Objective To construct and identify the transgenic plant vector recombinant pBI-Eg95 plasmid of Echinococcus granulosus. Methods Total RNA was extracted from hydatid cyst protoscoleces of Echinococcus granulosus after sonication. A couple of specific primers were designed on the basis of known sequences of Eg95 gene. The desired gene was amplified by PCR technique from the cDNA, and then was cloned into the plant expression vector pBI121 to construct the recombinant pBI-Eg95 plasmid. The recombinant plasmid was electroporated into Agrobocterium tumefaciens (At) LBA4404 strain. The positive recombinant clones were confirmed by restriction endonuclease digestion and characterized by PCR. Results For RT-PCR, a specific band around 471 bp was amplified. The result of DNA sequencing of Eg95 showed the identity with the published sequence. The same band was obtained by restriction endonuclease digestion and PER from the plasmids of positive recombinant At(rAt). Conclusions The recombinant pBI-Eg95 plasmid was successfully constructed, and it provides the basis to further research of the transgenic plant vaccine of Echinococcus granulosus.

Objective To investigate the protective immunity in mice immunized with recombinantBifidobacteria bifidum(Bb)-Eg95 vaccine of Echinococcus granulosus (Eg) and challenged with Eg protoscoleces.Methods Fifty-six female BALB/c mice 12-14 weeks old and weighed 20-25 g were vaccinated with the recombinant Bb-Eg95 vaccine subcutaneously,intramuscularly,intranasally and orally,respectively,with blank vector,Bb and medium of solution(MRS) as control,8 mice in each group.Mice were challenged with Eg protoscoleces on week 8 after immunization and killed on week 25 after infection.The weight of hydatid cyst was measured and the decreased larva rate was calculated.Sera were collected to determine the levels of IgE,IgG and its subclasses by enzyme linked immunosorbent assay(ELISA).Splenocytes were collected and cultivated to test the proliferation of splenocytes using methyltetrazolium (MTT) assay under EgAg and concanavalin A (ConA) stimulation.The results were compared with analysis of variance and the comparison between two groups was performed with LSD-t test.Results There was significant difference in the weight of hydatid cyst between groups (F =11.062,P ＜ 0.05).Compared with MRS control group[(0.075 ± 0.019)g],the hydatid cyst weight decreased in subcutaneous group [(0.050 ± 0.013)g],intramuscular group[(0.050 ± 0.019)g],intranasal group[(0.028 ± 0.016)g] and oral group [(0.031 ± 0.018)g,all P ＜ 0.01).Compared with subcutaneous and intramuscular groups,the hydatid cyst weight decreased in intranasal and oral groups(all P ＜ 0.05).The decreased larva rate was inversely proportional to the weight of hydatid cyst.There was significant difference in the levels(obsorbancy,A) of IgG,IgG2a,IgG2b,IgG1,IgG3 and IgE between these groups(F =21.774,36.977,27.071,14.746,10.131,9.444,P ＜ 0.05 or P ＜ 0.01).Compared with MRS control group (0.015 ± 0.002,0.002 ± 0.001,0.003 ± 0.001),the levels of IgG,IgG2a and IgG2b increased in subcutaneous group(0.022 ± 0.004,0.007 ± 0.002,0.008 ± 0.002),intramuscular group (0.023 ± 0.003,0.008 ± 0.002,0.007 ± 0.002),intranasal group(0.032 ± 0.007,0.012 ± 0.002,0.013 ± 0.004)and oral group(0.028 ± 0.006,0.010 ± 0.003,0.010 ± 0.002,P ＜ 0.05 or P ＜ 0.01).Compared with subcutaneous and intramuscular groups,the levels of IgG,IgG2a and IgG2b increased in intranasal and oral groups(P ＜ 0.05 or P ＜ 0.01).Compared with MRS control group(0.009 ± 0.001,0.009 ± 0.002,0.009 ± 0.001),the levels of IgG1,IgG3 and IgE decreased in subcutaneous group(0.022 ± 0.004,0.007 ± 0.002,0.008 0.002),intramuscular group(0.004 ± 0.001,0.004 ± 0.001,0.004 ± 0.002),intranasal group(0.005 ± 0.002,0.005 ± 0.003,0.005 ± 0.002)and oral group(0.005 ± 0.001,0.004 ± 0.002,0.004 ± 0.003,all P ＜ 0.01).There was significant difference in the proliferation of splenocytes in the supernatant of cultured splenocyte,of cultured splenocyte + EgAg and of cultured splenocyte + ConA(F =63.975,359.833,167.399,P ＜ 0.01).There was significant difference in the proliferation of splenocytes inside groups(F =6741.955,4953.667,869.320,201.235,175.413,139.653,169.994,all P ＜0.01).Compared with the cultured splenocyte the proliferation of splenocytes increased in the cultured splenocyte +EgAg and splenocyte + ConA (all P ＜ 0.01).Compared with the cultured splenocyte + EgAg,the proliferation of splenocytes increased in the cultured splenocyte + ConA(P ＜ 0.01).Conclusion An effective and protective immunity is induced by the recombinant Bb-Eg95 vaccine of Eg in mice.

This article is to explore the practical application of concept maps in nursing teaching practice to make it as a learning tool to promote undergraduates to make a meaningful study. Besides, the results is applied in research on improving the teaching method so as to provide an effective teaching policy and evaluation tools to promote the scientific research and clinical practice in nursing care.

