Thymidine phosphorylase in human epidermal keratinocytes.
Thymidine phosphorylase in human epidermal keratinocytes.
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人表皮角质形成细胞中的胸苷磷酸化酶。
DOI：
10.1016/0006-2952(88)90740-x
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发表时间：
1988
影响因子：
5.8
通讯作者：
Milstone,LM
中科院分区：
文献类型：
--
作者：
Schwartz,PM;Milstone,LM
材料和方法将从新生儿包皮分离的人角质形成细胞在培养皿中生长，所述培养皿用如前所述的经辐照的3 T3细胞的饲养层制备[3]。在原代培养物变得汇合之前，将细胞传代到35 mm培养皿中的经辐照的3 T3细胞上;将5-7个细胞层的汇合的次级培养物用于实验。显微镜下，没有成纤维细胞的证据。对代表性角质形成细胞培养物进行支原体检测，结果为阴性。使细胞在Dulbecco改良Eagle培养基（Gibco Laboratories.格兰德岛用20%胎牛血清、氢化可的松、表皮生长因子、霍乱毒素、青霉素、链霉素等作对照。由20%胎牛血清提供的完全培养基中胸苷的浓度约为0.2 PM。如前所述制备培养细胞和包皮的可溶性提取物[3]。简要将培养的细胞洗涤除去培养基，刮入含有5 mM二硫苏糖醇的磷酸盐缓冲盐水中，并通过超声破碎。切开包皮以尽可能多地去除皮下物质和真皮。用Polytron匀浆器（Brinkmann Instrument Co.，韦斯特伯里，纽约）。将来自细胞或组织的粗提取物离心（20，000 g 4”，30分钟）以除去颗粒物质并储存在-70”下。Bradford测定法用于测定蛋白质浓度[8]。在含有0.5至10 μ g来自磷酸盐缓冲盐水中的粗提物的蛋白质的反应混合物中测定胸苷催化活性（9.6 mM磷酸盐，oH 7.2），在37 '处含有5 mM二硫苏糖醇：通过加入用胸苷稀释的[3 H]胸苷（20 Ci/mmol，新英格兰核，Boston，MA）开始反应，得到终浓度范围为0.2 - 100 μ M，4 μ Ci/mmol。
Materials and methodsHuman keratinocytes, isolated from neonatal foreskin, were grown in culture dishes prepared with a feeder layer of irradiated 3T3 cells as previously described [3]. Before the primary culture became confluent, cells were passaged onto irradiated 3T3 cells in 35mm dishes; confluent secondary cultures of 5-7 cell layers were used for experiments. Microscopically, there was no evidence of fibroblasts. Representative keratinocyte cultures were tested for the presence of mycoplasma, and the results were negative. Cells were grown in Dulbecco’s Modified Eagle Medium (Gibco Laboratories. Grand Island. NY) suoblemented with 20% fetal bovine’serum, hydrdcortisonk: epidermal growth factor, cholera toxin, penicillin and streptomycin. The concentration of thymidine in complete medium contributed by 20% fetal bovine serum was approximately 0.2 PM. Soluble extracts of cultured cells and foreskin were prepared as previously described [3]. Briefly. cultured cells were washed free of medium, were scraped into phosphatebuffered saline containing 5 mM dithiothreitol, and were disrupted by sonication. Foreskins were dissected to remove as much subcutaneous material and dermis as possible. The minced tissue was homogenized in phosphatebuffered saline, 5 mM dithiothreitol with a Polytron homogenizer (Brinkmann Instrument Co., Westbury, NY). The crude extract from cells or tissue was centrifuged (20,000 g 4”, 30 min) to remove particulate material and stored at-70”. The Bradford assay was used to measure the protein concentration [8].Thymidine catabolism was measured in reaction mixtures containing 0.5 to 1Opg of protein from crude extracts in phosphate-buffered saline (9.6 mM phosphate, oH 7.2) containing 5 mM dithiothreitol at 37’: the reactibn was started by addition of [jH] thymidine (20 Ci/mmol, New England Nuclear, Boston, MA) diluted with thymidine to give final concentrations ranging from 0.2 to 100 PM, 4 pCi/