中枢神経系に於けるHCNPの存在意義の解明

阐明HCNP在中枢神经系统中存在的意义

• 批准号: 04268212
• 负责人: 小鹿 幸生
• 金额: $ 0.96万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04268212/
• 关键词: Chorinergic neuron; Cholineacetyl Trans Perase; Neurotrophic Pactor; HCNP; free-HCNP; HCNP前駆体

Hippocampal cholinergic neurostimulating peptide(HCNP)はラット培養中隔核のChATase産生を誘導してAcCho産生を促進する新規ペプチドである。このHCNPおよびNCNPのN末端アセチル基を除いたfree-HCNP,PAGEで23KDa近傍に移動するHCNP前駆体の各々を定量する測定系の確立を目指した。free-HCNPで家兎を免疫して得た抗HCNP抗体は、ELISAでこれらペプチドと反応を示すのみならず、海馬抽出液のWestern blot解析からHCNP前駆蛋白とも反応を示すが、他の成分との交叉反応性は少ないとことが確認された。この抗体を用いRIAの緒条件を検討し、以下の方法を最終測定法とした。緩衝液には1mM EDTAを含む50mM Tris-HCl(pH7.8)を用い、[ ^3H]-HCNP(5000DPM/0.1ml)+緩衝液(0.1m)又は資料(既知又は未知0.1ml)+1/200希釈抗HCNP抗血清(50μl)→incubation(4℃,overnight)→Amerlex-M0.5ml添加→incubation,30℃,15min→遠心上清0.3ml中の放射活性を測定、既知free-HCNP量を用いた検量線から未知資料中のHCNP量を算定した。このRIA系を用い14日齢ラット大脳皮質、海馬、小脳、橋を0.1%TFA溶液中で超音波破壊し、遠沈上清中に含まれるHCNP様物の定量を試みると、それぞれ15.0,7.0,4.9,30.0Pmol/mg蛋白(fluorescamin法)であった。また、C_4の逆相系カラケとA液:0.1%TEA,B液:0.7%TFAを含む90%アセトニトリルを用いたHPLCでfree-HCNP,HCNPおよびHCNP前駆蛋白は、それぞれB液濃度40%(RT14分16秒)、42%(RT16分16秒)、51%(RT25分4秒)に溶出される。各組織抽出液をHPLCで分画し、free-HCNP,HCNP前駆蛋白のRT近傍の画分に含まれるHCNP様物量をRIAで測定すると、各々の量比は大脳皮質で3:3:1,海馬で1:6:1,小脳で1:1:1.5,橋で2:3:0.2であった。

Hippocampal cholinergic neurostimulating peptide (HCNP) induces ChATase production in the septal nucleus and promotes AcCho production in the septal nucleus. HCNP's N-terminal chromium-free NCNP's free-HCNP, PAGE's 23KDa close proximity and mobile HCNP front body's each 々をquantitative するdetermination system has been established. free-HCNP's immune system, anti-HCNP antibody, ELISA's anti-HCNP antibody, ELISA's anti-HCNP antibody, hippocampal extract fluid's Western Blot analysis showed that the HCNP pro-protein and cross-reactivity of other components were confirmed and confirmed. The antibody was tested using the RIA conditions and the following method was used as the final measurement method. The buffer contains 1mM EDTA and 50mM Tris-HCl (pH7.8). ^3H]-HCNP (5000DPM/0.1ml) + buffer (0.1m) and information (known and unknown 0.1ml) + 1/200 anti-HCNP antiserum (50μl) → incubation (4℃, overnight )→Add Amerlex-M0.5ml→incubation, 30°C, 15min→Determine the radioactivity in 0.3ml of the telecentric supernatant, and use the measurement line to calculate the amount of HCNP in unknown data. The RIA system uses 14-day-old ultrasonic decontamination of large cortex, hippocampus, small intestine, and bridge in 0.1% TFA solution, and HCNP in the supernatant of the far sedimentation Quantitative testing of 様objects was carried out using みると and それぞれ15.0, 7.0, 4.9, 30.0 Pmol/mg protein (fluorescamin method).また、C_4 reverse phase system カラケとA solution: 0.1%TEA, B solution: 0.7%TFA む90% アセトニトリルを Used いたHPLCでfree-HCNP, HC NPおよびHCNP pre-protein solution and それぞれB liquid concentration are 40% (RT14 minutes and 16 seconds), 42% (RT16 minutes and 16 seconds), and 51% (RT25 minutes and 4 seconds). Each tissue aspirate was separated by HPLC, free-HCNP, and the HCNP pre-protein was separated by RT and the amount of HCNP was analyzed by RIA. The measured ratio of each test was 3:3:1 for the large cortex, 1:6:1 for the hippocampus, 1:1:1.5 for the small fish, and 2:3:0.2 for the bridge.