Objective To cultivate and identify the transgenic affalfa containing Echinococcus granulosus Eg95 gene. Methods The alfalfa plants were transformed by co-cultivating alfalfa cotyledons via recombinant Agrobacterium tumefaciens LBA4404 harboring pBI-Eg95. The transgenic alfalfa explants were selected by kanamyein after calli formation, shoots and roots regeneration in the selective medium, the seedlings of transgenic plants were obtained which were finally transplanted into pots containing nutrient soil. After 2-3 months growth, the complete transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene were obtained. To identify the transgenic alfalfa plants, the total DNA, RNA and leaf protein were extracted from fresh leaf tissue of the transgenic alfalfa plants and confirmed by PCR, RT-PCR, SDS-PAGE and Western blot assay. Results A specific band around 471 bp was amplified by PCR with total DNA, and the same band was obtained by RT-PCR with total RNA, which confirmed that the Eg95 gene was stably integrated into the transformed alfalfa genome. SDS-PAGE analysis showed that the relative molecular mass(Mr) of the expressed protein was about 16.5×103, consistent with the Eg95 protein, and the level of Eg95 expression was up to 0.06% of total soluble leaf protein by Bio-Rad Quantity one assay. Western blot verified the expressed protein was reactive with the sera of mice infected with Echinococcus granulosus. Conclusion The transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene are successfully cultivated.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma, Small Cell ; metabolism ; pathology ; therapy ; Cardia ; Chemotherapy, Adjuvant ; Chromogranin A ; metabolism ; Female ; Follow-Up Studies ; Gastrectomy ; methods ; Humans ; Male ; Middle Aged ; Phosphopyruvate Hydratase ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; therapy ; Survival Rate

Objective To determine the weight coefficient of the customer requirements of emergency infusion equipment.Methods Customer requirements were made clear using methods of literature research, personal interviews and expert meeting. Analytic hierarchy process approach was adopted to establish the hierarchical structure model, construct paired comparison matrix structure, calculate the weight coefficient and make logic consistency check.Results The weight coefficients were determined for 8 primary and 36 secondary customer requirements, which all met the logical consistency check (CR<0.1).Conclusion The research and development of emergency infusion equipment has to pay attention to such key requirements firstly as safety, efficacy and portability, and the intelligence has to be supported by the key requirements and intermediate needs.

Objective To explore the inhibition role of 1 ,25 dihydroxyvitamin D3 on laryngeal cancer Hep‐2 cell proliferation and its influence on mTOR signal pathway .Methods Hep‐2 cells were treated with different concentrations of 1 ,25 dihydroxyvitamin D3 (10-8 ,10-7 ,10-6mol/L) for 24 ,48 ,72 h respectively .The proliferation situation of Hep‐2 cells was detected by the MTT meth‐od and the inhibition rate was calculated .The effect of 1 ,25 dihydroxyvitamin D3 on Hep‐2 cell cycle distribution was analyzed by flow cytometry .The influence of 1 ,25 dihydroxyvitamin D3 on mTOR signaling pathway was detected by Western blot .Results Different concentrations of 1 ,25 dihydroxyvitamin D3 could inhibit the proliferation of Hep‐2 cells ,changed the cell cycle distribu‐tion and increased the proportion of Hep‐2 cells in G0/G1 phase .The expressions of TSC1 and TSC2 protein after 1 ,25 dihydroxyvi‐tamin D3 intervention were increased compared with the control group (P<0 .01) ,while the Rheb protein expression was signifi‐cantly decreased(P<0 .01):mTOR protein and phosphorylation level were significantly decreased compared with the control group (P<0 .01) ,the decrease of mTOR protein phosphorylation was especially obvious (P<0 .01);4EBP‐1 protein expression was in‐creased compared with the control group (P<0 .01) .Conclusion 1 ,25‐dihydroxyvitamin D3 alters the Hep‐2 cell cycle distribution , affects the protein expression of mTOR signaling pathway ,thus inhibits the cell proliferation .

Objective To detect levels of serum platelet activating factor(PAF),thrombomodulin(TM) and white blood cell(WBC),platelet count(PLT) in neonates with meconium aspiration syndrome(MAS),and observe changes of mediators of inflammation and function of endotheliocute.Methods All cases were taken vein blood in 24 h and 72-96 h after birth.Surm PAF and TM were detected by EILSA technique,at the same time,blood cell counts were determined.Results PAF and WBC in neonates with MAS increased,which were relevant to the patients′ condition.TM of neonates with MAS increased significantly,especially in 72-96 h after birth and(aggrava)-ted with the patients′ condition.Conclusion Neonates with MAS have inflammatory reaction and injured endotheliocyte,which are(inte)-raction.

To investigate the effect of NVP-DPP728, a DPP-Ⅳ inhibitor on new-onset diabetes and the autoimmune response in non-obese diabetic ( NOD ) mice. Diabetes could be reversed in 75% of NVP-DPP728 treated 20 NOD mice. In these 15 mice with remission, insulitis scores were significantly lower than those of the control group. The percentage of Tregs was increased in the thymus and celiac lymph nodes, plasma TGF-β1 and GLP-1 were also significantly increased ( P＜0. 01 ). NVP-DPP728 treatment may reverse new-onset diabetes in NOD mice by reducing insulitis and increasing Tregs.

Objective　To compare the Immunoreactivity of various HEV Antigen with Anti-HEV IgM. Methods Solid-phase enzyme immunoassay( EIA ) was developed for detecting anti-HEV IgM by using synthetic peptides E30, E42, E33, and recombinant antigen from HEV ORF-2. Results Of 60 anti-HEV positive sera by using E30, E42, E33 and recombinant antigen as coating antigen, Anti-HEV IgM positive rates were 76.7%, 26.6%, 18.3% and 66.7% respectively. In Acute-phase and convalescence-phase sera of the patients with Hepatitis E, Anti-HEV IgM positive rate was 90% and 3.3% respectively. Conclusions The HEV E30-based EIA will be very useful in the early diagnosis of Hepatitis E.