To establish an HPLC-MS/MS method for the analysis of quercetin, kaempferid and isorhamnetin in rats plasma and study its pharmamacokinetics after an intragastrical administration of Hippophae rhamnoides extracts. Five healthy male Sprague-Dawley (SD) rats were given single doses of H. rhamnoides extracts (quercetin 26.35 mg x kg(-1), kaempferid 4.040 mg x kg(-1), isorhamnetin 31.37 mg x kg(-1)), and then their orbital sinus blood samples were collected at different time points. The drug plasma concentration of the three flavonoids was determined by HPLC-MS/MS method. After that, the main pharmacokinetics parameters were calculated by using Kinetica 5. 0. 11 software. The methodological test showed that the linear concentration ranges of quercetin, kaempferid and isorhamnetin were 7.500-600.0 μg x L(-1) (R2 = 0.998 5), 1.000-80.00 μg x L(-1) (R2 = 0.998 5 ) and 10.00-800.0 μg x L(-1) (R2 = 0.998 0), respectively. The inner and inter-days precisions were both less than 14.0%. The plasma samples showed a good stability and consistency with the requirement of biological sample analysis after the samples were frozen once and placed at - 20 degrees C for 15 d and room temperature for 6 h and the treated analytes were placed at -20 degrees C for 24 h. For quercetin, the pharmacokinetic parameter t(½β), AUC(0-∞), MRT(0.∞), C.(max) and T(max) were (113.3 ± 19.37) min, (12 542.14 ± 3 504.05) μg x h x L(-1), (119.6 ± 13.29) h, (164.6 ± 27.33) μg x L(-1) and (5.199 ± 0.840 3) h, respectively. For kaempferid, the pharmacokinetic parameters t(½β), AUC(0-t), MRT(0-∞), C(max) and T(max) were (79.85 ± 17.15) min, (934.51 ± 94.59) μg x h x L(-1), (81.50 ± 13.75) h, (80.15 ± 14.24) μg x L(-1) and (3.827 ± 0.902 7) h, respectively. For isorhamnetin, the pharmacokinetic parameters t1,2,, AUC(0-t), MRT(0-∞), C(max) and T(max) were (118.3 ± 20.73) min, (26 067.77 ± 4 124.60) μg x h x L(-1), (129.0 ± 16.30) h, (269.6 ± 29.32) μg x L(-1) and (6.513 ± 1.450) h, respectively. The HPLC-MS/MS analysis method established in this study was proved to be sensitive and accurate and could be applied in the pharmacokinetic study of quercetin, kaempferid and isorhamnetin in rat plasma.
Animals ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; pharmacokinetics ; Hippophae ; chemistry ; Kaempferols ; blood ; pharmacokinetics ; Male ; Quercetin ; analogs & derivatives ; blood ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; methods

Hepatitis B virus (HBV) is a major cause of chronic liver disease, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. HBV polymerase (Pol) is an essential viral protein that is important for HBV replication and might be involved in the development of hepatocellular carcinoma. Protein-protein interactions appears to be crucial for its role. The aim of this study was to screen and identify the proteins that interact with Pol using a co-immunoprecipitation-based LC-MS/MS identification technique. The HBV Pol gene was amplified by polymerase chain reaction (PCR) and cloned into pCDNA3.1(+). The recombinant plasmid pCDNA3. 1(+)-Pol-flag was transfected into HeLa cells. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 45 proteins that co-immunoprecipitated with flag-tagged HBV Pol. Eleven of these have previously been reported as proteins that interact with HBV Pol. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 45 identified proteins and HBV Pol. Among these proteins, four proteins may play a role in three major molecular cellular networks, and are therefore worthy of further investigation.
Cell Line, Tumor ; Chromatography, Liquid ; methods ; Gene Products, pol ; chemistry ; genetics ; metabolism ; Hepatitis B ; genetics ; metabolism ; virology ; Hepatitis B virus ; chemistry ; enzymology ; genetics ; Humans ; Immunoprecipitation ; methods ; Protein Interaction Maps ; Software ; Tandem Mass Spectrometry ; methods

