AIM: To construct eukaryotic expression vector of human vascular endothelial growth factor (VEGF) gene, and to explore the effects of VEGF gene on proliferation and chemosensitivity of leukemia cells. METHODS: The recombinant eukaryotic express plasmid pEGFP-C1-hVEGF_(165) was constructed with conventional gene engineering methods. The pEGFP-C1-hVEGF_165 was transfected into leukemia cell K562. The proliferation of the recombinant eukaryotic expression plasmid was analyzed by CCK8 method. The level of VEGF mRNA was detected by reverse transcription and quantitative polymerase chain reaction (RT-PCR). The expression level of VEGF protein was determined by ELISA in cell culture medium and cells. The cell cycle was detected by flow cytometry. RESULTS: VEGF_(165) eukaryotic expression vector was successfully constructed, confirmed by enzyme digestion and sequencing. Compared with the K562 cells transfected pEGFP-C1, the K562 cells transfected with pEGFP-C1-hVEGF_(165) grew faster and expressions of VEGF mRNA and protein increased significantly. In addition, K562 cells transfected with pEGFP-C1-hVEGF_(165) had relatively higher cell survival rate to chemotherapy drugs homoharringtonine(HHT) and fluorouracil(FU). CONCLUSION: VEGF_(165) eukaryotic expression vector increases the level of VEGF mRNA and protein expression, accordingly promotes the proliferation of leukemia cells and decreases the sensitivity of leukemia cells to HHT and FU.

In this article we built formula database of health food containing Gardeniae Fructus with Traditional Chinese Medicine Inheritance Support System (V2.0). And on this basis, use data mining method such as association rules of the software, to analyze commonly used formula raw materials or materials combination of formula containing Gardeniae Fructus and raw material application having assisted function formula to protect chemical liver injury. The result shows that of the 71 health food formulas containing Gardeniae Fructus, most used materials are Gardeniae Fructus, Lycii Fructus, Angelica Sinensis Radix, Poria and so on. Commonly used materials combination mostly are Gardeniae Fructus and Lycii Fructus, Gardeniae Fructus and Angelica Sinensis Radix, Gardeniae Fructus and Poria, Gardeniae Fructus and Paeonia. There are nearly 18 healthcare functions of the health food containing Gardeniae Fructus, and most of these are assisted functions to protect chemical liver injury, and then immune modulating function. Of 23 formulas containing Gardeniae Fructus having assisted function formula to protect chemical liver injury, Gardeniae Fructus usually combined with traditional Chinese medicine which nourishs blood and liver such as Pueraria, Lycii Fructus, Hawthorn, Paeonia and Turnjujube. Analyzing formula raw materials application of health food containing Gardeniae Fructus contributes a lot to the further development and utilization.
Chemical and Drug Induced Liver Injury ; drug therapy ; Data Mining ; methods ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Food, Organic ; Fruit ; chemistry ; Gardenia ; chemistry ; Medicine, Chinese Traditional ; methods

Objective To explore the interaction between serum melatonin and the status of disease and probe the effect factor of serum melatonin change in asthmatic children.Methods Serum melatonin was measured in asthmatic children with 15 cases of mild persistent asthma,15 cases of moderate persistent asthma,15 cases of severe persistent asthma,15 cases of stable asthma and 15 cases of normal subjects by enzyme-linked immunosorbent assay(ELISA).Results The levels of serum melatonin in the 5 groups of mild persistent asthma,moderate presistent asthma,Severe Persistent asthma,Stable asthma,control subject were(22.76?5.16)ng/L,(16.79?3.35)ng/L,(11.54?1.45)ng/L,(22.06?3.36)ng/L,(28.72?4.32)ng/L,respectively.There were significant differences between any of them(Pa

