Plant expression cassette for TaDREB from wheat was constructed into plasmid pBIR1.Aloe stems were used as explants for the transformation mediated by Agrobaterium.Infected tissues were selected using G418 to generate transformants.In total,58 resistant plantlets to the antibiotics were obtained from the infected explants.The designed primers according to the selective gene npt II and the target gene TaDREB were used to analyze all of the G418 resistant plantlets.PCR results demonstrated that TaDREB were successful transferred into aloe genomic with the transformation efficiency of 0.5%.The transgenic aloe plants were treated under 4℃ for two weeks and then at-20℃ for 30min.The treatment showed that the leaves of negative plants appeared severe evidence of freeze injury with brown,withered and translucent,while the positive plants appeared good growing condition.The activities of enzymes such as peroxidase(POD)and superoxide dismutase(SOD)of transgenic plants which were stressed for 14 days under low temperature were analyzed.The results indicated that the trend of SOD and POD activities in transgenic plants was down-up-up-up,and that in non-transgenic plants was down-up-down-down.The average value of relative electrical conductivity in the positive plants was 0.456 which was lower than 0.685 in the negative plants.It is supposed that transformation of the kind of gene could improve the resistant ability of aloe to low temperature.

Animals ; Male ; NF-kappa B ; metabolism ; Otitis Media with Effusion ; immunology ; metabolism ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Th1 Cells ; cytology ; Th2 Cells ; cytology

OBJECTIVE:To observe the clinical efficacy and safety of azithromycin combined with Reduning injection in the treatment of children with mycoplasma pneumoniae infection. METHODS:80 children with mycoplasma pneumoniae infection were randomly divided into control group and research group. All the children were given routine treatment,including oxygen inha-lation,defervescence,nutritional support,reducing sputum and relieving asthma,etc. Based on it,children in control group were orally treated by Azithromycin enteric coated tablets 10 mg/kg,once a day. Children in research group were treated by Reduning in-jection 10 ml and 5% glucose injection 100 ml by intravenous infusion,once a day,based on the treatment in control group. The course of both was 14 d. The clinical data was observed,including the clinical efficacy,interleukin-6 (IL-6),interleukin-8 (IL-8),tumor necrosis factor-α(TNF-α)and the incidence of adverse reactions(ADR)before and after treatment. RESULTS:The total effective rate in research group was significantly higher than control group,with significant difference(P<0.05). After treat-ment,the IL-6,IL-8 and TNF-α in 2 groups were significantly lower than before,and research group was lower than control group,with significant difference(P<0.05). There were no significant differences in the incidence of ADR in 2 groups(P>0.05). CONCLUSIONS:Based on the routine treatment,azithromycin combined with Reduning injection has more obvious efficacy than only azithromycin in the treatment of children with mycoplasma pneumoniae infection with similar safety.

OBJECTIVETo explore the molecule mechanism of Salidroside inducing directional differentiation of mouse mesenchymal stem cells (MSCs) into neuronal cells.

METHODSThe mouse multipotent mesenchymal precursor cell line (D1) was taken as the objective. Cultured MSCs were divided into the negative control group (complete culture solution), the positive control group (containing 1 mmol/L β-mercaptoethanol), the Salidroside induced group (20 mg/L Salidroside), and the blocked group (20 ng/ ml DKK1, a special inhibitor of Wnt/β-catenin signal pathway). All cells were inoculated in a 6-well plate (1 x 10(4) cells/cm2) and grouped for 24 h. The expression of p-catenin was detected by fluorescence Immunochemistry in the negative control group, the positive control group, and the Salidroside induced group. The expression of neuron-specific enolase (NSE), beta 3 class III tubulin (β-tubulin III), nuclear receptor related factor 1 (Nurr1), glial fibrillary acidic protein (GFAP) mRNA, Wnt3a, β-catenin, low-density lipoprotein receptor-related protein6 (LRP6), Axin mRNA were detected using reverse transcrip- tion PCR (RT-PCR). The expression of β-catenin and NSE protein were analyzed by Western blot in the negative control group, the positive control group, and the Salidroside induced group. Ca2+ chelating agents (EGTA), L-type Ca2+ channel blocker (Nifedpine), and IP3Ks special inhibitor (LY294002) were used to block Ca2+ signal pathway respectively. The expression of Wnt3a, LRP-6, Axin, glycogen syn- thase kinase (GSK-3), and β-catenin mRNA were detected by RT-PCR. The β-catenin protein expression was analyzed using Western blot.

