Objective: To chine and express the recombinant human endothelial monocyte-activating polypeptide-Ⅱ(EMAP-Ⅱ)and identify its anti-tumor biological activities.Methods: EMAP-Ⅱ_(147-312)was expressed by the expression vector pMAL-p2x and E.coli BL-21 and the product was purified.The production of tissue factor(TF)in human umbili- cal vein endothelial cell ECV-304 mediated by the recombinant EMAP-Ⅱwas determined by chemiluminescence sub- strate.The promoting effect of recombinant EMAP-Ⅱon TNF?-induced ECV-304 cell.Apoptosis was determined by flow cytometry.Its inhibitory effect on human pancreaic cancer cell SW1990 proliferation was determined by MTT method. Results:DNA sequencing verified that EMAP-Ⅱwas correctly cloned.The molecular mass of the protein identified by SDS-PAGE was consistent with the theoretic value.The productivity of recombinant EMAP-Ⅱwas 500?g per 1 g bacteria (wet mass).The purified product induced expression of tissue factor(TF)in ECV-304 cells;it also enhanced the sensi- tivity of ECV-304 cells to the apoptotic effect of TNF?([16.6?2.5]% vs[25.6?2.3]%,P

2 years),seizure frequency(≥1 time/month),persistence time(≥60 s),gene-ralized seizure were all associated with the incidence of the loss of neuron in ammonias.Multivariate Logistic regression analysis showed that the independent influencital factors for the loss of neuron in ammonias in children with temporal epilepsy included seizure frequency,persis-tence time and tape of seizure. Conclusions The loss of neuron in ammonias though 1H-MRS can be detected.The results of multivariate analysis verify that the development of the loss of neuron in ammonias may be associated with many factors including age of onset,course of di-sease,seizure frequency,persistence time and generalized seizure.In order to lower the incidence of the loss of neuron,early intervening treatment is very important.

Objective:To express Tumstatin_(183-230)-TRAIL fusion protein and to observe its biological functions.Methods: SOE-ing PCR was employed to amplify the recombinant sequence of Tumstatin_(183-230)and TNF-related apoptosis-inducing ligand (TRAIL_(114-281)).An expression vector pMAL-Tu-T was constructed by inserting Tu-T sequence into pMAL-c_2;the vector was used to transfect E.coli BL21(DE3)and expression of MBP-Tu-T fusion protein was induced by IPTG.Amylose Resin columns were employed to purify the fusion protein.The biological functions of MBP-Tu-T protein was examined by inhibitory test of endothelial cell proliferation,standard tumor cell cytotoxic assay,in vitro tube formation inhibition,and electron microscopic observation(apoptosis).Results:The expression rate of MBP-Tu-T fusion protein in E.coli was about 20%. Purified recombinant protein obviously inhibited endothelial cell proliferation(IC_(50)12.5?g/ml),induced apoptosis of pancreatic cancer cells,and inhibited tube formation.Conclusion:Constructed MBP-Tu-T fusion protein is bifunctional,which lays a solid foundation for further investigation of antitumor effect of Tumstatin_(183-230)-TRAIL in vivo.

Objective: To prepare a nanoprobe, anti-human melanoma ganglioside single chain variable fragment (GD/ScFvMEL) antibody conjugated with CdTe quantum dot, and to observe its ability to specifically bind human malignant melanoma cells. Methods: The GD/ScFvMEL gene was cloned into pET32a (+), and the plasmid was then transformed into E. coli BL21 (DE3) for GD/ScFvMEL protein antibody expression. The expressed GD/ScFvMEL antibody was purified by denaturing method and further refolded by modified dialysis method. The purified GD/ScFvMEL antibody was analyzed by SDS-PAGE. The GD/ScFvMEL-QDs nanoprobe was prepared by conjugating GD/ScFvMEL antibody with CdTe quantum dot, and its specificity was observed by incubating with MGC-803 cells and melanoma A375 cells. Results: The recombinant pET32a-GD/ScFvMEL was constructed and confirmed by PCR, restriction endonuclease analysis and DNA sequencing. The proportion of expressed GD/ScFvMEL antibody in total bacteria proteins was about 40% as detected by SDS-PAGE. The purified- and refolded-GD/ScFvMEL antibody was effectively conjugated with CdTe quantum dot, and the resulting GD/ScFvMEL-QDs nanoprobe was successfully prepared. The GD/ScFvMEL-QDs nanoprobe could specifically bind melanoma A375 cells, but could not bind stomach cancer MGC-803 cells. Conclusion: We have successfully prepared an anti-human melanoma ganglioside single-chain antibody-CdTe quantum dot nanoprobe, which can specifically bind melanoma cells.

