THE TRIAL STUDY FOR THE MINIMUM INVASIVE DIAGNOSTIC TEST FOR MHS -THE EFFECTS OF CAFFEINE AND HALOTHANE ON CULTURED HUMAN SKELETAL MUSCLE CELL MESURING INTRA CELLULAR Ca CONCENTRATION CONCENTRATION AND GENETIC ANALYSIS-

MHS微创诊断试验的试验研究-咖啡因和氟烷对培养的人体骨骼肌细胞测定细胞内Ca浓度的影响及遗传分析-

• 批准号: 08407052
• 负责人: YUGE Osafumi
• 金额: $ 16万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08407052/
• 关键词: Malignant Hyperthermia; Ca-induced-Ca Release Rate; Halothane; Caffeine; [CaィイD12+ィエD1]i; Caltured human skeletal muscle cell; Screening test of MH; Genetic linkage study; 骨格筋リアノジン遺伝子; スクリーニングテスト

1. CICR rate and genetic linkage studiesWe have performed genetic linkage analysis with CICR test in 63 subjects who were referred to our hospital for investigation of MHS.Eleven of them had a history of f-MH episode and 33 had a family history of f-MH, 9 had a history of mild MH reaction and I O had a family history of mild MH reaction.We performed CICR test in 23 subjects. Ten of 11 f-MH subjects, 3 family members with f-MH and 2 unrelated subjects with mild MH reactions showed the accelerated changes in CICR test.We isolated genomic DNA from all of 63 subjects. Analysis for a substitution of cytosine 1840 to thymine which is same mutation of porcine MHS, was performed in all 63 subjects. We also checked the several other one point mutations in some but not all. However, we din not find any alterations. From our results, we can say there remains a possibility of other point mutations. However, we found one MHS family, which carried infomative polymorphisms. The proband, blue square o … More ne, manifested f-MH during a neurosurgical operation. CICR test in his daughter and son showed acceralation.In genetic linkage study they showed informative polymorphisms relating to MHS diagnosed with CICR test. In this family, linkage analysis of the RYR1 gene was very useful for further examination, including muscle biopsy2. The pilot stud of screening test using caltured human skeletal muscle cellWe cultured normal human skeletal muscle cell (SRMC 2874, CloneticsィイD1RィエD1) on glass cover slips and loaded with 5 μ M Fura-2/AM. Cover slips were mounted on perfusion system which was perfused with HEPES solution. Caffeine (0.25-10mM) and Halothane (0.1-2mM), which were dissolved in HEPES solution in a gas-tight vial, was connected to this perfusion system just before these stimulations. The cover slip was placed on the stage of a fluorescence inverted microscope, combined with ARGUS-50 (Hamamatsu PhotonicsィイD1RィエD1). The fluorescence intensity ratio with excitation at 340/380 nm and emission at 510 nm was recorded every 5 sec.As the results, 1)Caffeine (0.25-10mM) stimulations increased [CaィイD12+ィエD1]i in a concentration- dependent manner. 2)Also ,halothane (0.1-2mM) stimulations increased [CaィイD12+ィエD1]i in a concentration-dependent manner. Less

1. CICR率和遗传连锁研究我们对转诊至我院进行MHS检查的63名受试者进行了CICR检测的遗传连锁分析。其中11名受试者有f-MH发作史，33名有f-MH家族史，9名有轻度MH反应史，10名有轻度MH反应家族史。我们对23名受试者进行了CICR试验。 11 名 f-MH 受试者中的 10 名、3 名患有 f-MH 的家庭成员和 2 名具有轻度 MH 反应的无关受试者在 CICR 测试中显示出加速变化。我们从所有 63 名受试者中分离了基因组 DNA。对所有 63 名受试者进行了 1840 号胞嘧啶替换为胸腺嘧啶的分析，胸腺嘧啶与猪 MHS 的突变相同。我们还检查了一些但不是全部的其他几个单点突变。然而，我们没有发现任何改变。从我们的结果来看，我们可以说仍然存在其他点突变的可能性。然而，我们发现了一个MHS家族，它携带信息多态性。先证者，蓝色方块 o … 更多 ne，在神经外科手术中表现出 f-MH。他的女儿和儿子的 CICR 测试显示加速。在遗传连锁研究中，他们显示出与 CICR 测试诊断的 MHS 相关的信息多态性。在这个家族中，RYR1 基因的连锁分析对于进一步检查（包括肌肉活检）非常有用。使用培养的人骨骼肌细胞进行筛选试验的试点我们在玻璃盖玻片上培养正常人骨骼肌细胞（SRMC 2874，CloneticsィイD1RィエD1）并加载5μM Fura-2/AM。将盖玻片安装在灌注有 HEPES 溶液的灌注系统上。咖啡因 (0.25-10mM) 和氟烷 (0.1-2mM) 溶解在气密小瓶中的 HEPES 溶液中，在进行这些刺激之前将其连接到该灌注系统。将盖玻片放置在荧光倒置显微镜的载物台上，并结合ARGUS-50(Hamamatsu PhotonicsィイD1RエD1)。每5秒记录一次340/380 nm激发和510 nm发射的荧光强度比。结果，1)咖啡因(0.25-10mM)刺激以浓度依赖性方式增加[CaiiD12+ィD1]i。 2)此外，氟烷（0.1-2mM）刺激以浓度依赖性方式增加[CaィイD12+ィエD1]i。较少的

项目成果

奥史郎、他: "生検筋による悪性高熱症診断のための出張検査"日本臨床麻酔学会誌. 19. 574-578 (1999)

Okushiro 等人：“使用活检肌肉诊断恶性高热的现场检查”日本临床麻醉学会杂志 19. 574-578 (1999)。

O Yuge, M Morio, H Kikuchi, K Mukaida, Y Maehara, M Nakao, M Kawamoto: "Clinical Classification and Incidence of Malignant Hyperthermia in Japan"Proc. of the 3nd Symposium on MH. 49-56 (1996)

O Yuge、M Morio、H Kikuchi、K Mukaida、Y Maehara、M Nakao、M Kawamoto：“日本恶性高热的临床分类和发病率”Proc。

Y Maehara, K Mukaida, E Hiyama, M Morio, M Kawamoto, O Yuge: "Genetic Analysis with Calcium-inducede Calcium Release Test in Japanese Malignant Hyperthermia Susceptible(MHS) Families"Hiroshima J of Medical Science. 48(1). 9-15 (1999)

Y Maehara、K Mukaida、E Hiyama、M Morio、M Kawamoto、O Yuge：“日本恶性高热易感 (MHS) 家族中钙诱导钙释放试验的基因分析”广岛医学科学杂志。

前原康宏 弓削孟文 他: "一般手術症例における術中体温変化" 麻酔と蘇生. 34・3. 182- (1998)

Yasuhiro Maehara、Menofumi Yuge 等：“一般外科病例的术中体温变化” 麻醉与复苏 182- (1998)。

向田圭子 前原康宏 弓削孟文: "4chloro-m-cresolのヒト骨格筋CICR機構に対する影響" 麻酔と蘇生. 34・3. 183- (1998)

Keiko Mukoda、Yasuhiro Maehara、Mengfumi Yuge：“4 氯间甲酚对人体骨骼肌 CICR 机制的影响”麻醉与复苏 34・3。