except for degree was superior to the control group (P＜0.01).No new onset GO or deterioation oc curred in the two groups;the experimental group had 42 cases(71.2 ％) of GO improvement and 17 cases(28.8 ％) of stability,while the control group had 39 cases(68.4％) of GO improvement and 18 cases(31.6％) of stability,and the curative effects had no sttis-tical difference between the two groups(P＞0.05).The side effects in the control group were more than those in the experimental group(63.1％ vs.30.5％,P＜0.05).the body mass increase was more obvious [(3.8±1.8) kg vs.(1.4±1.2)kg,P＜0.01],and SBP and DBP in the two groups were slightly increased[(10±4)mm Hg vs.(9±5)mm Hg P＞0.05].Conclusion Using the initial dose of oral prednisone(about 0.4 mg/kg) for 2 months and simultaneously replenishing glutamine (2 g/d)can effectively improve mild-moderate GO,and effectively reduce the adverse reactions of GC.However it is needed to extend the follow-up time to assess whether it can truly prevent the deterioration of GO,and to conduct a further study for the role of glutamine.

Objective Based on middle cerebral artery occlusion (MCAO) model,to investigate the effects of melatonin (MT) on DWI and expression of Fas,FasL and cleaved Caspase-3 proteins in rat model with focal cerebral ischemia.Methods Eighty SD rats were randomly divided into Sham group (n=16),MCAO group (n=32) and MT group (n=32).The rats in sham group were treated with sham-operation.And the rats in MCAO and MT groups were peritoneally injected with saline and MT respectively.The behavioral scores were assessed in the three groups.The rats in MCAO and MT group with the behavioral scores of 1 3 points were selected in the study.The DWI relative signal intensity (rDWI-SI),Fas,FasL and cleaved Caspase-3 proteins were respectively examined by MR scaning and immunohistochemical staining in all rats of each group at 6 h,24 h,72 h and 7 days after ischemia reperfusion (IR) or sham-operation.And the DWI and immunohistochemical results for each group were compared.Results At last,there were 16 rats in sham group,29 rats in MCAO group and 30 rats in MT group,respectively.There was significant difference of the behavioral scores among the three groups (x2 =50.125,P＜0.01).The behavioral scores of MT and MCAO groups were higher than those of sham group (all P ＜0.05).And the behavior scores of the MT group were lower compared with MCAO group after IR.Compared with the rDWI-SI values measured at 6 h,24 h and 72 h,7 days in sham group,the rDWLSI values of MT and MCAO groups were significantly higher (all P＜0.01).And the rDWI-SI was higher in MCAO group than those in MT group at 6 h,24 h and 72 h after IR (all P＜0.01).And there was no significant difference of rDWI-SI at 7 days after IR between MT and MCAO groups (P＞0.05).The immunohistochemical staining results showed that the number of Fas,FasL and cleaved Caspase-3 positive cells in MCAO and MT groups were significantly higher than those in sham group (all P＜0.01).And there were less Fas,FasL and cleaved Caspase-3 positive cells in MT groups compared with MCAO group (all P＜0.05) at 6 h,24 h and 72 h after IR.There was no significant difference of Fas,FasL and cleaved Caspase-3 positive cells among the three groups at 7 days after IR (P＞0.05).Conclusion MT can effectively alleviate the rDWI-SI value and inhibit the expression of Fas,FasL and cleaved Caspase-3 proteins in rats of focal cerebral ischemia.