Grant-in-Aid for Scientific Research (A)
|Allocation Type||Single-year Grants|
|Research Institution||Hiroshima University|
YUGE Osafumi Faculty of Medicine, Anesthesiology, Hiroshima University, Professor, 医学部, 教授 (40034128)
OHOSAWA Yasuhiro Faculty of Medicine, Anesthesiology, Hiroshima University, Research Associate, 医学部(平成11年9月30日付辞職), 助手 (00263682)
HUJII Kohoku Faculty of Medicine, Anesthesiology, Hiroshima University, Associate Professor, 医学部(平成11年3月31日付辞職), 講師 (60034021)
KAWAMOTO Masashi Faculty of Medicine, Anesthesiology, Hiroshima University, Professor, 医学部, 助教授 (40127642)
MAEHARA Yasuhiro Faculty of Medicine, Anesthesiology, Hiroshima University, Assistant Professor, 医学部・附属病院, 講師 (20238877)
|Project Period (FY)
1996 – 1999
Completed(Fiscal Year 1999)
|Budget Amount *help
¥25,000,000 (Direct Cost : ¥25,000,000)
Fiscal Year 1999 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1998 : ¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1997 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1996 : ¥18,500,000 (Direct Cost : ¥18,500,000)
|Keywords||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 studies
We 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
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 biopsy
2. The pilot stud of screening test using caltured human skeletal muscle cell
We 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