Cellular pharmacology of 2',3'-didehydro-2',3'-dideoxythymidine (D4T) in human peripheral blood mononuclear cells.
Cellular pharmacology of 2',3'-didehydro-2',3'-dideoxythymidine (D4T) in human peripheral blood mononuclear cells.
复制标题
人外周血单核细胞中 2,3-二脱氢-2,3-二脱氧胸苷 (D4T) 的细胞药理学。
DOI：
10.1016/0006-2952(90)90418-k
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发表时间：
1990
影响因子：
5.8
通讯作者：
Sommadossi,JP
中科院分区：
文献类型：
--
作者：
Zhu,Z;Ho,HT;Hitchcock,MJ;Sommadossi,JP
材料和方法化学品。[sH]D4 T（20 Ci/mmol）购自Moravek Biochemicals（Brea，CA）。所有其他化学品和试剂均为现有的最高分析等级。Ql&.从健康的HIV和B肝炎病毒血清阴性志愿者的全血中获得的人外周血单核细胞（PBMC）通过在LeucoPREP细胞分离管中在23'下沉淀肝素化的新鲜全血或通过单步Ficoll-Hypaque不连续梯度离心来分离。收集单核细胞层并用冷磷酸盐缓冲溶液（PBS）洗涤。然后将细胞沉淀以1 × 106个细胞/ml的浓度重悬于补充有10%胎牛血清、1%丙酮酸钠、1%非必需氨基酸和1%青霉素-链霉素-两性霉素B的RPM 11640培养基中。对于用植物血凝素（PHA）刺激的PBMC进行的实验，将PHA以5 μ g/ml的终浓度添加到培养物中。t&.所有培养物在37 ℃、5%CO2下孵育规定的时间。为了检测受刺激PBMC中D4 T的活化及其在不同个体中的变化程度，通过与PHA预孵育48-72小时制备培养物。从培养物中除去PHA后，然后加入药物和新鲜培养基并孵育长达48小时。在静息PBMC中，将药物加入到新鲜制备的培养物中并温育24小时。用2、10或25 pM [sH] D4 T孵育后，通过离心沉淀细胞，并用冷PBS洗涤，然后在-20 ℃下用1 mL冷60%甲醇提取沉淀两次。移除之后
MATERIALS AND METHODSChemicals.[sH] D4T (20 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA). All other chemicals and reagents were of the highest analytical grade available. Ql&. Human peripheral blood mononuclear cells (PBMC) obtained from whole blood of healthy HIV and hepatitis B virus-seronegative volunteers were separated either by sedimentation of heparinized fresh whole blood in a LeucoPREP cell separation tube at 23’or by single-step Ficoll-Hypaque discontinuous gradient centrifugation. The mononuclear cell layer was collected and washed with cold phosphate-buffered solution (PBS). The cell pellet was then resuspended in RPM1 1640 medium supplemented with 10% fetal calf serum, 1% sodium pyruvate, 1% non-essential amino acids and 1% Penicillin-Streptomycin-Fungizone at a concentration of 1 x 106 cells/ml. For experiments performed with phytohemagglutinin(PHA)-stimulated PBMC, PHA was added to the cultures at a final concentration of 5 pg./t&. All cultures were incubated at 37’under 5% CO2 for specified time periods.To examine the activation of D4T in stimulated PBMC and its degree of variation among different individuals, cultures were prepared by preincubation with PHA for 48-72 hr. Following removal of PHA from culture, drug was then added with fresh medium and incubated for time periods up to 48 hr. In resting PBMC, drug was added to the freshly prepared culture and incubated for 24 hr.@ After incubation with 2, 10 or 25 pM [sH] D4T, cells were pelleted by centrifugation and washed with cold PBS, and then pellets were extracted twice at-20’with 1 mL of cold 60% methanol. Following removal of