Objective To investigate the effect of recombinant adenovirus mediatied human endostatin (rAD-GFP-ES)on rats with collagen typeⅡinduced arthritis(CIA),and explore the mechanism of inflamma- tion and cytokines inhibition on rats CIA.Methods The rAD-GFP-ES was amplified and purified.The model of rat CIA was induced by intradermal injection of typeⅡcollagen combined with complete Freund's adjuvant(CFA). On the second day after the injection,the therapeutic administration of rAD-GFP-ES(1?10~(11)pfu?kg~(-1)?week~(-1)?4 weeks)were performed to the rats.The mean arthritis index(AI)was scored every week since then.The relative concentrations of ES,IL-I?,TNF-?in sera collected at the fourth week were evaluated by western blotting. Results①The titer of the purified rAD-GFP-ES and rAD-GFP was 6.6?10~(12)pfu/ml and 4.8?10~(12)pfu/ml,re- spectively(A_(260nm)/A_(280nm)＞1.3).②The concentration of ES in sera of the group treated with rAD-GFP-ES was 2.4-lold higher compared to the normal group.③The mean arthritis index of the group treated with rAD-GFP- ES was much lower than that of the model group.The administration of rAD-GFP-ES could significantly de- creas the production of IL-1?and TNF-?in sera.Conclusions①The rAD-GFP-ES is efficiently expressed in vivo.②The rAD-GFP-ES has an inhibitory effect on the arthritis index of rat CIA.③IL-1?and TNF-?are involved in the pathogenesis of RA.The rAD-GFP-ES has an inhibitory effect on the expression of IL-1?and TNF-?in rat CIA.

OBJECTIVETo investigate the effect of stromal interaction molecule 1 (STIM1) silencing on EPCs cell cycle.

METHODSRat bone marrow derived endothelial progenitor cells (EPCs) were isolated and cultured in L-DMEM with 20% FBS. Ad-si/rSTIM1 and Ad-hSTIM1 were then transfected into EPCs and the expression of STIM1 mRNA was detected by RT-PCR. The cell cycle was determined using flow cytometry analysis and intracellular free Ca2+ was measured using LSCM. Co-immunoprecipitation was performed to examine the interaction between STIM1 and TRPC1. Protein levels of inositol 1, 4, 5-trisphosphate were analyzed with ELISA assay.

RESULTSForty-eight hours after transfection, the expression of STIM1 mRNA was significantly downregulated (0.37 +/- 0.02 vs. 1.00 +/- 0.02, P < 0.05) and intracellular free Ca2+ level was significantly reduced (34.07 +/- 4.10 vs. 86.51 +/- 14.12, P < 0.05) in Ad-si/rSTIM1 group compared with control group. The cell cycle was arrested at G1 phase [(90.91 +/- 1.10)% vs. (77.10 +/- 0.56)%, P < 0.05] and the store-operated channel entry was strikingly inhibited in EPCs after treatment with Ad-si/rSTIM1. However, cotransfection of Ad-hSTIM1 with Ad-si/rSTIM1 significantly reversed these responses. Interestingly, co-immunoprecipitation study showed that STIM1 co-precipitated with TRPC1, and IP3 levels measured by ELISA were similar among three groups (P > 0.05).

CONCLUSIONsiRNA-mediated knockdown of STIM1 inhibited EPCs proliferation by reducing intracellular free Ca2+ through TRPC1-SOC signaling pathway.

Adenoviridae ; genetics ; Animals ; Cell Cycle ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; Gene Silencing ; Genetic Vectors ; Membrane Proteins ; genetics ; Neoplasm Proteins ; genetics ; RNA, Small Interfering ; Rats ; Stem Cells ; cytology ; Stromal Interaction Molecule 1 ; Transfection ; Transient Receptor Potential Channels ; metabolism

OBJECTIVETo investigate the effect of vascular endothelial growth factor 165 (VEGF 165) gene on vascularization of dermal substitute in vivo.

