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31.
HT Syyong HHC Yang G Trinh C Cheung KH Kuo C van Breemen 《British journal of pharmacology》2009,156(4):587-600
Background and purpose:
Uridine 5''-triphosphate (UTP) is a potent vasoconstrictor of cerebral arteries and induces Ca2+ waves in vascular smooth muscle cells (VSMCs). This study aimed to determine the mechanisms underlying UTP-induced Ca2+ waves in VSMCs of the rat basilar artery.Experimental approach:
Isometric force and intracellular Ca2+ ([Ca2+]i) were measured in endothelium-denuded rat basilar artery using wire myography and confocal microscopy respectively.Key results:
Uridine 5''-triphosphate (0.1–1000 µmol·L−1) concentration-dependently induced tonic contraction (pEC50 = 4.34 ± 0.13), associated with sustained repetitive oscillations in [Ca2+]i propagating along the length of the VSMCs as asynchronized Ca2+ waves. Inhibition of Ca2+ reuptake in sarcoplasmic reticulum (SR) by cyclopiazonic acid abolished the Ca2+ waves and resulted in a dramatic drop in tonic contraction. Nifedipine reduced the frequency of Ca2+ waves by 40% and tonic contraction by 52%, and the nifedipine-insensitive component was abolished by SKF-96365, an inhibitor of receptor- and store-operated channels, and KB-R7943, an inhibitor of reverse-mode Na+/Ca2+ exchange. Ongoing Ca2+ waves and tonic contraction were also abolished after blockade of inositol-1,4,5-triphosphate-sensitive receptors by 2-aminoethoxydiphenylborate, but not by high concentrations of ryanodine or tetracaine. However, depletion of ryanodine-sensitive SR Ca2+ stores prior to UTP stimulation prevented Ca2+ waves.Conclusions and implications:
Uridine 5''-triphosphate-induced Ca2+ waves may underlie tonic contraction and appear to be produced by repetitive cycles of regenerative Ca2+ release from the SR through inositol-1,4,5-triphosphate-sensitive receptors. Maintenance of Ca2+ waves requires SR Ca2+ reuptake from Ca2+ entry across the plasma membrane via L-type Ca2+ channels, receptor- and store-operated channels, and reverse-mode Na+/Ca2+ exchange. 相似文献32.
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34.
脂肪干细胞在体外特定培养液中向软骨细胞表型的分化 总被引:3,自引:1,他引:3
目的:应用转化生长因子β1,体外诱导脂肪干细胞向成软骨细胞表型分化,探讨其作为组织工程化软骨种子细胞的可行性。方法:实验于2005-04/2006-06在华中科技大学同济医学院公卫实验室完成。①取大鼠腹股沟处脂肪,酶消化法分离、培养脂肪干细胞,体外传代培养。②取第3代细胞通过转化生长因子β1、地塞米松和维生素C诱导脂肪干细胞向软骨细胞分化。③诱导后14d观察细胞形态变化,进行阿辛蓝染色检测软骨基质的分泌、免疫组织化学检测细胞Ⅱ型胶原的表达,采用Western-blot和反转录-聚合酶链反应检测诱导前后成软骨相关的Sox9,蛋白聚糖与Ⅱ型胶原的表达。结果:①细胞接种的最初几日,细胞呈圆形,1周后贴壁细胞呈长梭形,体积增大;14d后贴壁生长细胞基本长满单层,中心细胞排列紧密,形态与骨髓间充质干细胞相似。诱导培养后,细胞形态逐渐由梭型向多角形、多边形转变。诱导14d后多数细胞呈平坦的多边角形状细胞;其夹杂多角突起状或多角纺锤状细胞。②诱导后阿辛蓝染色示糖胺聚糖均匀分布于基质中。③免疫组织化学染色示基质中Ⅱ型胶原表达阳性。④反转录-聚合酶链反应检测成软骨相关的Sox9、蛋白聚糖、Ⅱ型胶原mRNA表达阳性。⑤Western-blot印迹检测细胞诱导后Ⅱ型胶原蛋白表达阳性。结论:脂肪干细胞在特定培养液的诱导下可向成软骨细胞表型分化,并能分泌软骨细胞特异性基质,有望成为软骨组织工程新的细胞来源。 相似文献
35.
Normal human platelets were shown to contain the enzyme dihydropteridine reductase. The enzyme was not found in a variety of other cells of hematogenous origin. Partial purification and kinetic and physical data indicated that the platelet enzyme is similar to that previously characterized from liver. Dihydropteridine reductase is important for the regeneration of tetrahydrobiopterin, a required cofactor in hydroxylation reactions involved in biogenic amine formation. The presence of the enzyme may indicate that some synthesis de novo of serotonin and/or catecholamines occurs in platelets, as opposed to a purely storage and transport function. In addition, screening for hyperphenylalaninemia due to dihydropteridine reductase deficiency may become feasible by assaying platelets for enzyme activity. 相似文献
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37.
Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y- containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers. 相似文献
38.
Lester J. Layfield M. D. Debbi H. Conlon HT Richard Dodge M. S. Elizabeth Saria M. D. Billie-Jo Kerns B. S. 《The breast journal》1996,2(2):141-149
Abstract: Traditionally, estrogen and progesterone receptor levels have been determined by biochemical ligandbinding assays, but more recently immunohistochemical techniques have become available. They have gained popularity due to their low cost, smaller sample size requirements, and direct visualization capability of reaction location. Several antibody clones are commercially available and antibodies directed against the estrogen receptor (ER) are supplied by Ventana Medical Systems (Tucson, AZ), Abbott Laboratories (Abbott Park, IL), and lmmunotech Westbrook, ME). Antibodies directed against the progesterone receptor (PgR) are supplied by Ventana Medical Systems (Tucson, AZ), lmmunotech (Westbrook, ME), and Becton-DickinsonKelI Analysis Systems (San Jose, CA). Computer-assisted image analysis using the CAS ZOOTM (Becton-DickinsonKIS, San Jose, CA) allows quantitation of immunohistochemically determined receptor levels. Correlation of quantitated immunohistochemical ER levels with values determined by ligand-binding assay revealed the Ventana antibody to most closely predict the ligand-binding results (wk = .667). The Ventana anti-progesterone antibody quantitation most closely correlated with the ligand-binding results (wk = 435) for determination of PgR. Progesterone receptor level as determined by any of the tested methods did not stratify patients into favorable and unfavorable prognostic groups. Estrogen receptor level as determined by the Ventana antibody was the most predictive of patient outcome but this relationship did not reach statistical significance (p = .09). Most discrepancies between the ligand-binding assay and the immunohistochemical assays were associated with one of three factors: (a) low volume of neoplastic cells present due either to small sample size or high stromal content, (b) premenopausal status with circulating endogenous estrogens potentially occupying receptor sites, (c) presence of benign breast epithelium resulting in a false-positive ligand-binding assay. 相似文献
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吸收度线性组合分光光度法的初步探讨 总被引:11,自引:0,他引:11
本文在联立方程法(包括双波长法、系数倍率法)、三波长法、正交函数法的基础上提出一种新的计算分光光度测定法——吸收度线性组合法。文中对上述诸法进行了比较,并从理论和实验上证明了新法只要合理组合,随着测试点数的增加,测定精度可望改善。 相似文献