RESULTSCompared with the positive control group, β-catenin protein was strong positively expressed; the expression of Wnt3a, β-catenin, LRP6, Axin, NSE, β-tubulin III, Nurr1 mRNA, and NSE protein were obviously up-regulated in the Salidroside induced group (P < 0.01). Compared with the positive control group and the Salidroside induced group, β-catenin, NSE, Nurr1, and β-tubulin III mRNA expression decreased; β-catenin and NSE protein expression were also down-regulated in the blocked group (P < 0.01). Compared with the Salidroside induced group, the expression of Wnt3a, LRP-6, β-catenin, and Axin mRNA were down-regulated in the Ca2+ signal blocked group and the salidroside induced group (P < 0.01, P < 0.05).

CONCLUSIONSalidroside affected directional differentia- tion of MSCs into neuronal cells through Wnt/β-catenin and Ca2+ signal pathway.

Animals ; Cell Differentiation ; drug effects ; Glucosides ; pharmacology ; Glycogen Synthase Kinase 3 ; Lipoproteins, LDL ; Low Density Lipoprotein Receptor-Related Protein-6 ; Mesenchymal Stromal Cells ; physiology ; Mice ; Neurons ; Phenols ; pharmacology ; Phosphopyruvate Hydratase ; RNA, Messenger ; Signal Transduction ; Wnt Signaling Pathway ; physiology ; beta Catenin ; metabolism

ObjectiveTo investigate the influence of cobalt chloride ( CoCl2 )-mimetic hypoxia on theproliferation,apoptosis and migration of human pancreatic cancer cell fine PANC1.MethodsPANC1 cells were treated with 0(control),100,200,400,800 μmol/L CoCl2 respectively for 24 h.Real-time RT-PCR and Western blot were used to determine hypoxia induced factor ( HIF)-1o mRNA and protein expression respectively,and cell counting kit-8(CCK-8) assays,flow cytometry and cell scratch test were used to examine the proliferation,apoptosis and migration of PANC1 cells,respectively.ResultsIn the control group and 100,200,400 and 800 μmol/L CoCl-2 groups,the expressions of HIF-1t mRNA were 1,1.08 ±0.12,1.12 ± 0.09,1.04±0.11,0.66 ±0.07,and the expressions of VEGF mRNA were 1,2.69±0.35,4.81 ±0.54,2.19 ± 0.21,0.79 ± 0.08,while the expressions of HIF-1 α protein were 0.23 ± 0.03,0.36 ± 0.04,1.15 ± 0.11,1.08 ± 0.09,0.44 ± 0.04; and the expressions of VEGF protein were 0.14 ± 0.02,0.12 ± 0.01,0.95 ±0.09,0.87 ±0.09,0.55 ±0.06; and cell viability rates were 100％,(98.43 ±2.88)％,(76.15 ± 0.70)％,(53.87 ±0.77)％,(35.23 ±0.67)％ ; while cell apoptotic rates were (5.2 ±1.12)％,(5.74 ± 1.07)％,(6.82 ± 1.85)％,(12.09 ±3.53)％,(31.88 ±6.95)％ ; the cell migration distance of PANC1 cells were (43.24 ±3.67)％,(59.46 ±5.39)％,(80.56 ±8.05)％,(63.89 ±5.96)％,(9.09 ± 1.59 ) ％.Compared with those of control group,the expressions of VEGF mRNA,VEGF and HIF-1 α protein,cell migration distance showed a two-way variation ( ascending first and descending later) (P ＜0.05 ),and the expression of HIF-1α mRNA and cell proliferation rate was decreased in a dose-dependent manner,while the cell apoptosis was increased in a dose-dependent manner.Conclusions CoCl2 significantly inhibits the proliferation and promotes apoptosis of PANC1 cells at certain level.CoCl2 has a two-way effect on the migration of PANC1 cells,and it may be related to the direct injury of high concentration of CoCl2 on cells.

