KISEP Experimental Research J Korean Neurosurg Soc , 1998 일과성뇌허혈후 Amyloid Precursor Protein mrna 와 Amyloid Precursor Protein 발현의변화 * 이상형 김

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1 KISEP Experimental Research J Korean Neurosurg Soc , 1998 일과성뇌허혈후 Amyloid Precursor Protein mrna 와 Amyloid Precursor Protein 발현의변화 * 이상형 김혜선 ** 정성진 ** 이영재 *** 서유헌 ** 양희진 정영섭 한대희 조병규 = Abstract = The Change of Expression in Amyloid Precursor Protein mrna and Amyloid Precursor Protein by Transient Ischemia Sang Hyung Lee, M.D., Hye Sun Kim, Ph.D.,** Sung Jin Jeong, BSc,** Young-Jae Lee, Ph.D.,*** Yoo Hun Suh, M.D.,** Hee Jin Yang, M.D., Young Seob Jung, M.D., Dae Hee Han, M.D., Byung-Kyu Cho, M.D. Departments of Neurosurgery and Pharmacology,** Seoul National University College of Medicine, Seoul, Korea Department of Veterinary Medicine,*** Cheju National University, Cheju, Korea KEY WORDS 서 론 J Korean Neurosurg SocVolume 27November,

2 일과성뇌허혈후 Amyloid Precursor Protein mrna 와 Amyloid Precursor Protein 발현의변화 연구범위및방법 1. 뇌허혈동물모델제조법 뇌조직에서 total RNA의분리 3. APP의 PCR 시발체 (Primer) 의설계 Table 1. Time schedule of experimental groups Group Occlusion time Reperfusion time A* B** 1 hr 0 hr C 1 hr 1 hr D 1 hr 3 hr E 1 hr 8 hr F 1 hr 1 day G 1 hr 3 day H 1 hr 7 day *normal control, **sham control, hrhour J Korean Neurosurg SocVolume 27 November, 1998

3 이상형 김혜선 정성진 이영재 서유헌 양희진 정영섭 한대희 조병규 4. 역전사중합효소연쇄반응 (RT-PCR(Reverse Transcription-Polymerase Chain Reaction)) 방법 Fig. 1. Amyloid Precursor Protein primer for PCR and their product size. 5. 단백질분리와 Western Blot 연구결과 1. 뇌허혈이유발된백서의대뇌피질에서의 APP mrna 의발현 J Korean Neurosurg SocVolume 27 November,

일과성 뇌허혈후 Amyloid Precursor Protein mrna와 Amyloid Precursor Protein 발현의 변화 는 더이상 증가하지 않아 30회로 고정하여 이후 실험을 시 후 실험에서는 DMSO를 첨가하지 않았다(Fig. 4 D-). 행하였다(Fig. 3).

현되었다. 반면, 신장과 간조직에서는 APP770과 APP751 결과, APP695의 mrna는 1일, 3일째에는 감소하는 것으로 이 주로 발현되었으며, 특히 신장에서는 APP751이 주로 보였으나 수술하기 전과 비교할 때 3일째에만 유의하게 감소 발현되는 것으로 나타났다(Fig. 4 D ). RT-PCR에 대한 하였다(p<0.05).

APP770의 mrna의 증가는 1일째에는 수술하기 전과 단순히 폐색시킨 것과 비교할 때 증가의 차이가 있었으며(p<0.01), 3일째에는 APP751 및 APP770의 mrna가 모두 통계적으로 유의한 증가를 보였다(p<0.01)(Fig. 5A, 5B, 7). 2.

PCR products of 331, 274, 197bp equivalent to APP770, APP751, APP695 were obtained. Fig. 3. Amplication cycle-dependent PCR amplication of APP695 cdna. Fig. 4.