METHODSHuman umbilical vein endothelial cells (HUVECs) were cultured in M199 medium containing FBS in the volume fraction of 10% (briefly called complete medium). (1) HUVECs were divided into non-transfection group (without transfection), empty vector group [transfected with pIRES2-enhanced green fluorescent protein (EGFP) plasmid], and VEGF plasmid group (transfected with pIRES2-EGFP-VEGF plasmid) according to the random number table, with 6 wells in each group. At post transfection hour (PTH) 24, the expression of green fluorescent protein (GFP) in each group was observed under inverted phase contrast fluorescence microscope, and the expression rate of GFP was detected with flow cytometer. Cells in non-transfection group were tested with the same methods as listed above. The cells in stable transfection in empty vector group and VEGF plasmid group were sifted by neomycin. The mRNA and protein expression levels of VEGF 165 in cells and the protein amount of VEGF 165 in the supernatant of cell culture medium in 3 groups were respectively determined by real-time fluorescent quantitation PCR, Western blotting, and enzyme-linked immunosorbent assay. (2) Forty-eight male nude mice were divided into 4 groups according to the random number table, with 12 mice in each group. Mice in saline group were subcutaneously implanted with dermal substitutes which had been cultured in saline for 2 days on both sides of back (the same site below); mice in medium group were subcutaneously implanted with dermal substitutes which had been cultured in complete medium for 2 days; mice in non-transfected cells group were subcutaneously implanted with dermal substitutes that had been cultured in complete medium with non-transfected HUVECs for 2 days; mice in transfected cells group were subcutaneously implanted with dermal substitutes that had been cultured in complete medium with HUVECs stably transfected with VEGF plasmid for 2 days. The dermal substitutes in every group were taken out on post operation day (POD) 3, 7, 14, and 21. Distributions of microvessels and HUVECs in dermal substitutes were observed by immunohistochemical staining, and the microvessel number was counted on POD 14; the expression level of VEGF 165 protein in dermal substitutes was determined by Western blotting. The experiments were all done in triplicate. Data were processed with one-way analysis of variance and LSD method.

RESULTS(1) Obvious green fluorescence was only observed in the two groups with transfected cells at PTH 24. Expression rates of GFP in the cells of non-transfection group, empty vector group, and VEGF plasmid group were respectively 0, (85.2 ± 3.2) %, and (93.1 ± 2.4) %. In the non-transfection group, empty vector group, and VEGF plasmid group, the relative expression amounts of VEGF 165 mRNA were respectively 1, 1.05 ± 0.09, and 3.02 ± 0.13 (F = 5.28, P < 0.05); the relative expression amounts of VEGF 165 protein were respectively 0.78 ± 0.16, 0.76 ± 0.13, and 1.92 ± 0.18 (F = 7.62, P < 0.05); the protein quantity of VEGF 165 in cell supernatant was respectively (62.4 ± 2.7), (73.1 ± 3.8), (117.5 ± 3.1) pg/mL (F = 15.08, P < 0.05). The mRNA and protein levels of VEGF 165 and VEGF 165 protein amount in supernatant were significantly higher in VEGF plasmid group than in the other two groups, with P values all below 0.05. (2) The number of HUVECs in dermal substitutes of transfected cells group was significantly higher than that of the other three groups on POD 14. The numbers of microvessels of dermal substitutes on POD 14 in saline group, medium group, non-transfected cells group, transfected cells group were respectively 4.2 ± 1.1, 5.2 ± 1.1, 6.6 ± 0.9, 13.8 ± 0.8 per 200 times visual field (F = 17.96, P < 0.01). The microvessel number in transfected cells group was significantly higher than that of the other three groups, with P values all below 0.05. The relative expression ratio of VEGF 165 protein of dermal substitutes in transfected cells group was significantly higher than that in saline group as of POD 7. On POD 14 and 21, the relative expression ratios of VEGF 165 proteins in non-transfected cells group (1.652 ± 0.086, 2.152 ± 0.062) and transfected cells group (2.403 ± 0.091, 2.879 ± 0.047) were significantly higher than those of saline group (1.299 ± 0.027, 1.362 ± 0.103), with P values all below 0.05. And the index level of transfected cells group was significantly higher than that in non-transfected cells group (with P values below 0.05). The VEGF 165 protein content in dermal substitutes increased with time extension in all groups.

CONCLUSIONSTransfection of VEGF 165 gene in HUVEC could effectively facilitate vascularization of dermal substitutes in vivo by high expression of VEGF 165 protein.

Animals ; Cells, Cultured ; Dermis ; blood supply ; Human Umbilical Vein Endothelial Cells ; Humans ; Male ; Mice ; Mice, Nude ; Plasmids ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Objective To study the correlation between fractional anisotropy(FA)and tumor microarchitecture(MVD,VEGF and celluarity).Methods Fouteen gliomas(5 grade Ⅰ and Ⅱ,4 grade Ⅲ, 5 grade Ⅳ)confirmed histo-pathologically were performed on diffusion tensor imaging(DTI)using a GE Signa Excite Ⅱ 3.0 T MR scanner(8-channel head coil,SE echo planner imaging(EPI),thickness:5 mm, spacing:0,directions:25,B values:0 and 1000 s/mm~2,TR 6000 ms,TE minimum,FOV:240 mm? 240 mm,image matrix 128?128,NEX 2).Postprocessing was done using a DTI specific software to gain FA image.ROIs were drqwn in tumor parenchyma and the value of FA was recorded.The positive expression of VEGF and CD34 was shown using immuno-histochemistry method.The VEGF,MVD,and cellularity of every slices were recorded.Pearson correlation analysis was used.Results FA(which is 0.102?0.080 in grade Ⅰ and Ⅱ,0.171?0.037 in grade Ⅲ,0.200?0.021 in grade Ⅳ)has the trend to raise with the increasing grade of astrocytomas.FA has significant positive correlation to MVD(40/HP in grade Ⅰ and Ⅱ, 86/HP in grade Ⅲ,101/HP in grade Ⅳ),VEGF(8% in grade Ⅰ and Ⅱ,47% in grade Ⅲ,55% in grade Ⅳ),and cellularity(104/HP in grade Ⅰ and Ⅱ,160/HP in grade Ⅲ,265/HP in grade Ⅳ).The correlation coefficients between FA and VEGF,MVD,and cellularity were 0.748,0.668,0.625 respectively.Conclusion As a new imaging method,DTI can reveal the microarchitecture in gliomas and be value of distinguishing gliomas of different grade.DTI provides a new method of precise diagnosis to glioma preoperatively.