Objective To explore the effects of thyroid hormone on the expression of homeobox gene Nkx6.1 in offspring of hypothyroidism rats and the relationship between gene expression and hormone level by supplying their hypothyroidism pregnant mother with thyroid hormone. Method A total of 240 Wistar rats were half nude and half female. Female rats were randomly divided into eight groups: control, hypothyroidism group, hypothyroidism groups which were supplied with thyroid hormone in high, medium and low dosage in early stage(1- 17 d) and in late stage( 18 - 20 d). According to 100 grams of body weight, the concentration of thyroid hormone were 3.5,2.0,0.5 μg/d in high, medium and low dosage group. All the rats were fed with low-iodine food. The normal control group was given KIO_3 solution and the other groups were given deionized water. After three months female rats were mated with male rats. The content of Nkx6.1 mRNA in brain tissue of 17-day fetal rats, new-born and 20- day old offspring by real-time fluorescence quantitative PCR techniques. Results①A rat model of hypothyroidism was successfully established, there were statistical significance between 8 groups in TT_3,TT_4,FT_3,FT_4(F=4.08,31.99,5.79,26.34, all P < 0.01 ). ② The expression of Nkx6.1 mRNA had significant difference(F = 758.720, 1121.589,144.716, all P < 0.01 ) between groups in 17-day fetal rats, new-bern and 20-day old offsprings and intra- groups in different time (F=2898.863,325.605,716.285,56.329,236.727,196.678,7115.752,9152.306, all P < 0.01 ). ③The time factor and dosage factor had influence on Nkx6.1 mRNA expression(F = 1176.655,246.530, all P < 0.01 ). There were interaction between time and dosage factor(F = 1249.934, P < 0.01 ). ④Comparison of Nkx6.1 mRNA expression between hypothyroidism group and normal control group had significant difference in the above three time points(all P < 0.01 ). ⑤Comparisons of Nkx6.1 mRNA expression between 6 hypothyroidism groups which were supplied with thyroid hormone and hypothyroidism group had significant difference(all P < 0.01 ) in new-bern and 20-day old offspring; comparisons of Nkx6.1 mRNA expression between hypothyroidism groups which were supplied with high and medium thyroid hormone and hypothyroidism group had significant difference in 17-day fetal rats(all P < 0.01 ). ⑥Comparison of Nkx6.1 mRNA expression between hypothyroidism groups which were supplied with medium thyroid hormone in early stage and normal control group had no statistical significance (all P > 0.05), while between the other 5 groups which were supplied with thyroid hormone and normal control group had significant difference(all P < 0.01 ) in the above three time points.⑦Multiple comparison of early stage groups which were supplied with thyroid hormone showed that the expression of Nkx6.1 mRNA had significant difference(all P < 0.01) between high, low dosage groups and medium group in 17-day fetal rats, new-bern and 20-day offspring(all P< 0.01). ⑧Multiple comparison of late stage groups supplied with thyroid hormone showed that old offspring and between high dosage groups and low dosage groups in 17-day fetal rats and 20-day the expression of Nkx6.1 mRNA had significant difference(all P < 0.01 ) between three groups in new-bern and 20- day old offspring. Conclusion The expression of Nkx6.1 in rats offspring is highly related to the supply dosage and supply time of thyroid hormone in hypothyroidism pregnant rats.

The establishment of high specificity and sensitivity method of small molecule monoclonal antibody-based immunoassay has a great importance in the study of small molecule compounds in Chinese medicine, wherein synthesis of small molecule artificial antigen is a critical step in the preparation of small molecule antibodies. Oxidation method using sodium iodide was used to synthesize immunogenic antigen (FRn-BSA) and coating antigen (FRn-OVA) of forsythin. UV spectroscopy and thin layer chromatography showed that forsythin was successfully conjugated with BSA and OVA. After immuned FRn-BSA, the mice could specifically produce anti-forsythin antibodies with titer up to 1:8 000, and the linear range was from 1 mg x L(-1) to 100 mg x L(-1). In this paper, the artificial antigen of forsythin was successfully synthesized, which can be applied for preparation of monoclonal antibodies and establishment of appropriate immune method.
Animals ; Antibodies ; immunology ; Antigens ; chemistry ; immunology ; Bridged Bicyclo Compounds, Heterocyclic ; chemistry ; immunology ; Drugs, Chinese Herbal ; chemistry ; Furans ; chemistry ; immunology ; Male ; Mice ; Mice, Inbred BALB C

