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THE CLINICAL FEATURES AND PATHOGENESIS IN MITOCHONDRIAL DNA MUTATION : BASIC STUDY FOR GENE THERAPY

线粒体DNA突变的临床特征和发病机制:基因治疗的基础研究

基本信息

批准号：
09670487
负责人：
SATOH Shinobu
金额：
$ 1.98万
依托单位：
Yokohama City University Hospital
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Mitochondrial DNA (mtDNA) mutations are associated with various disease. Recent studies have shown that there is a close association between the nt 3243 of t RNA^<Leu(UUR)> gene (A3243G mutation) and maternally inherited diabetes mellitus (DM). Multiorgan involvement has been suggested. As a test of exocrine pancreatic function, we performed a bentiromide test using benzoyl-tyrosyl-p-aminobenzoic acid (PABA). % recovery of PABA decreased at a range 19-68% (cf normal 81.9 8.5% ; DM 77.0 12.6%). The percentage of mutant mtDNA ranged from 6-63%. This result indicated that exocrine pancreatic dysfunction may be associated with the A3243G mutation. We have calculated the prevalence of spontaneous abortion in 379 women who delivered children in our hospital. The prevalence of spontaneous abortion was 14.4% (spontaneous abortion/total pregnancy=96/717). In our study, the prevalence of spontaneous abortion was higher in the population with the mutation than that of general population (25.0 vs 13.4%). In the basic study, we used C_2C_<12> myoblast treated with ethidium bromide 250 ng/ml for 10 days in medium. The cell line C2C12 was treated with EB for 10 days, and total RNA extract from cells from the cells. After 10 days incubation with EB, no mtRNA was detectable in C_2C_<12> cells, but the contents of beta-actin were not affected. After 60% confluent, we changed medium to 2% horse serum, 50 mug/ml uridine and 0.1 mg/ml pyruvate. Differentiation to myotubes in C_2C_<12> cells with EB was remarkably impaired. We also study the effect of EB in differentiated C_2C_<12> myotubes on insulin stimulated glucose transport. C_2C_<12> myotubes were treated with EB 3 days, and 2-deoxyglucose transport was measured. Insulin-stimulated glucose transport activity was decreased in C_2C_<12> myotubes treated with EB.In conclusion, we observed variable clinical symptoms in the patients and lacking of mtDNA induced a impaired muscle differentiation and its function in culture cells.

线粒体DNA(MtDNA)突变与多种疾病有关。最近的研究表明，tRNA^&lt；Leu(UUR)&Gt；基因的nt3243(A3243G突变)与母系遗传性糖尿病(DM)密切相关。已有多器官受累的说法。作为胰腺外分泌功能的测试，我们用苯甲酰-酪氨酰-对氨基苯甲酸(PABA)进行了苯妥胺试验。PABA回收率下降19-68%(正常81.9-8.5%，DM 77.0-12.6%)。突变型mtDNA的百分率为6-63%。这一结果提示胰腺外分泌功能障碍可能与A3243G突变有关。我们统计了379例在本院分娩的产妇的自然流产发生率。自然流产发生率为14.4%(自然流产/妊娠总数=96/717)。在我们的研究中，携带该突变的人群中自然流产的患病率高于普通人群(25.0%对13.4%)。在基础研究中，我们使用含250 ng/ml溴化乙锭的C_2C_(12)成肌细胞在培养液中孵育10天。将EB作用于C2C12细胞10d，提取细胞总RNA。EB作用10天后，C2C&lt；12&gt；细胞中未检测到mtRNA，但β-肌动蛋白含量未受影响。60%融合后，换成2%马血清+50ug/ml尿苷+0.1 mg/ml丙酮酸。EB对C_2C_(12)细胞向肌管的分化有明显的抑制作用。我们还研究了分化C_2C_(12)肌管中EB对胰岛素刺激的葡萄糖转运的影响。用EB处理C_2C_(12)肌管3天，测定2-脱氧葡萄糖转运。EB处理的C2C12-gt；肌管中胰岛素刺激的葡萄糖转运活性降低。结论：我们观察到患者有不同的临床症状，mtDNA缺乏导致肌肉分化及其培养细胞功能受损。