보고서 정보
주관연구기관 |
한국과학기술연구원 Korea Institute Of Science and Technology |
연구책임자 |
오태환
|
참여연구자 |
김기환
,
박찬웅
,
배은희
,
임혜원
,
윤태영
,
신영희
,
김영미
,
백승연
,
이동윤
,
정재원
,
강승모
,
김윤희
,
나승열
,
오영준
,
정광철
|
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 1999-12 |
주관부처 |
국무조정실 |
연구관리전문기관 |
한국과학기술연구원 Korea Institute Of Science and Technology |
등록번호 |
TRKO200200048054 |
DB 구축일자 |
2013-04-18
|
초록
신경세포의 발생, 퇴화, 재생 및 기능 유지 연구의 당해 연도 연구내용 및 연구 범위는 다음과 같다. 1. 신경세포 발생 및 분화 연구 : 핵심 factor screening과 관련 cDNA 준비 2. 외상에 의한 중추신경 퇴화 및 재생 연구 : Reactive gliosis 형성기전 및 세포 퇴화의 신호전달 기전 연구 Ⅰ 3. 신경세포사멸 (apoptotic neuronal cell death)의 신호전달 기전연구 4. 신경세포의 기능유지에 필요한 신경전달물질 및 그 조절인자 연구 : 에스트로젠의 뇌 기능 유지 효과
Abstract
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The project of "Studies on CNS neurons: Development, Degeneration, Regeneration and Function" are summarized by separated subtitles based on each experiment's background, significance and results. Studies on neuronal development, apoptosis, and differentiation by BMP-4: BMP-4 is a muitifunctional cy
The project of "Studies on CNS neurons: Development, Degeneration, Regeneration and Function" are summarized by separated subtitles based on each experiment's background, significance and results. Studies on neuronal development, apoptosis, and differentiation by BMP-4: BMP-4 is a muitifunctional cytokine, which belongs to TGF-β superfamily. Several reports suggested that BMP-4 had key roles in migration, proliferation, and differentiation of neuronal stem cell. However, temporal and spacial signals directed from BMP-4 has not been exactly known. Here we used Pl9 embryonal carcinoma cells as a model system to understand mechanism of neuronal differentiation by BMP-4 and search the possibility to apply BMP-4 in neuronal regeneration after traumatic injury. BMP-4 could maintain neuronal differentiation whereas astrocyte differentiation had been blocked by BMP-4 at concentration of 20 ng/ml. Inhibition of p38 MAPK mimics the effect of BMP-4, which suggests that SMAD pathway and p38 pathway had related in certain point during astrocyte and neuronal differentiation. These collective data gave a way to application of BMP-4 to neuronal regeneration, where the reactive gliosis is a one of the major impediment to axonal regeneration. Cloning and Analysis of a Neuron Specific ARPiN (nARPiN), a Component of hSWI/SNF Complex: Development of embryo is the result of the sequential turn-on and -off of master transcription factors, which is programmed in developmental process. However, chromatin structure as well as transcription factors has a great influence on gene expression. These results suggest that changes in chromatin structure closely regulate gene expression pattern, and accordingly developmental process. hSWI/SNF is a chromatin remodeling factor that controls developmental process. We cloned a neuron specific ARPiN (nARPiN), a component of hSWI/SNF complex. The difference between ARPiN and nARPiN resides in domain 2 exclusively. Domain 2 of actin provides myosin binding site. We made a HA-tagged nARPiN and over-expressed HA-nARPiN in HeLa cells. HA-nARPiN is localized in nucleus. We showed HeLa cells express both ARPiN and nARPiN. Since nARPiN is expressed exclusively in neuronal cells in Northen blotting analysis, it is under investigation whether the expression of ARPiN and nARPiN is a characteristic of immortalized cells. Characterization of the effect of intracellular calcium ion, Src kinase and MAP kinase on the expression of pip92 induced during the differentiation of immortalized CNS hippocampal cells: Calcium is a critical second messenger mediating intracellular signaling and regulating gene expression in neuronal cells. Src tyrosine kinase family are involved in a broad range of cellular responses ranging from cell division and cytoskeletal rearrangement to the differentiation of neuronal cells. Immediate early gene pip92 is known to be selectively induced by differentiating factors during the neuronal differentiation or cell death in H19-7 cells. In the present we have examined the effect of intracellular calcium mobilization and Src kinase on the activation of pip92 expression in immortalized rat hippocampal H19-7 cells. Increase of intracellular calcium ion levels induced by thapsigargin or ionomycin stimulated the expression of pip92 in neuronal H19-7 cells. Transient transfection of the cells with kinase-inactive MAPK kinase (MEK) and Src kinase, or pretreatment with chemical MEK inhibitor, PD98059, significantly inhibited pip92 expression induced by thapsigargin. When constitutive-active v-src or MEK was overexpressed, the transcriptional activity of pip92 gene was markedly increased. Taken together, these results suggest that an increase of intracellular calcium ion levels stimulates pip92 expression via MEK-ERK- as well as Src kinase-dependent signaling pathways. Role of Tumor Necrosis Factor- α in Neuronal and Glial Apoptosis After Spinal Cord Injury: We investigated the role of tumor necrosis factor (TNF)- α in mediating the onset of neuronal and glial apoptosis after traumatic spinal cord injury in rats. We examined the appearance of TNF- α and apoptotic cells in the spinal cord of rats subjected to crush injury. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive neurons were first observed within and surrounding the lesion area 16 hr after injury, with a highest number at 24-48 hr after injury. TUNEL-positive neurons disappeared after 4 days and then reappeared again 1 week after injury. TUNEL-positive glial cells were observed 24 hr after injury and present throughout 1 week period studied. An intense TNF- α immunoreactivity at the lesion center was observed as early as 1 hr after crush and remained high until 8 hr after injury. Thereafter, TNF- α staining gradually spreaded out from the lesion area to both roastrally and caudally, and was detectable 1 week after injury. Injection of a neutralizing TNF- α antibody into the lesion site immediately after crush injury significantly reduced apoptotic cells and inducible nitric oxide synthase (iNOS) at 24 hr after injury. Furthermore, an iNOS inhibitor, NG-monomethyl-L-arginine acetate (L-NMMA), significantly reduced the number of apoptotic cells as well. These results support the hypothesis that TNF- α serves as an external signal triggering apoptosis in neurons and oligodendroglia after spinal cord injury, and that nitric oxide (NO), produced by TNF- α -induced iNOS expression, is a mediator of this apoptosis. Caspase-dependent and independent calpain activation in dopaminergic neuronal cell death: Activation of proteases such as caspase and calpain has been shown to regulate cell death in a variety of cell types. To investigate whether there exists a stress-specific protease activation pathways and to what extent Bcl-2 plays a role for regulating protease activity, we used a dopaminergic neuronal cell line, MN9D. As determined by morphological criteria, staurosporine (STS) induced a prototypic apoptosis while a dopaminergic neurotoxin, N-methyl-4-phenylpyridinium (MPP+) largely induced necrosis in MN9D dopaminergic neuronal cell line. In these two distinct types of cell death, caspase activity increased following treatment with STS but not MPP+ as determined by a fluorogenic caspase substrate assay and immunoblot analysis for a specific cleavage of poly( ADP-ribose )polymerase (PARP). Consequently, cotreatment with a broad-spectrum caspase inhibitor (BAF or Z-VAD-fmk) or overexpression of viral protein p35 in MN9D cells attenuated STS- but not MPP+-induced cell death. In these cell death paradigms, Bax protein was found to be cleaved both in STS- and MPP+-induced cell death at the later stage of cell death as determined by immunoblot analysis. Proteolytic cleavage of Bax was mediated by calpain but not caspase-1, -3 and 9 as determined by incubating 35S-methionine-labelled Bax. Interestingly, cotreatment of cells with calpain inhibitor (calpeptin) blocked all of the drug-induced cleavage of Bax while cotreatment of caspase inhibitors or overexpression of viral protein p35 blocked only STS-induced Bax and PARP cleavage. Thus, our data indicate that calpain is activated in a caspase-dependent or -independent manner depending on types of stresses applied. Effects of Ginsenosides on the Performance of Female Mice during Acquisition of the Morris Water Maze: Estrogen can influence on the expression of behaviors not associated directly with reproduction, including learning and memory. Recently estrogen has received considerable attention for its effects on neuroprotection and neural circuits in brain areas associated with cognition. Although estrogen replacement therapy may be helpful to postmenopausal women, it also results in a number of harmful side effects. Ginseng also has steroidal qualities and contains several ginsenoside components which have similar backbone structure to estrogen. The objectives of this experiment were 1) to examine the effects of estrogen and 2) to investigate the effects of ginsenosides as estrogenic agent on learning and memory using the Morris water maze, a traditional experimental task for spatial memory. In the experiments designed here, ovariectomized mice were implanted subcutaneously with Silastic capsules containing 17 β -estradiol (100-250㎍/㎖), panaxadiol (PD) and panaxatriol (PT) saponins (15-100㎍/㎖) diluted with sesame oil. In the first set of experiment, we examined the effects of estradiol on learning and memory during the Morris water maze when estradiol was delivered via Silastic capsules following training improved