OBJECTIVETo develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.

METHODSThe heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.

RESULTSThe baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.

CONCLUSIONThe recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Antibodies, Monoclonal ; biosynthesis ; immunology ; Baculoviridae ; genetics ; Hepatitis A virus ; immunology ; Hepatitis Antibodies ; biosynthesis ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; immunology ; Immunoglobulin G ; biosynthesis ; immunology ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Recombinant Proteins ; biosynthesis ; immunology

OBJECTIVETo explore the relationship between serum creatinine (Scr) reference values in healthy adults and geographic factors and provide evidence for establishing Scr reference values in different regions.

METHODSWe collected 29 697 Scr reference values from healthy adults measured by 347 medical facilities from 23 provinces, 4 municipalities and 5 autonomous regions. We chose 23 geographical factors and analyzed their correlation with Scr reference values to identify the factors correlated significantly with Scr reference values. According to the Principal component analysis and Ridge regression analysis, two predictive models were constructed and the optimal model was chosen after comparison of the two model's fitting degree of predicted results and measured results. The distribution map of Scr reference values was drawn using the Kriging interpolation method.

RESULTSSeven geographic factors, including latitude, annual sunshine duration, annual average temperature, annual average relative humidity, annual precipitation, annual temperature range and topsoil (silt) cation exchange capacity were found to correlate significantly with Scr reference values. The overall distribution of Scr reference values featured a pattern that the values were high in the south and low in the north, varying consistently with the latitude change.

CONCLUSIONThe data of the geographic factors in a given region allows the prediction of the Scr values in healthy adults. Analysis of these geographical factors can facilitate the determination of the reference values specific to a region to improve the accuracy for clinical diagnoses.

Alveolar echinococcosis is a zoonotic parasitic disease caused by an infection with Echinococcosis multilocularis.The liver is the primary organ of alveolar echinococcosis.Alveolar echinococcosis is usually characterized by invasive growth and consequently iscalled"parasitic cancer."Resection of radical lesions is a preferred and effective treatment for hepatic alveolar echinococcosis.End-stage hepatic alveolar echinococcosis often occurs with parasiticcirrhosis,such as secondary biliary cirrhosis,congestive liver cirrhosis or Budd-Chiari syndrome.Few studies have examined hepatic multilocular echinococcosis leading to cirrhosis.This article reviews the aspects of hepatic alveolar echinococcosis involving the invasion of important blood vessels and bile ducts,thereby leading to secondary biliary cirrhosis and congestive liver cirrhosis caused by hepatic alveolar echinococcosis.

OBJECTIVETo observe the influence on prognosis and possible side-effects of arginine in

METHODSMulti-center clinical trial, randomized double blinded patients with severe trauma and burns. and placebo control methods were employed in the study. Eighty-six patients with severe trauma and burns were randomly divided into control (C, n = 45) and arginine treatment (Arg, n = 41) groups. The patients in Arg group received arginine in dose of 0. 4 g x kg(-1) x d(-1) orally, while those in C group received same dose of placebo (tyrosine) for 7 days. All the patients in both groups were given diet with equal calories and equal nitrogen content. The changes in the wound healing time, hospital stay, and the incidence of side-effects of the medication in both groups of patients were observed and compared before and after the supplementation of arginine.

RESULTSThe wound healing time and hospital stay days of severe trauma patient in Arg group (n = 29) were 11. 1+/-2. 8 d and 19+/-6 d, which were all obviously shorter than those in C group (13. 2+/-5. 5 d, 22 +/-6 d, n =33, P <0.05). On the other hand, in severe burn patients there were no significant difference of the wound healing time (20+/-5 d vs 22+/-8 d, n = 12, P > 0. 05) and hospital stay days (28+/-6 d vs 29+/-8 d, n = 12, P >0. 05) between the Arg and C groups. In addition, in C and Arg groups, the occurrence of the side-effects were seldom (2. 44% vs 2. 22% , P = 1. 000) and it disappeared when the supplementation of drugs was stopped.