Objective To observe the therapeutic effect ofbotulinum toxin type A (BTX-A) on spasticity of the upper extremity after stroke. Methods Fifty-four patients with upper extremity spasticity after stroke were randomly divided into treatment group and control group. The patients in treatment group were treated with BTX-A and rehabilitation training, while those in control group only with rehabilitation training. The efficacy was assessed with Modified Ashwonh Scale, Fugi-Meyer Assessment and Bartbel index before and 2, 4 and 12 weeks after treatment. Results The Ashworth scale score in treatment group was reduced significantly as compared with that in control group after 2- and 4-week treatment (P<0.01). And there was significant difference in FugI-Meyer Assessment and Barthel index between treatment group and control group after 4- and 12-week treatment (P<0.01). Conclusion The local injection of BTX-A for the muscle spasticity after stroke is effective and safe, and its combination with rehabilitation training can improve rapidly Fugl-Meyer scores and activities of daily living of patients.

Radiotracer techniques were employed to characterize 65Zn adsorption and desorption in root-cell-wall of hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) species of Sedum alfredii Hance. The results indicated that at the end ofa 30 min short time radioisotope loading period, comparable amounts of 65Zn were accumulated in the roots of the two ecotypes Sedum alfredii, whereas 2.1-fold more 65Zn remains in NHE root after 45-min desorption. At the end of 60 min uptake period, no difference of 65Zn accumulation was observed in undesorbed root-cell-wall of Sedum alfredii. However, 3.0-fold more 65Zn accumulated in desorbed root-cell-wall ofNHE. Zn2+ binding in root-cell-wall preparations of NHE was greater than that in HE under high Zn2+ concentration. All these results suggested that root-cell-wall of the two ecotypes Sedum alfredii had the same ability to adsorb Zn2+, whereas the desorption characteristics were different, and with most of 65Zn binding on root of HE being available for loading into the xylem, as a result, more 65Zn was translocated to the shoot.

Objective To investigate the ostengenie potential of adipose-derived stem cells(AD- SCs)when exposed to adenovirns containing hBMP-2 cDNA(Adv-hBMP-2)and offer a choice of cell source for gene therapy and tissue engineering.Methods Human adipose tissues were obtained from patients who received orthopaedic surgery or liposuction.ADSCs were obtained by digesting the adipose tissues.Firstly,flowcytometric analysis was performed for the confirmation of mesenchymal stem cell ori- gin and the surface markers including CD34,CD44,CD68,CD71,CD90,and CD105.The ADSCs were transfected by Adv-hBMP-2 and the effects were tested in vitro,lmmunoprecipitation and Western blotting and ELISA were performed for confirming BMP gone transduction and its stable expression.The transform of ADSCs was assessed by extracellular ALP staining,intracellular ALP spectrophotometry,von Kossa staining and RT-PCR.In the in vivo experiment ADSC-Adv-hBMP-2 cells were injected into the hind limb of nude mice and analyzed radiographically and histologically.Results ADSCs were successfully isolated from human adipose tissues.The isolated ADSCs expressed CD44,CD71,CD90 and CD105 and CD34 and CD68 were absent.The result confirmed the mesenchymal stem cell origin of the cells.West- ern blotting and ELISA confirmed successful and persistent hBMP-2 production by ADSC-Adv-hBMP-2 cells.Extracellular ALP staining,intracellular ALP spectrophotometry,yon Kossa staining and RT-PCR revealed that ADSCs treated with Adv-hBMP-2 had a tendency of transfering into osteoblast.X-ray and H&E sections from hind limb of nude mice injected with ADSC-Adv-hBMP-2 cells confirmed bone forma- tion at 2 weeks.Conclusions Liposuction aspirates contain abundant ADSCs that can be transduced with hBMP-2 gene,and the tranduced ADSCs differentiate into the osteoblast.ADSCs may be an ideal source of mesenchyme-lineage stem cells for gone therapy and tissue engineering.