Objective To investigate the expressions of RGC-32 and E-cadherin in pancreatic cancer and analyze their clinicopathological significance and the correlation with each other.Methods SP immunohistochemistry was used to detect the expressions of RGC-32 and E-cadherin in 42 cases of pancreatic cancer tissues,12 cases of chronic pancreatitis tissues and 8 cases of normal pancreatic tissues.Results The positive staining for RGC-32 was predominantly observed in the cytoplasm of pancreatic acinar cells.The positive staining for E-cadherin was mainly observed in the cytomembrane of normal pancreatic and chronic pancreatitis acinar cells,but aberrant expression ( cytoplasm expression and ( or ) weaker expression) could be found in pancreatic cancer cells.The positive expression rate of RGC-32 and aberrant expression rate of E-cadherin were 78.6％ (33/42) and 54.8％ (23/42),respectively,in pancreatic cancer tissues,which were significantly higher than those in normal pancreatic tissues [37.5％ (3/8) and 0] and chronic pancreatitis [41.7％ (5/12)and 8.3％ (1/12) with statisticai significance,P ＜0.05].The expression of RG C-32 in pancreatic cancer was associated with lymph node metastasis and TNM staging (P =0.016,0.025,respectively),but not with age,gender and differentiation degree ( P =0.831,1.000,0.629,respectively).The aberrant expression of E-cadherin was associated with differentiation degree,lymph node metastasis and TNM staging ( P =0.024,0.004,0.004,respectively),but not with age and gender ( P =0.970,1.000,respectively).A significantly positive correlation was found between positive expression rate of RGC-32 and aberrant expression rate of E-cadherin (r =0.458,P ＜0.01 ).Conclusions Both positive expression rate of RGC-32 and aberrant expression rate of E-cadherin are up-regulated significantly in pancreatic cancer tissues and RGC-32 may be involved in the invasion and metastasis of pancreatic cancer by regulating epithelial mesenchymal transition.

A double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers was established. In the reaction system of xanthine oxidase, WST-1 works as the probe for the ultra oxygen anion generation, and product uric acid works as xanthine oxidase activity indicator. By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector, the changes of these indicators' concentration were observed and the influence factors of this reaction system to establish the high throughput screening model were studied. And the model is confirmed by positive drugs. In the reaction system, the final volume of reaction system is 50 μL and the concentrations of xanthine oxidase is 4 mU x mL(-1), xanthine 250 μmol x L(-1) and WST-1 100 μmol x L(-1), separately. The Z'-factor of model for xanthine oxidase inhibitors is 0.537 4, S/N is 47.519 9; the Z'-factor of model for superoxide anion scavengers is 0.507 4, S/N is 5.388 9. This model for xanthine oxidase inhibitors and superoxide anion scavengers has more common characteristics of the good stability, the fewer reagent types and quantity, the good repeatability, and so on. And it can be widely applied in high-throughput screening research.
Enzyme Inhibitors ; pharmacology ; Free Radical Scavengers ; pharmacology ; High-Throughput Screening Assays ; Superoxides ; Uric Acid ; Xanthine ; Xanthine Oxidase ; antagonists & inhibitors