Objective To explore the effect of Melatonin(MT) on CD4+CD25+ regulatory T cell (CD4+CD25+Tr)and airway inflammation in asthmatic rat.Methods Thirty-two SD rats were randomly divided into 4 groups,8 rats in each group.Asthmatic group:rats were immunized on day 1 and 7 by intraperitoneal inject of mixture of ovalbumin(OVA) and aluminumhydroxide.From day 14,the animals were allenged with aerosolized OVA for 20 min per day for 7 consecutive days.MT group:OVA-sensitized rats were injected intraperitoneally with 0.1 mg/kg MT 30 min before each OVA challenge.Dexamethasone group:OVA-sensitized rats were injected intraperitoneally with 0.5 mg/kg Dexamethasone 30 min before each OVA challenge.Control group:OVA for inhalation and MT for intraperitoneal injection was replaced with saline.After the last challenge,peripheral blood was stained to count the percentage of eosinophil(EOS).Then the rats were lavaged and total leukocytes counts in bronchoalveolar lavage fluid(BALF) were performed after staining with Wright-Giemsa staining.The EOS counts around the airway was counted after the histological section of lung staining with hematoxylin and eosin staining.The serum level of immunoglobulin E(IgE) was detected by immunoenhancement.The change of CD4+CD25+Tr was assessed with flow cytometry.SPSS 10.0 software was applied to analyze data. Results In asthmatic rats,the CD4+CD25+ Tr/ CD4+T cells ratio had significant negative relationship with the EOS counts around the airway and the total leukocytes counts in BALF (r=-0.73 P0.05).There was a significant decrease in the percentage of the eosinophils in peripheral blood,the eosinophil counts around the airway,the total leukocytes counts in BALF and the serum level of IgE in MT group compared with asthmatic group (Pa

Objective To investigate the function of transferrin-DNA complex,transported by transferrin(Tf)and trans-arterial injection via interventional approach be the duel-target-orientated delivery and the transferring into malignant cells to get more effective therapy.Methods p53-LipofectAMINE ligand with different concentrations of Tf(0,10,25,50,100?g)transfected the 4 strains including LM6、Hep3B、YY and L02 in vitro to evaluate the gene transfeetion efficiency through western blot.Then,after setting up the VX2 hepatocarcinoma models,we delivered the Tf-p53-LipofeetAMINE complex into the hepatic arteries via interventional techniques to analyse the transfection efficiency in vivo.Results Tf,within the range of 10 100?g,could increase gene transfection efficiency mediated by liposome,and the efficiency increases with the raise of Tf concentration.Combination with interventional technique to inject Tf-DNA complex into tumor arteries,gene transfeetion efficiency was enhanced in rabbit models.Conclusion Tf can enhance gene- liposome transfection efficiency,furthermore with combination of interventional catheter technique,there would be a potential duel-target-orientated gene therapy method.(J Intervent Radiol,2007,16:99-103)

hMR-1 (Homo Myofibrillogenesis Regulator 1, AF417001) is a novel homo gene, which was firstly cloned in our laboratory. The former studies revealed that hMR-1 is a transmembrane protein which shows protein interaction with sarcomeric proteins like myomesin I, myosin regulatory light chain, alpha-enolase and some cell regulator proteins such as eukaryotic translation initiation factor3 subunit 5 (eIF3S5) and etc. In this work, we focused on cloning the homologous gene of hMR-1 from mouse C57BL/6J and exploring its expression using Pichia pastoris yeast system. Two pairs of primers were synthesized according to the hMR-1 gene homologous sequence on mouse genome chromosome 1. The mouse MR-1 gene (mMR-1) was cloned by PCR following the first round RT-PCR from mouse C57BL/6J spleen total RNA. Sequence analysis verified that mMR-1 gene and amino acids sequence showed 90.4% and 90.1% identity with hMR-1, respectively. The prediction of hydrophobic transmembrane structure of mMR-1 suggested it is also a transmembrane protein. The mMR-1 Pichia pastoris expression vector pPIC9-mMR-1 was constructed by fusion of the flanking mMR-1 ORF in the pPIC9 plasmid. After linearization of pPIC9-mMR-1 with Sal I, the 8.5kb DNA fragment was transformed into Pichia pastoris GS115 strain by electroporation. GS115/Mut+ pPIC9-mMR-1 transformants were selected on minimal methanol medium. Integration of mMR-1 gene into the yeast genome in the recombinants was verified by PCR from the transformants total DNA. The mMR-1 protein was expressed by induction under the concentration of 0.5 % methanol. The specific induced protein of 25 kD molecular mass in SDS-PAGE was confirmed to be the mMR-1 protein by Western blot rsing hMR-1 polyclonal antibody. The expression level of this recombinant mMR-1 protein was about 50 mg/L. The successful expression of mMR-1 in the Pichia pastoris GS115 will facilitate the further functional analysis of the novel gene MR-1 in animal model.
Amino Acid Sequence ; Animals ; Cloning, Molecular ; Humans ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Muscle Proteins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

