Regulation of methotrexate polyglutamate accumulation in vitro: effects of cellular folate content.
Regulation of methotrexate polyglutamate accumulation in vitro: effects of cellular folate content.
复制标题
体外甲氨蝶呤聚谷氨酸积累的调节：细胞叶酸含量的影响。
DOI：
10.1016/0006-2952(83)90211-3
复制
发表时间：
1983
影响因子：
5.8
通讯作者：
Balinska,M
中科院分区：
文献类型：
--
作者：
Galivan,J;Nimec,Z;Balinska,M
方法与结果胰岛素来源于美国礼来公司。来自Sigma化学公司的溶卵磷脂（I型）和ICN制药公司的亚叶酸[3 ', 5 '。7-甲氨蝶呤和[3]。5 '。7- ' Hlfolic acid来自Amersham， [G- ' HJfolinic acid来自Moravek Chemicals。已公布的方法被用于纯化放射性标记叶酸[171]和亚叶酸[181]。H35肝癌细胞按先前描述的方法进行培养[2,9]，并在生长96小时后进行融合。然后在规定的条件下培养细胞。用纯化的[3 '。5 '。7-甲氨蝶呤（S-SO × lO ' dpm/nmol） 12.91。提取细胞内容物，并按上文[2.9]分析甲氨蝶呤池的细胞内浓度。结果表示为基于1 mg细胞蛋白等于3.15的摩尔浓度。所有的细胞都是水。采用Fry等人[5]描述的高压液相色谱法和DEAE纤维素色谱[9]描述的高压液相色谱法确定两个60mm培养皿的甲氨蝶呤和甲氨蝶呤多谷氨酸盐的组成。通过对溶卵磷脂技术[19]的改进，制备了通透性H35细胞。通过在溶血卵磷脂溶液中加入1 mM的CaClZ并将孵育时间缩短至60秒，改变了这一过程，这使得溶血卵磷脂处理后24小时的细胞产量比先前报道的增加了33% [IY]。溶卵磷脂治疗后。在无叶酸的Swims -77培养基中加入10 mU胰岛素/ml密封24小时，然后加入[3 ',5 ']。7——“Hlmethotrexate。像以前一样。用台盼蓝[19]分析，封闭后的细胞存活率为100%。
Methods and resultsInsulin was obtained from Eli Lilly 8: Co.. lysolecithin (Type I) from the Sigma Chemical Co.. and folinic acid from ICN Phamaceuticals.[3’, 5’. 7-‘HlMethotrexatc and [3’. 5’. 7-‘Hlfolic acid were from Amersham and [G-“HJfolinic acid was from Moravek Chemicals. Published procedures were utilized for the purification of radiolabeled folic acid [171 and folinic acid [181. H35 hepatoma cells were cultured as described previously] 2, 9] and utilized at confluency, which occurs after 96 hr of growth. The cells were then incubated under the conditions specified. with purified [3’. 5’. 7-‘Hlmethotrexate (S-SO x lO’dpm/nmole) 12.91. The cellular contents were extracted, and the intracellular concentration of the methotrexate pool was analyzed as described previously [2.9]. The results are expressed as molar concentration based upon I mg cell protein being equivalent to 3.15! ll of cell water [2]. The composition of the methotrexate and methotrexate polyglutamates with the contents of two 60 mm culture dishes was established by the high-pressure liquid chromatography described by Fry er al.[5] and DEAE cellulose chromatography[9].Permeabilized H35 cells were prepared by a modification of the lysolecithin technique[19]. This procedure was altered by including 1 mM CaClZ in the Iysolecithin solution and reducing the incubation time to 60 sec. which resulted in a 33% increase in cell yields over that previously reported 24 hr after lysolecithin treatment[IY]. Following lysolecithin treatment. the cultures were allowed to seal in folate-free Swims S-77 medium with 10 mU insulin/ml for 24 hr before adding [3’, 5’. 7-“Hlmethotrexate. As before. the viability of the sealed cells was 100% when analyzed with Trypan blue [19].