Introduction of Artifitial Translocator into Plasma membrane in Cultured Human Cell-Line

将人工易位器引入培养的人类细胞系的质膜中

基本信息

批准号：
61870016
负责人：
KAGAWA Yasuo
金额：
$ 13.44万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1988
项目状态：
已结题

项目摘要

A cell is surrounded by biomembranes, which permeate specific substrates via specific translocators. Therefore, even if the cultured cell, it is hard to control the cellular conditions by changing the extra culture medium. We have attempted to control the cellular energy state by two methods. First, we have tried to isolate the mutant cell lines which are deficient in oxidative phosphorylation. Second, we have tried to incorporate an artificial tanslocator by introducing the manipulated gene; in this case, we use a fusion protein of ADP/ATP translocator with a retro virus envelop protein, which target plasma membrane. The following results were obtained by several trials.1) To detect the ATP concentration easily, an ATP sensor was made by using thermo-stable proton translocating ATPase purified from a thermophilic bacterium.2) The cytochrome C oxidase deficient cell line were established from a patient with mitochondrial myopathy. The cell survive could be controlled by substrate for oxidative phospholylation.3) The pyruvate dehydrogenase deficient cell line was established from a patient with its deficiency. The defective gene were analyzed in detail.4) The mitochondrial deficient cell were made by treating the human promyelocytic leukemia cell HL-60 with retinol. In addition, the gene expression of mitochondrial proteins were also examined by Northern blotting method.5) The human cDNA encoding ADP/ATP translocator was cloned and sequenced.6) The ADP/ATP translocator cDNA was fused to a retro virus envelop gene under SV40 promoter. The constructed gene was transfected into mouse fibroblast cells. The resulting transformant was unstable and could not be maintained. Therefore, neomycin resistant gene was connected directly and then transfected to HeLa cells. The experiment is just on the way.

细胞被生物膜包围，生物膜通过特定的转运蛋白渗透特定的底物。因此，即使是培养的细胞，也很难通过改变额外的培养基来控制细胞条件。我们尝试通过两种方法来控制细胞能量状态。首先，我们尝试分离氧化磷酸化缺陷的突变细胞系。其次，我们尝试通过引入操纵基因来整合人工移位器。在这种情况下，我们使用 ADP/ATP 易位蛋白与逆转录病毒包膜蛋白的融合蛋白，该蛋白靶向质膜。经过多次试验，得到以下结果：1）为了方便地检测ATP浓度，利用从嗜热细菌中纯化的热稳定质子转位ATP酶制作了ATP传感器。2）从细胞色素C氧化酶缺陷细胞系中建立了细胞色素C氧化酶缺陷细胞系。线粒体肌病患者。氧化磷酸化底物可以控制细胞的存活。3)丙酮酸脱氢酶缺陷细胞系是从丙酮酸脱氢酶缺陷患者中建立的。对缺陷基因进行了详细分析。4)用视黄醇处理人早幼粒细胞白血病细胞HL-60制备线粒体缺陷细胞。此外，还通过Northern印迹法检测了线粒体蛋白的基因表达。5）克隆并测序了编码ADP/ATP易位子的人cDNA。6）将ADP/ATP易位子cDNA与SV40下的逆转录病毒包膜基因融合发起人。将构建的基因转染至小鼠成纤维细胞中。所得转化体不稳定且无法维持。因此，直接连接新霉素抗性基因，然后转染HeLa细胞。实验刚刚开始。