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

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

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 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.

Objective To investigate the effects of intestinal ischemia reperfusion (IIR) on the progression of inflammatory reaction in hemorrhagic necrosis pancreatitis (HNP).Methods Eighty rats were randomly divided into sham operation (SO) group,acute edematous pancreatitis (AEP) group,AEP + IIR group and HNP group according to the random number table.Erythrocyte velocity (EV),functional capillary density (FCD) and leukocyte adherence (LA) were observed at 0,1,2,3 and 6 hours after the models were completed.The serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected.All data were analyzed by using the analysis of variance or the t test.Results The level of EV in the AEP group significantly decreased at 1 hour,and got increased at 3 hours,while the level of EV in the AEP group was still significantly lower than that in the SO group ( t =9.60,P ＜ 0.05 ).The levels of EV in the AEP + IIR group and HNP group constantly decreased,and increased at 6 hours,but were continually lower than that in the AEP group ( t =6.03,6.12,P ＜0.05 ).The level of FCD in the AEP group was significantly lower than that in the SO group at 3 hours ( t =8.20,P＜0.05).The levels of FCD in the AEP + IIR group and HNP group were significantly lower than that in the AEP group at 3 hours (t =35.60,23.80,P ＜ 0.05 ).Compared with AEP group,the level of LA in the AEP group was significantly increased at 1 hour ( t =75.00,P ＜ 0.05 ) and reached peak at 3 hours.The levels of LA in the AEP + IIR group and HNP group were significantly higher than that in the AEP group at 1,2,3,6 hours (t =23.00,29.50,53.00,38.70,23.10,48.20,39.20,47.50,P＜0.05).Compared with SO group,the level of TNF-α in the AEP group significantly increased since l hour (t =77.00,P ＜ 0.05),and began to decrease at 3 hours; the levels of TNF-α in the AEP +IIR group and HNP group at 2 hours were significantly higher than that in the AEP group (t =23.50,18.10,P＜0.05).The levels of IL-6 in the AEP group at 1,2,3,6 hours were significantly higher than those in the SO group ( t =93.50,146.00,243.60,209.20,P ＜ 0.05 ).The levels of IL-6 in the AEP + IIR group and HNP group at 1 hour were not significantly different from that in the AEP group ( t =2.30,2.03,P ＞ 0.05),while the levels of IL-6 in the AEP + IIR group and HNP group at 2 hours were significantly higher than that in the AEP group (t =35.63,29.80,P ＜ 0.05 ).Conclusion IIR may enhance the inflammatory reaction of AEP and IIR might be one of the factors to exaggerate the inflammatory reaction of HNP.

Objective To observe the influence ofgypenosides on hydrogen sulfide in liver tissue and plasma of rat with type 2 diabetes mellitus and nonalcoholic fatty liver disease.Methods 58 SPF male SD rats,with body mass 220～250 g,were randomly divided into a blank control group (group N,n=7),and a NAFLD and T2DM model group (Group M,n=51).Group N was fed with ordinary diet in the first four weeks,group M was fed with diets of high fat and sugar,injected with 40 mg/kg STZ overnight,and the same diets for the next four weeks.The rat model with T2DM and NAFLD was build.NAFLD and T2DM model group were divided into three groups:a high dose GPS group (JH,n=9) injected with 1 g/kg · d-1 GPS,a low dose GPS group (JL,n=9) injected with 0.5 g/kg · d-1 GPS,and a model group (M,n=9) injected with the same volume of water,and high fat diet at the same time.The treatment period was six weeks,and the experiment period was fourteen weeks.TG,TC,BS,and H2S in the plasma of rat were tested,and H2S in the liver tissue of the rat was tested.Results ①The changes of H2S in plasma:group JH [(4.30±0.43) μmol/L] and JL [(3.83 ±0.47) μmol/L] was lower than group M [(2.67 ± 0.41) μmol/L],there was a significant difference.②The changes of H2S in the liver tissue:group JH [(333.52±37.94) pmol/min/mg/protein] and JL [(275.81 ±36.07)pmol/min/mg/protein] was lower than group M [(237.8± 33.05) pmol/min/mg/protein],there was a significant difference.③BS levels:group JH(10.86±3.46)mmol/L,group JL (14.78±3.39)mmol/L,group M(18.84±4.24) mmol/L,group JH and JL was lower than group M,there was a significant difference (P＜0.01).④The plasma TG level:group N (0.96±0.09) mmol/L,group JH (2.82± 0.66) mmol/L,group JL (1.83± 0.56) mmol/L,group M (3.97 ± 0.64) mmol/L.group JH and JL was lower than group M,there was a significant difference (P＜0.01).Conclusion Gypenoside can reduce the blood sugar,triglycerides,and total cholesterol in rat with with type 2 diabetes mellitus and nonalcohol fatty liver disease.H2S concentrations in plasma and liver tissue of the rats with T2DM and NAFLD were increased by GPS,showing dose dependence.Gypenosides can also improve metabolism of blood glucose and lipid in rats with T2DM and NAFLD.

Objective To explore the temporal and spatial expression pattern of tenascin-c(tnc) and tenascin-w(tnw) in zebrafish early development,to further explore the role of tenacsin in zebrafish embryo development,and the association between them.Methods Zebrafish embryos at 2 hours post fertilization(hpf),4 hpt,8 hpt,10 hpf,24 hpt,48 hpr,72 hpf and 7 days post fertilization(dpf) were collected to extract RNA for reverse transcription-polymerase chain reaction(RT-PCR) and fix the embryos at different stages for in situ hybridization.Temporal and spatial expression pattern of tnc and tnw on different stages of zebrafish early development was observed.Results tnc and tnw all expressed in zebrafish from 24 hpf to 7 dpf,but did not expressed from 2 hpf to 10 hpf.Tnc expressed at pharyngeal arch,notochord,somite in 24 hpf,then weakly expressed at somite,but highly expressed at otic vesicle,pectoral fin and hindbrain in 48 hpf,and tnc was expressed at hindbrain,pharyngeal and notochord and disappeared at somite and pectoral.tnw expressed at hindbrain,midbrain and otic vesicle in 24 hpf,expressed at somite,notochord,hindbrain,otic vesicle and pharyngeal in 48 hpf.In 72 hpf,tnw expressed weakly at somite and notochord.Conclusions Zebrafish tnc and tnw have special temporal and spatial expression pattern,and share partial overlapping expression pattern.

Objective To explore the expression pattern of tenascin-c(tnc)gene in zebrafish embryo abnormal development which was induced by ethanol,and to further understand the function of tnc gene in embryo develepment.Methods Zebrafish were treated with ethanol at different concentration from 100 to 500 mmol/L,and embryos at 24 and 48 hours were collected and fixed,then tnc expression pattern was observed by in situ hybridization and reverse transcriptase-polymerase chain reaction(RT-PCR).Results The result of RT-PCR showed that ethanol at 100 and 200 mmol/L could increase the expression of tnc,while the result of in situ hybridization showed that,while ethanol at 300 mmol/L and above decrease the expression of tnc in presumptive position at 24 hours,and ethanol at 100 mmol/L and above caused increase expression of tnc in zebrafish heart.Conclusions tnc is increased when treated with 100 and 200 mmol/L ethanol and is presented in the abnormal development of hearts of zebrafish,which can promote the normal development of embryos in some degrees.The expression pattern of tnc in pathologic state is highly conserved in all vertebrate,and in adult and embryos as well.