To investigate the resources of medicinal plant, such as wild Apocynum, supervised classification based on Principal Component Analysis (PCA) and texture feature were used to monitor wild medicinal plants from image captured by ZY-3 and World-view-2 and compare which satellite Image are more appropriate to monitor the wild medicinal plants. The research results shows that: for more complex growth conditions wild medicinal plants Apocynum, high-resolution images Worldview-2 is more suitable for its remote identification, the low-resolution satellite ZY-3 can only recognizes the wild medicinal plants which distributed intensively. If the study target distribution is more intensive and larger scale, and cultivated type medicinal plants, the use of satellite ZY-3 in low resolution remote sensing data to identify the target can be a good choice, it is not necessary to buy high-resolution data, in order to avoid waste of expenditure, for the scattered distribution, the high-resolution satellite imagery data may be indispensable to identify targets.
Apocynum ; chemistry ; growth & development ; China ; Conservation of Natural Resources ; Geographic Information Systems ; Plant Dispersal ; Plants, Medicinal ; chemistry ; growth & development ; Remote Sensing Technology ; methods

To confirm the mechanism of exosomes as tumor vaccines inducing immunity response, dendritic cells (DCs) were induced from human peripheral blood mononuclear cells, while exosomes were isolated from DC loaded tumor antigen. The effect of exosomes on priming T cell proliferation was analysed under conditions with or without DCs, or DCs at different mature stages. The function of exosomes in immunity was detected through block test after blocking some molecules (CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83). The effect of DCs on embedded exosomes was observed by confocal microscopy, the effect of blocking surface molecules on exosomes on DC-embedding exosomes was assayed by flow cytometry. The results indicated that both exosomes derived from imDC (imDex) and exosomes derived from mDC (mDex) could not prime T cells without DC or with imDC. The exosomes derived from mDC induced with different cytokines (LPS, TNF-alpha, CpG, CD40L) were no significant difference in concentrations but were different in effect. The immunity function of exosomes depended on CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83 molecules, the effect of priming T cells is reduced when these molecules were blocked. Confocal microscopy and FACS assay showed that blocking CD11a and CD54 could inhibit exosome-targeted DC and DC-embedded exosomes. It is concluded that the exosomes target DCs through their surface molecules, therefore results in immune response of T cells.
Antigens, Neoplasm ; immunology ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; secretion ; Exosomes ; immunology ; Humans ; K562 Cells ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; cytology ; immunology

The aim of this study was to examine the priming effect of sphingosine 1-phosphate (S1P) on fMLP-activated neutrophils, mainly to detect the neutrophil respiratory burst products, and to investigate the signaling pathway involved in S1P activity. Flow cytometry was used to evaluate the new isolated neutrophil; the superoxide anion output was detected indirectly by cytochrome C reduction in respiratory burst; the dihydro-rhodamine 123 was used to detect the intensity of respiratory burst; the signal transduction pathways of neutrophil respiratory burst were explored by Western blot. The results showed that after pretreated with S1P, the level of superoxide anion released by fMLP-activated neutrophils significantly increased; the Rhodamine 123 mean fluorescence intensity in S1P primed fMLP-activated neutrophils group was significantly higher than that in fMLP treatment group; PI3K and Akt proteins involved in the signal pathway of neutrophil respiratory burst. It is concluded that S1P is a new priming reagent, which primes respiratory burst of fMLP-activated neutrophils; this signal pathway may be that S1P interacts with its receptor, activates PI3K, then activates Akt-transmitting signals through NADPH oxidase, finally results in the respiratory burst.
Cells, Cultured ; Humans ; Lysophospholipids ; metabolism ; NADPH Oxidases ; metabolism ; Neutrophils ; metabolism ; physiology ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptors, Lysosphingolipid ; metabolism ; Respiratory Burst ; Signal Transduction ; Sphingosine ; analogs & derivatives ; metabolism ; Superoxides ; metabolism

OBJECTIVETo evaluate the efficacy and safety of N-acetylcysteine (NAC) and glutathione (GSH) in treating chronic hepatitis B patients.

METHODSSeventy-five patients with chronic hepatitis B were treated daily with an injection containing the same basic therapeutic drugs and randomly divided into a NAC group (50 patients) and a GSH group (25 patients). A daily dose of 8 grams of NAC and 1.2 grams of GSH was added to the injections of the two groups respectively. The trial lasted 28 days. Hepatic function and other biochemistry parameters (TBil, PTA, ALB et al) were tested on experimental day 0 and on days 7, 14, 21 and 28. The evaluation on the total effective rates of the NAC and GSH groups was based on the decreases of serum TBil and the increases of PTA.

RESULTSBoth NAC and GSH have therapeutic effects. The total effective rate was 84% in the NAC group and 72% in the GSH group. The rate of side effects was 13% in the NAC group.

CONCLUSIONNAC and GSH can decrease the level of serum TBil and increase PTA, but NAC was more effective in decreasing TBil than GSH. Serious adverse effects of NAC were not observed during the period of our treatment.

Acetylcysteine ; therapeutic use ; Adult ; Bilirubin ; blood ; Female ; Glutathione ; therapeutic use ; Hepatitis B, Chronic ; blood ; drug therapy ; Humans ; Male ; Middle Aged ; Treatment Outcome