This paper is to evaluate the biocompatibility and cytotoxicity of a new Ni-free Zr-based bulk metallic glass (BMG), Zr60.14 Cu22.31 Fe4.85 Al9.7 Ag3, by comparing it with conventional Ti6Al4V alloy. According to ISO 10993-5: 1999 and GB/T 16886.5-1997 standards, Zr60.14 Cu22.31 Fe4.85 Al9.7 Ag3, pure Zr and Ti6Al4V materials were extracted with surface area of sample/volume of medium ratio being 1 cm2/mL and 0.5 cm2/mL, respectively. The viabilities of MG-63 cells (Human osteosarcoma cell line) cultured in the BMG medium extracts for 1, 3 and 5 days were determined by CCK-8 assay. The cellular morphology of MG-63 cells cultured on the surface of samples for 3 days was tested through laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM). The relative growth rate (RGR) of MG-63 cells cultured in Zr60.14 Cu22.31 Fe4.85 Al9.7 Ag3 and pure Zr were both more than 85%, indicating that the cytotoxicity of BMG was relatively low and met the national biomedical material eligibility standard. There was insignificant difference in the morphology of MG-63 cells cultured in the BMG medium extracts and the control group through LSCM and SEM, which showed the BMG had excellent biological compatibility. The Zr-based bulk metallic glass Zr60.14 Cu22.31 Fe4.85 Al9.7 Ag3 and the conventional Ti6Al4V alloy both had no obvious cytotoxicity to MG-63 cells. These results provided evidence that the new Zr-based bulk metallic glass could be potential replacement material for the orthopedic surgical implant.
Alloys ; Biocompatible Materials ; Cell Line ; Glass ; Humans ; Microscopy, Electron, Scanning ; Nickel ; Titanium ; Zirconium

Background Retinal development continues during the early postnatal period in mammals.Correct arrangement of layers and precise location of various cells in the retina are vital for forming normal visual function during critical period plasticity.Spectral-domain optical coherence tomography(SD-OCT)provides highquality in vivo retinal imaging and the possibility to measure retinal thickness longitudinally. Objective The present study was to investigate the changes of retinal thickness during critical period plasticity in rats. Methods In vivo consecutive scanning of retinal image was performed in 10 SPF Sprague-Dawley rats at postnatal day 14(P14),P18,P21,P24 and P42 with SD-OCT,and retinal histopathological examination was used to detect retinal morphologic changes at the same postnatal ages in 20 matched rats.The whole retinal thickness,the thickness from inner limiting membrane(ILM)to inner plexiform layer(IPL),the thickness of inner nuclear layer(INL)and the thickness from outer nuclear layer(ONL)to retinal pigment epithelium(RPE)were measured using Cirrus HD-OCT system and HMIAS-2000 Imaging System in retinal sections.The measurement parameters by Cirrus HD-OCT and those by hematoxylin-eosin staining were compared.The use of animals followed the Statement of National Institute of Health (USA). Results In vivo high-resolution images of rat retinas with SD-OCT compared well with histology,which enabled quantitative comparison of the SD-OCT and histological data during critical period plasticity in rats.From P14 to P42,the retinal thickness gradually decreased with the increase of rat ages(F=15.425,P=0.000),and so were the thickness from ILM to IPL,the thickness of INL and the thickness from ONL to RPE(F=3.973,P=0.007;F=17.529,P=0.000;F=7.038,P=0.000).The retinal thickness,thickness of INL.thickness from ONL to RPE measured by Cirrus HD-OCT were significantly correlated with those measured by retinal sections among P14,P18,P21,P24 and P42 rats(r=0.794,P=0.000;r=0.784,P=0.000;r=0.681,P=0.000). Conclusion SD-OCT is a demonstratably valuable technology to study the structure of retinas in rats.The retinal thickness is shown to reduce in thickness throughout the development of the retina during critical period plasticity due to the decrease in thickness of INL and the distance from the ONL to RPE,as illustrated by OCT scanning.

A new method of identification and study of diversity of endophytes wa s described in this paper. The seedlings of tomato were treated using four meth o ds, sterile water, chemical reagents, ultraviolet radiation and mechanical to pe el off, respectively. Then endophytes were isolated using plates of beef- pept on e, Gaos I and PDA respectively. The optimal method of wiping off non-endophyte s was determined. We had obtained 148 isolates of endophytes with different size, shape and color. 43 strains of 148 were amplified using method of ERIC-PCR. T he results showed that 32 strains with amplified bands could fall into 28 classes. The purified bacteria were cultured confronting to leaf blight pathogen of tomat o to screen high resistant strain. Three bacteria strains with high resistance to pathogen were obtained.