In this paper, the five strains of Polygonatum odoratum were used as the experimental materials to test the supercooling point, freezing point, the degree of supercooling, the transition stage time, cooling time and water composition of the plant tissue. The cold resistance of P. odoratum was analyzed with the Gray Correlation Method. The results showed that the cold resistances of the five strains of P. odoratum were different, and the water content of plant tissue had some relevance with freezing point and supercooling point, whereas, it could not be measured when the moisture content was too low. The order of cold resistance of the five strains of P. odoratum was ZJCY, DYYZ, XYYZ, CYYZ and JZ I.
Cold Temperature ; Plant Roots ; chemistry ; physiology ; Polygonatum ; chemistry ; classification ; physiology ; Water ; analysis

OBJECTIVETo study the therapeutic effect of agonistic CD40 monoclonal antibody combined with tumor specific cytotoxic T lymphocyte (CTL) on B lymphoma.

METHODSHuman B lymphoma cell line, Daudi cells, were cultured with CD40 mAb (5C11) for 24 and 48 hours, respectively. Annexin V/PI-binding assay was employed to analyze apoptosis, and FCM to analyze Fas (CD95) expression. Human peripheral monocyte-derived DC were loaded with apoptotic Daudi cells and stimulated by SC11 for further maturation. Tumor specific CTL were generated in vitro by co-culture of mature DC with autologous T lymphocytes. DNA fragmentations of Daudi cells treated with 5C11, CTL or 5C11 combined with CTL were determined by JAM assay. To establish the B lymphoma model, Daudi cells were subcutaneously injected into humanized SCID mice (hu-SCID). 1 or 3 weeks after tumor transfer. tumor-bearing mice were respectively treated with SC11, CTL, 5C11 combined with CTL by intraperitoneal injection. Tumor volume in differently treated mice was measured every week after therapy, and the survival of tumor-bearing mice was recorded.

RESULTS5C11 significantly up-regulated FAS expression in Daudi cells, but had no significant effect on apoptosis rate of Daudi cells. Tumor-specific CTL could effectively kill Daudi cells. Fragmentation of Daudi cells co-cultured with CTL was remarkably enhanced by combination with SC11. Tumor growth in hu-SCID mice was apparently delayed by treatment with SC11, CTL, or SC11 combined with CTL. Moreover, minimal tumor burden mice got 30.0% or 70.0% complete remission (CR), respectively, when received CTL treatment or combination treatment of SC11 with CTL, and the lifespan of tumor bearing mice was also prolonged significantly.

CONCLUSIONSC11 may enhance the sensitivity of Daudi cells to apoptosis by up-regulation of Fas expression and promote cytotoxicity of CTL in vitro and therapeutic effect in vivo.

