In vitro molecular reconstitution of the respiratory burst in B lymphoblasts from p47-phox-deficient chronic granulomatous disease.
In vitro molecular reconstitution of the respiratory burst in B lymphoblasts from p47-phox-deficient chronic granulomatous disease.
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p47-phox 缺乏的慢性肉芽肿病 B 淋巴母细胞呼吸爆发的体外分子重建。
DOI：
10.1172/jci116171
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Lin,Y
中科院分区：
文献类型：
--
作者：
Volpp,BD;Lin,Y
爱泼斯坦-巴尔病毒转化的淋巴细胞在与刺激的吞噬细胞中发生的酶反应类似的酶反应中产生超氧化物。我们从对照组、四名P47Phox缺陷的慢性肉芽肿疾病患者和三名父母那里产生了转化的B淋巴母细胞系。对照组和亲本的细胞每30分钟产生7.0-35nmoL的O2-/10(7)细胞，对佛波醇肉豆蔻酸酯作出反应。患者细胞系中没有一种产生任何可检测到的超氧化物。免疫印迹法检测到对照细胞和亲本细胞的胞浆中均有p47-Phox和p67-Phox的表达，与中性粒细胞一样，这些蛋白与GTP-琼脂糖有亲和力。通过免疫印迹检测，患者的细胞系中没有检测到P47-Phox。在所有细胞系中均检测到胞浆蛋白的mRNA。我们通过聚合酶链式反应从两名患者身上获得了多个克隆。1例患者为复合杂合子，每个等位基因导致早期终止密码子。来自另一名患者的克隆只显示了碱基75处的GT缺失。将p47-Phox基因插入EBV表达载体，潮霉素B筛选获得稳定表达的细胞系。来自p47Phox缺陷患者的转基因细胞系产生正常水平的超氧化物，并很容易检测到胞浆中的p47Phox。因此，B淋巴母细胞为研究NADPH氧化酶，表达功能重组形式的氧化酶成分，以及在CGD图像中进行遗传重建的初步实验方法提供了一个很好的模型系统
Epstein-Barr virus-transformed lymphocytes generate superoxide in response to various agonists in an enzymatic reaction similar to that which occurs in stimulated phagocytes. We generated transformed B lymphoblast cell lines from controls, from four patients with p47-phox-deficient chronic granulomatous disease, and from three parents. The cells from controls and from the parents generated 7.0-35 nmol of O2-/10(7) cells per 30 min in response to phorbol myristate acetate. None of the patient cell lines generated any detectable superoxide. Both p47-phox and p67-phox were detected by immunoblot in the cytosol of control and parent cell lines and, as in neutrophils, these proteins had affinity for GTP-agarose. The patients' cell lines contained no detectable p47-phox by immunoblot. mRNA for both cytosolic proteins was detected in all cell lines. We generated cDNA and obtained multiple clones from two patients by polymerase chain reaction. One patient was a compound heterozygote with each allele resulting in an early stop codon. Clones derived from the other patient demonstrated only a GT deletion at base 75. The cDNA for p47-phox was inserted into an EBV-expression vector and stably transfected cell lines were obtained using hygromycin B selection. Transfected cell lines from a p47-phox-deficient patient generated normal levels of superoxide and had readily detectable cytosolic p47-phox. Thus, B lymphoblasts provide an excellent model system for studies of the NADPH oxidase, for expression of functional recombinant forms of oxidase components, and for initial experimental approaches to genetic reconstitution in CGD.Images