4 일과성 뇌허혈후 Amyloid Precursor Protein mrna와 Amyloid Precursor Protein 발현의 변화 는 더이상 증가하지 않아 30회로 고정하여 이후 실험을 시 후 실험에서는 DMSO를 첨가하지 않았다(Fig. 4 D-). 행하였다(Fig. 3). APP의 mrna의 발현은 뇌를 포함한 대 APP 유전자발현에 대한 일과성 허혈의 영향을 알아보기 부분의 조직에서 발현되는데 특히 APP695는 뇌에서 주로 위해 재관류후 1시간, 3시간, 8시간, 1일, 3일, 7일후에 백서의 나타났으며 APP770과 APP751는 매우 낮은 수준으로 발 대뇌피질을 적출하여 RNA를 추출한 후 RT-PCR을 시행한 현되었다. 반면, 신장과 간조직에서는 APP770과 APP751 결과, APP695의 mrna는 1일, 3일째에는 감소하는 것으로 이 주로 발현되었으며, 특히 신장에서는 APP751이 주로 보였으나 수술하기 전과 비교할 때 3일째에만 유의하게 감소 발현되는 것으로 나타났다(Fig. 4 D ). RT-PCR에 대한 하였다(p<0.05). APP751 및 APP770의 mrna는 1일 이 dimethyl sulfoxide(dmso)의 영향을 확인한 결과 DMSO 후에 증가하기 시작하여 3일째 최고 수준으로 증가하였으며, 를 첨가하지 않았을 때 PCR이 더 잘되는 것으로 나타나 이 7일째에는 다시 재관류하기 전으로 감소하였다. APP770의 mrna의 증가는 1일째에는 수술하기 전과 단순히 폐색시킨 것과 비교할 때 증가의 차이가 있었으며(p<0.01), 3일째에는 APP751 및 APP770의 mrna가 모두 통계적으로 유의한 증가를 보였다(p<0.01)(Fig. 5A, 5B, 7). 2. 뇌허혈이 유발된 백서의 대뇌피질에서의 APP단백질발현 허혈이 APP의 단백질의 발현에 대한 영향을 관찰하기 Fig. 2. Confirmation of the primer. PCR was performed using the synthesized primer with cdna. PCR products of 331, 274, 197bp equivalent to APP770, APP751, APP695 were obtained. Fig. 3. Amplication cycle-dependent PCR amplication of APP695 cdna. Fig. 4. The expression pattern of APP mrna isoforms in rat brain, liver and kidney(d Dimethylsulfoxide) Fig. 5. RT-PCR analysis of APP isoform mrna levels in cerebral cortex after I hour of ischemia. Lane M PCR marker, 1 group A(control), 2 group B(sham control), 3 group C, 4 group D, 5 group E, 6 group F, 7 group G, 8 group H. (A) Changes in the expression of APP 3 isoforms detected by RT-PCR. (B) Autoradiogram showing the changes in the expression of APP 3 isoforms, using 32P-α dctp β-actin band was weakened by reducing the exposure time for exact comparison with 3 APP isoform bands. Fig. 6. Immunoblotting analysis of the cortex homogenates after ischemia using 22C11. Lane 1 group A(control) 2 group B(sham control), 3 group C, 4 group D, 5 group E, 6 group F, 7 group G, 8 group H. J Korean Neurosurg Soc/ Volume 27 / November, 1998

5 이상형 김혜선 정성진 이영재 서유헌 양희진 정영섭 한대희 조병규 Fig. 7. The changes of ratio of mrna of three isoforms. APP770, APP751 and APP695 in cerebral cortex. Each point represent s the means.e.m.n5 percentage of the total of three isoforms. The intensity of the bands of five autoradiograms were quantitated by densitometer and statistical analysis was made by ANOVA**p0.01. Fig. 8. The effect of transient ischemia on the level of amyloid precursor protein in rat cerebral cortex. There was no significant difference by ANOVA. 고찰 J Korean Neurosurg SocVolume 27 November,

6 일과성뇌허혈후 Amyloid Precursor Protein mrna 와 Amyloid Precursor Protein 발현의변화 1486 J Korean Neurosurg SocVolume 27 November, 1998