Animals ; Antibodies, Monoclonal ; immunology ; therapeutic use ; Apoptosis ; immunology ; CD40 Antigens ; immunology ; Cell Line, Tumor ; Coculture Techniques ; Female ; Flow Cytometry ; Humans ; Immunotherapy, Adoptive ; methods ; Lymphoma, B-Cell ; immunology ; pathology ; therapy ; Mice ; Mice, SCID ; Remission Induction ; Survival Analysis ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Xenograft Model Antitumor Assays ; fas Receptor ; immunology

Deep brain stimulation (DBS) of the pedunculopontine nucleus (PPN) is a novel therapy developed to treat Parkinson's disease. We report a patient who underwent bilateral DBS of the PPN and subthalamic nucleus (STN). He suffered from freezing of gait (FOG), bradykinesia, rigidity and mild tremors. The patient underwent bilateral DBS of the PPN and STN. We compared the benefits of PPN-DBS and STN-DBS using motor and gait subscores. The PPN-DBS provided modest improvements in the gait disorder and freezing episodes, while the STN-DBS failed to improve the dominant problems. This special case suggests that PPN-DBS may have a unique role in ameliorating the locomotor symptoms and has the potential to provide improvement in FOG.
Deep Brain Stimulation* ; Freezing ; Gait ; Humans ; Hypokinesia ; Parkinson Disease* ; Subthalamic Nucleus ; Tremor ; Weather

OBJECTIVETo explore the anti-tumor mechanism of the combination of cisplatin with DC vaccine in tumor-bearing mice.

METHODSB16 melanoma cells were treated with cisplatin at the final concentration of 20 µg/ml in vitro for 24 h. The expression of HMGB1, Hsp70 and TGF-β were detected by Western blot. B16 tumor-bearing mouse models were generated. The therapeutic effect of the combination of cisplatin (100 µg/mouse i.p., for sequential 3 days) and intratumoral injection of DC cells (3×10(6)/mouse, twice with a 7-day interval) in the tumor-bearing mouse models was evaluated. Expression of MHC II, ICAM-1 and CD86 was analyzed by flow cytometry. The mice were sacrificed at 28 days after tumor cell inoculation. The tumors were removed and weighed, and tissue samples were taken for pathological examination. Tumor infiltrating lymphocytes (TIL) were isolated by discontinuous gradient centrifugation. The distribution of T-reg and CD8(+) T cells in the TIL was analyzed by flow cytometry, and the ratio of CD8(+) T/T-reg was determined. The activity of cytotoxic lymphocytes (CTL) was determined by microcytotoxicity assay.

RESULTSCisplatin enhanced both the B16 cell apoptosis and HMGB1 expression. After loading with cisplatin-treated cell lysate, the expression of MHC II, ICAM-1 and CD86 on DC cells were (47.5 ± 8.8)%, (35.5 ± 8.3)% and (36.2 ± 9.2)%, respectively. At 28 days after tumor cell inoculation, the tumor weight of the control group was (2.1 ± 0.6) g, that of the cisplatin group was (0.3 ± 0.2) g and that of cisplatin + DC vaccine group was (0.5 ± 0.2) g, showing a significant inhibition of tumor growth (P < 0.01). Furthermore, the CD8(+) T/T-reg ratio and CTL activity in TIL were also significantly enhanced in the tumor-bearing mice treated with cisplatin + DC vaccine. When the effector-to-target ratio was 20:1, 10:1 and 5:1, the CTL activity in the cisplatin + DC vaccine treated mice was (25.0 ± 5.0)%, (22.0 ± 6.0)% and (14.0 ± 4.0)%, respectively, significantly higher than (8.2 ± 3.6)%, (6.7 ± 1.8)% and (3.6 ± 1.9)%, respectively, in the control group (all P < 0.01).

CONCLUSIONCisplatin promotes the anti-tumor effect of DC vaccine by down-regulating T-reg cells and enhancing the CTL activity in tumors.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; B7-2 Antigen ; metabolism ; CD8-Positive T-Lymphocytes ; pathology ; Cancer Vaccines ; pharmacology ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Dendritic Cells ; immunology ; metabolism ; Female ; Genes, MHC Class II ; HMGB1 Protein ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Melanoma, Experimental ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; T-Lymphocytes, Cytotoxic ; immunology ; T-Lymphocytes, Regulatory ; pathology ; Tumor Burden ; drug effects