To explore the effect of arsenic trioxide (As2O3) on growth and secretion of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) of bone marrow stroma cells (BMSC) from the patients with multiple myeloma (MM). Specimens of bone marrow aspiration from MM patients were used to establish BMSC cultures. BMSC and human MM cell line CZ-1 were cultured together or alone in the absence or presence of As2O3 at various concentrations (1-20.0 micromol/L). Cell growth inhibition was assessed by MTT assay, cytokines in the culture supernatants were measured with ELISA. The results showed that As2O3 had cytostatic effect on CZ-1 with fifty percent growth inhibition (IC50) for 48 hours at 2.3 micromol/L. As2O3 did not inhibit the growth of BMSC. High levels of IL-6 and VEGF have been found in the culture supernatants of BMSC from MM patients. Cytokine production of BMSC treated with As2O3 significantly decreased as compared with controls (P < 0.05). Excitingly, even the increased cytokine production triggered by adhesion of MM cell and BMSC was also inhibited by As2O3. It is concluded that As2O3 has no inhibitory effect on cell growth of BMSC, but inhibit the production of IL-6 and VEGF by BMSC.
Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Bone Marrow Cells ; pathology ; Depression, Chemical ; Humans ; Interleukin-6 ; biosynthesis ; Multiple Myeloma ; pathology ; Oxides ; pharmacology ; Stromal Cells ; pathology ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; biosynthesis

This study was purposed to investigate the changes in quantum and function of gammadelta T cell subsets, and to explore its significance in pathogenesis of acquired pure red cell aplastic anemia (A-PRCA). Eleven patients were diagnosed as A-PRCA based on bone marrow smear and biopsy, and were treated with cyclosporine A and glucosidorum tripterygll totorum. The flow cytometry technique was used for analyses of T cells subsets and gammadelta T cells. Furthermore, peripheral mononuclear cells (MNC) isolated from A-PRCA patients were cultured in RPMI 1640 medium (10(5) cells/ml) containing 10% FCS, phytohemagglutinin (PHA, 10 microg/ml), and recombinant human interleukin-2 (rIL-2, 50 U/ml) for two weeks, then gammadelta T cells were isolated with the TCRgammadelta Microbead Kit from cultured cells. The collected gammadelta T cells were incubated with normal control bone marrow MNC in RPMI 1640 medium (37 degrees C, 5% CO2 atmosphere) for CFU-E, CFU-GM, and BFU-E colony assay. The result showed that compared with the control group, CD3(+), CD8(+) cells increased significantly in the patient group (P < 0.05), the CD4(+)/CD8(+) ratio decreased and reversed, and gammadelta T cells were significantly increased in patient group (P < 0.05). After treatment with cyclosporine A, 9 out of 11 patients got good response, and CD3(+), CD8(+) cells in the responding patient decreased, the ratio of CD4(+)/CD8(+) returned to normal, and gammadelta T cells also decreased to normal range. Moreover, in vitro culture, the gammadelta T cells isolated from A-PRCA patients showed an inhibiting action to CFU-E and BFU-E but not to CFU-GM in a dose-dependent manner. It is concluded that gammadelta T cells increase in A-PRCA patients, and decrease in parallel to normal range with significant improvement of anemia symptoms after immune suppressive therapy. The gammadelta T cells isolated from A-PRCA patients showed an inhibiting action to CFU-E and BFU-E but not to CFU-GM in vitro culture, suggesting that gammadelta T cells may bring an impact on the research of A-PRCA pathogenesis. Cyclosporine A demonstrated better therapeutic effect on A-PRCA patients.
Adult ; Aged ; CD4-CD8 Ratio ; Cells, Cultured ; Cyclosporine ; therapeutic use ; Female ; Flow Cytometry ; Humans ; Immunosuppressive Agents ; therapeutic use ; Male ; Middle Aged ; Receptors, Antigen, T-Cell, gamma-delta ; physiology ; Red-Cell Aplasia, Pure ; drug therapy ; etiology ; immunology ; T-Lymphocyte Subsets ; cytology ; T-Lymphocytes ; cytology ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo explore the correlation between hOCT1 polymorphism and imatinib mesylate (IM) effectiveness in chronic myelogenous leukemia(CML) patients, and to provide for the clinical individual personalized therapy.

METHODSFifty-three CML and 23 non-CML patients were enrolled in this study. Blood or bone marrow samples were collected. Amplification refractory mutation system (ARMS)-polymerase chain reaction was used to amplify the polymorphisms gene segment of hOCT1-P283L, R287G and M408V and their frequencies were statistically analysed. With clinical outcomes, the correlation between hOCT1 polymorphism and IM effectiveness in CML was analyzed.

