Transformation and characterization of mutant human fibroblasts defective in peroxisome assembly.
Transformation and characterization of mutant human fibroblasts defective in peroxisome assembly.
复制标题
过氧化物酶体组装缺陷的突变人类成纤维细胞的转化和表征。
DOI：
10.1016/0014-4827(92)90278-g
复制
发表时间：
1992
影响因子：
3.7
通讯作者：
T. Orii
中科院分区：
文献类型：
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作者：
H. Okamoto;Y. Suzuki;N. Shimozawa;S. Yajima;M. Masuno;T. Orii
利用电穿孔机将SV40ori−DNA导入人皮肤成纤维细胞，并对其生化、免疫细胞化学和细胞遗传学特性进行了分析。取1例Zellweger综合征患者和1例新生儿肾上腺脑白质营养不良患者的细胞(1×10~6)与2μg的SV40Ori−DNA悬浮于PBS中，然后产生2次高压脉冲(2000V，30μS)。转染4周后，可获得多个表达大T抗原的克隆。转化子的倍增时间约为亲本细胞的一半，饱和密度是亲本细胞的5~10倍。在转化子中保留了甘油酸氧化缺陷、磷酸二羟丙酮酰基转移酶缺陷和过氧化体β氧化酶生物合成障碍等生化异常。用抗过氧化氢酶免疫荧光染色检测，所有菌落中的过氧化物体都是缺陷的。细胞融合研究证实，转化子与亲本细胞属于相同的互补组。这些转化的突变细胞系有望成为研究与过氧化酶体生物发生缺陷相关的遗传病发病机制的有用工具。
Human skin fibroblasts deficient in peroxisome biogenesis were transformed by transfecting SV40 ori−DNA with the use of an electroporator, and the biochemical, immunocytochemical, and cytogenetic properties of the transformants were analyzed. Cells (1 × 106) from a patient with Zellweger syndrome and one with neonatal adrenoleukodystrophy were suspended with 2 μg of SV40 ori−DNA in PBS; then a high-voltage pulse (2000 V, 30 μs) was generated two times. Several colonies expressing large T-antigen were picked up 4 weeks after transfection. Doubling time of the transformants was about half of that and the saturation density was 5 to 10 times greater than that of the parental cells. Biochemical abnormalities including defective lignoceric acid oxidation, dihydroxyacetone phosphate acyltransferase deficiency, and disturbed biosynthesis of peroxisomal β-oxidation enzymes were preserved in the transformants. Peroxisomes were defective in all colonies, as determined by immunofluorescence staining using anti-catalase IgG. Cell fusion studies confirmed that the transformants belong to the same complementation groups as those of the parental cells. These transformed mutant cell lines are expected to be useful tools for investigating the pathogenesis of inherited diseases related to defects in peroxisome biogenesis.
Yasuyuki Suzuki、Nobuyuki Shimozawa、Seiji Yamaguchi、Shigehiro Yajima、Tadao Orii、Takashi Hashimoto：“2-[5-(4-氯苯基)戊基]环氧乙烷甲酸钠 (POCA) 对过氧化物酶体疾病患者成纤维细胞脂肪酸氧化的影响
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