spatial memory performance in ovariectomized female mice. In the second set of experiment, three different PD and PT saponin concentrations were delivered via Silastic implants to ovariectomized female mice and their effects were compared with estrogenic effects. Results of three separate experiments demonstrated that estradiol, PD and PT administrated by Silastic implants for 2 weeks prior to water maze training significantly improved spatial memory performance compared to ovariectomized (OVX) mice, as indicated by lower escape latency over trial. The positive effect of estradiol suggests that estrogen can affect performance on learning and memory. In addition, the positive effect of PD and PT saponins suggest that ginsenosides have an estrogen-like effects in mediating learning and memory related behavior action. 17 β -Estradiol Inhibits High-Voltage-Activated Calcium Currents in Rat Sensory Neurones via a Non-Genomic Mechanism: There is increasing evidence that estrogen influences electrical activity of neurons via stimulation of membrane receptors. Recently presence of estrogen receptor and its responsiveness in dorsal root ganglion (DRG) primary sensory neurons was reported. Therefore the current study was initiated to examine the rapid effects of estrogen on high-voltage-activated (HVA) Ca2+ channels and to determine its detail mechanism in female rat DRG neurons using whole-cell patch-clamp recordings. Application of 17β-estradiol (1 μM) caused a rapid inhibition of peak inward Ca2+ currents. This rapid estrogen-mediated inhibition was reproducible and occurred in a dose-dependent manner. The effects were sex- and stereo-specific; the inhibition was greater in cells isolated from intact female rats and was less by application of 17a-estradiol, the stereoisomer of the endogenous 17β-estradiol. Ovariectomy reduced the inhibition significantly but this effect was restored by administration of estrogen in ovariectomized subjects, which suggests that circulating serum estrogen may affect this rapid estrogen-mediated modulation. Occlusion experiments using selective blockers revealed 17b-estradiol mainly targeted both L-type and N-type currents. Overnight treatment with pertussis toxin profoundly reduced 17 β -estradiol -mediated inhibition in a dose-dependent manner On the other hand, estradiol conjugated to bovine serum albumin (EST-BSA) produced similar extent of inhibition as 17β-estradiol did. These results suggest that 17β-estradiol can modulate L- and N-type HVA Ca2+ channels in rat DRG neurons via an activation of pertussis-toxin sensitive G-protein and non-genomic signal pathways. It is likely that such effects are important in estrogen-mediated fast actions in sensory neuron function. Preparation of Monoclonal Antibody against Ginsenoside Rf from Panax Ginseng and its Application in Enzyme Immunoassay: A rapid and sensitive indirect competitive enzyme immunoassay method has been developed for quantitating ginsenoside Rf (Rf ) in crude total Panax ginseng saponins and in rat plasma using high titer mouse monoclonal antibody (mAb) raised against a conjugate of Rf and bovine serum albumin (BSA). The isotype of mAb against Rf was IgG3 with k chain. The presence of Rf inhibited the binding of the mouse anti-Rf mAb to a Rf-BSA solid phase coating antigen. The working range was 0.01-10 ng/assay and detection limits were 20 pg in various ginseng extract fractions or 34 pg in rat plasma per assay. The anti-Rf mAb cross-reacted with ginsenoside Rg2 by 57.5 % but not with other ginsenosides. However, this anti-Rf mAb did not cross-react with BSA and cellubiose, which is a carbohydrate component of Rf. Using this standard curve we could measure the amount of Rf in ginseng total extract, ginseng total saponins, protopanaxadiol saponins, propanaxatriol saponins, and commercial ginseng products. We could also measure the amount of Rf in rat plasma after oral administration of Rf and found that Rf reached a maximum level in rat plasma after 16 hr. These results indicate that the anti-Rf mAb could be useful for the quantitation of Rf in crude ginseng fractions and in body fluid.
목차 Contents
- 제 1 장 서론...21
- 제 1 절 연구개발의 필요성...21
- 제 2 절 연구개발의 목적 및 범위...23
- 제 2 장 국내외 기술개발 현황...24
- 제 3 장 BMP4에 의한 신경세포 발생, 사멸 및 분화 연구...26
- 제 4 장 신경세포의 발생과 재생과정에서 ARPiN의 기능 연구...33
- 제 5 장 중추신경계 해마신경세포의 분화시에 선택적으로 유도되는 pip92 유전자 발현에 대한 세포내 칼슘이온 및 Src kinase, MAP kinase 활성화의 영향 연구...39
- 제 6 장 척수 외상 후 신경 및 신경 교세포 사멸의 TNF-α의 역할...45
- 제 7 장 신경세포사멸에 관련된 caspase 및 non-caspase의 역할에 관한 연구...53
- 제 8 장 MORRIS WATER MAZE 습득에 대한 인삼성분의 효과...59
- 제 9 장 흰쥐의 감각신경세포에서 non-genomic 기전을 통한 17β-estradiol의 전압의존성 칼슘전류 억제효과...65
- 제 10 장 신경 세포안으로 칼슘 유입을 억제하는 진세노사이드 Rf에 대한 단클론 항체에 생성 및 응용에 대한 연구...73
- 제 11 장 참고문헌...80
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