RESULTS(1) For 74 Han Chinese, the allele frequencies of hOCT1-P283L, R287G and M408V were 39.86%, 29.05% and 45.27%, respectively. (2) The genotypes of hOCT1-P283L, R287G and M408V in 2 Tibetan Chinese were CC, CC, AG and CC, CG, AG, respectively. (3) In the CML patients with IM optimal response, the frequencies of 283T and 287G allele were predominant (P<0.05). No significant difference was found in the frequency distribution of hOCT1-M408V genotype and allele among the 3 different response groups (P>0.05).

CONCLUSION(1) Three single nucleotide polymorphisms (cSNP) P283L, R287G and M408V were found in the hOCT1 gene from 76 Chinese. (2) hOCT1 gene polymorphism is associated with the long-term molecular response of CML patients received IM therapy, indicating that the polymorphisms of hOCT1-283T, 287G may be good predictors for IM response. (3) There is no correlation between the polymorphisms of hOCT1-P283L, R287G, M408V and secondary IM resistance in CML patients.

Benzamides ; Female ; Gene Frequency ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; genetics ; Male ; Organic Cation Transporter 1 ; genetics ; Piperazines ; therapeutic use ; Polymorphism, Genetic ; Pyrimidines ; therapeutic use ; Treatment Outcome

Abstract?AIM: To investigate mechanism of bradykinin ( BK) on inflammations of retinal pigment epithelium ( RPE) cells.?METHODS: ARPE -19 cells were cultured in vitro, stimulated by 100nM BK for 24h. Cell morphology changes were observed by microscope, and BK receptor localization was detected through cell immunofluorescence. Changes of Ca2+in BK and BR antagonist stimuli were detected by laser scanning confocal microscopy.The expressions of COX-1, COX-2, eNOS and iNOS protein in control group and BK group were detected by Western Blot.?RESULTS: After the stimulation of BK, there was no significant changes of ARPE-19 cells in morphology.Kinin B1 receptors ( B1R ) and B2 receptors ( B2R ) could be detected in ARPE-19 cells.Compared with control group, Ca2+concentrations significantly increased in BK group; in B1R antagonist group and B2R antagonist group Ca2+concentrations increased less than BK group; B1R and B2R antagonist group showed no obvious changes in Ca2+concentrations.Compared with control group, COX-2 and iNOS protein concentrations were significantly increased in BK group (P<0.001).?CONCLUSION:BK induces the increasing expression of COX-2 and iNOS in the cultured ARPE cells through binding with either B1R or B2R.

To investigate the immune regulatory effects of human bone marrow mesenchymal stem cells on alloantigen T lymphocyte in vitro, human MSCs were isolated and expanded from bone marrow cells, and identified with cell morphology, and the phenotypes were assessed by immunohistochemistry and flow cytometry. As the stimulation factor of T lymphocytes proliferation, either PHA or dendritic cells isolated from cord blood were cocultured with CD2(+) T lymphocytes from peripheral blood mononuclear cells by magnetic beads with or without MSC in 96-well plats for seven days. T cell proliferation was assessed by [(3)H]-thymidine incorporation using a liquid scintillation counter. T cell subsets, Th1, Th2, Tc1 and Tc2 were analyzed by flow cytometry after co-culture of CD2(+) T cells with MSCs for 10 days. The results showed that a significant decrease of CD2(+) T cell proliferation was evident when MSC were added back to T cells stimulated by DC or PHA, and an increase of Th2 and Tc2 subsets were observed after co-culture of MSC with T lymphocytes. It is suggested that allogeneic MSC can suppress T cell proliferation in vitro and the cause of that was partly depend on interaction of cells and the alteration of T cell subsets.
Bone Marrow Cells ; cytology ; immunology ; CD2 Antigens ; immunology ; Cell Communication ; immunology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Flow Cytometry ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology ; immunology ; T-Lymphocyte Subsets ; cytology ; immunology ; T-Lymphocytes ; cytology ; immunology