CONCLUSIONOral feeding of arginine is beneficial in enhancing wound healing, reduction of hospital stay days in severe trauma patients and with little side-effects, but it is not beneficial to improve the prognosis of severe burn patients. Maybe this is due to inadequate number of case involved in the study.

Administration, Oral ; Adolescent ; Adult ; Aged ; Arginine ; administration & dosage ; adverse effects ; therapeutic use ; Burns ; diagnosis ; drug therapy ; Double-Blind Method ; Female ; Humans ; Length of Stay ; Male ; Middle Aged ; Prognosis ; Wound Healing

OBJECTIVETo evaluate Candesartan therapeutic effect against atherosclerotic plaque rupture and to explore the related mechanisms.

METHODSThirty-four New Zealand White male rabbits were randomly divided into three groups: the control group, the model control group and the Candesartan intervention group. The control group rabbits were fed with a normal diet. Rabbits of the latter two groups were fed with a 1% high-cholesterol diet and received a balloon catheter injury respectively one week after the cholesterol feeding. Candesartan (0.5 mgⁱkg⁻¹ⁱd⁻¹) was given to the Candesartan group rabbits 2 days before the performance of the balloon catheter injury. By the end of 12(th) week of the experiment, Russell's viper venom was used for rabbits of both the model control and the Candesartan groups in order to induce rupture of the plaques developed and followed by sacrifice of all the rabbits of the 3 groups. The aortas were removed and fixed for histological evaluation. Immunohistochemistry of MMP-9, macrophage markers and collagen were performed. The protein expression of MMP-9 was determined using Western blot analysis.

RESULTSIn the model control group, 7 of 9 rabbits with a total of 12 plaques developed rupture and thrombosis of the plaques after the induction. In contrast, only 2 of 10 rabbits with a total of 3 plaques demonstrated rupture and thrombosis in the Candesartan group (P < 0.05). The control group rabbits did not have plaque rupture and thrombosis. Compared with the model group, both the percentage area of MMP-9 and macrophages in the plaques were significantly decreased in the Candesartan group (12.35% ± 4.28% vs 32.58% ± 9.16%, P < 0.05; 13.87% ± 4.91% vs 23.8% ± 7.45%, P < 0.05). There was an increased percentage of collagen content in total plaques of the Candesartan group (30.27% ± 11.36% vs 4.18% ± 1.28%, P < 0.01). Compared with the model group, the protein expression of MMP-9 was significantly decreased in the Candesartan group (P < 0.01).

CONCLUSIONCandesartan has a preventive value against atherosclerotic plaque rupture in hypercholesterolemic rabbits, likely through its reduction of MMP-9 expression, inhibition of macrophage accumulation and increase of collagen content within the plaques.

Angiotensin II Type 1 Receptor Blockers ; therapeutic use ; Animals ; Antihypertensive Agents ; therapeutic use ; Aorta, Abdominal ; injuries ; Benzimidazoles ; therapeutic use ; Collagen ; metabolism ; Macrophages ; pathology ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Plaque, Atherosclerotic ; metabolism ; pathology ; Rabbits ; Random Allocation ; Rupture, Spontaneous ; prevention & control ; Tetrazoles ; therapeutic use ; Thrombosis ; etiology ; metabolism ; prevention & control

Isoquiritigenin,one of the active constituents in the Chinese herb liquorice,is found to have moderate inhibitory activity against rat monoamine oxidase B(MAO-B,IC5047. 2 μmol·L-1). However,the structure-activity relationship(SAR) remains unclear until now. In an attempt to reveal the SAR of inhibition by isoquiritigenin,and to identify more potent and selective inhibitors of MAOB,a series of 13 derivatives based on the scaffold of isoquiritigenin were prepared,and their purities and structures were confirmed by UPLC,1 H-NMR,13 C-NMR and HRMS. These compounds were then evaluated for their ability to inhibit the enzymatic activity of human MAO-B. The SAR of inhibition was summarized and a potent compound C8 with high inhibitory activity(IC501. 4 μmol·L-1) and selectivity(>57 folds over MAO-A) was identified. Enzyme kinetics studies suggested that C8 acted as a competitive inhibitor. In addition,C8 showed little cytotoxicity to glial cells in vitro,which could be a promising lead compound for further study.
Animals ; Drugs, Chinese Herbal ; Humans ; Monoamine Oxidase ; Monoamine Oxidase Inhibitors ; Plant Extracts ; Rats ; Structure-Activity Relationship