7 이상형 김혜선 정성진 이영재 서유헌 양희진 정영섭 한대희 조병규 결론 References 1) Abe K, Tanzi RE, Kogure KInduction of HSP70 mrna after transient ischemia in gerbil brain. Neurosci Lett , 1991a 2) Abe K, Tanzi RE, Kogure KSelective induction of Kunitztype protease inhibitor domain-containing amyloid precursor protein mrna after persistent focal ischemia in rat cerebral cortex. Neurosci Lett , 1991b 3) Araki W, Kitaguchi N, Tokushima Y, et altrophic effct of -amyloid precursor protein on cerebral cortical neurons in culture. Biochem Biophys Res Commun , ) Busciglio J, Lorenzo A, Yankner BA, et almethodological variables in the assessment of beta amyloid neurotoxicity. Neurobiol Aging , ) Card JP, Meade RP, Davis LGImmunocytochemic localization of the precursor protein for -amyloid in the central nervous system. Neuron , ) Checler FProcessing of the beta-amyloid precursor protein and its regulation in Alzheimer s disease. J Neurochem , ) Chen ST, Hsu CY, Hogan EL, et ala model of focal ischemic strokereprducible extensive cortical infarction. Stroke , 1996 J Korean Neurosurg SocVolume 27 November,

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9 이상형 김혜선 정성진 이영재 서유헌 양희진 정영섭 한대희 조병규 BO J , ) Seubert D, Cole G, Saitoh T, et alamyloid beta protein is a mitogern. Biochem Biophys Res Commun , ) Smith-Swintosky VL, Pettigrew LC, Craddok SD, et alsecreted forms of -amyloid precursor protein protect against ischemic brain injury. J Neurochem , ) Stephenson DT, Rash K, Clemens JAAmyloid precursor protein accumulates in regions fo neurodegeneration following focal cerebral ischemia in the rat. Brain Res , ) Tanaka S, Shiojiri S, Takahashi Y, et altissue-specific expression of three types of a protein precusor mrnaenhancement of protease inhibitor-harboring types in Alzheimer s disease. Biochem Biophys Res Commun , ) Tanzi R, Mcclalatchey AL, Lamperti ED, et alprotease inhibitor domain encoded by an amyloid protein precusor mrna associated with Alzheimer s disease. Nature , ) Tomimoto H, Akiguchi I, Wakita H, et alultrastuructural localization of amyloid precursor protein in the normal and post ischemic brain. Brain Res , ) Uemura Y, Kowall NW, Beal MFGlobal ischemia induces NMDA recepter mediated c-fos expression in neurons resistant to injury in gerbil hippocampus. Brain Res , ) Vass K, Welch WJ, Nowak TS JrLocalization of 70-kDa stress protein induction in gerbil brain after ischemia. Acta NeuropatholBerl , ) Waite J, Cole GM, Frautsch SA, et alsolvent effects on beta protein toxicity in vivo. Neurobiol Aging , ) Wakita H, Tomimoto H, Akiguchi I, et alregional accumulation of amyloid precursor protein in the gerbil brain following transient ischemia. Neuroscience Lett , ) Yankner BA, Dawes LR, Fischer S, et alneurotoxity of a fragment of the amyloid precursor associated with Alzheimer s disease. Science , ) Yankner BA, Duffy LK Kirssehner DANeurotrophic and neurotoxic effects of amyloid proteinreversal of by tachykinin neropeptides. Science , ) Yoneda K, Okada YEffects of anoxia and recovery on the neurotransmission and level of high energy phosphates in thin hippocampal slices from the guinea-pig. Neuroscience , 1989 J Korean Neurosurg SocVolume 27 November,

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untitled 3. 농업환경연구과 과제구분 기본연구 수행시기 전반기 연구과제 및 세부과제 수행 기간 소 속 책임자 농가에 적합한 부식성곤충 대량 사육기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 1) 부식성 곤충 먹이 제조 기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 색인용어 부식성곤충, 장수풍뎅이, 계통, 먹이제조 ABSTRACT In first check,