• Journal of Genetic Medicine
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• 제12권1호
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• pp.25-30
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• 2015

Purpose: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. Materials and Methods: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. Results: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. Conclusion: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

• 대한약리학회지
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• 제30권2호
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• pp.167-179
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• 1994

Recent evidence indicates that glial cells have a wide range of funtions which are critical for maintaining a balanced homeostatic environment in the central nervous system(CNS) peripheral nervous system(PNS). Morever, astrocytes are known to participate in the tissue repair and neuroimmunologic events within the CNS through many kinds of growth factors and cytokines. We investigated the effect of $TGF\;{\beta}_1$, on the growth and biochemical changes of rat glial cells in culture. The proliferative effect was determined by $^3H-thymidine$ uptake and the double immunostain with anti-cell-specific marker and anti-Bromodeoxyuridine(BrdU) antibody. To check the effect of biochemical changes we compared the amounts of glial fibrillar acidic protein(GFAP) and the activity of glutamine synthetase(GS) in astrocyte. And the amounts of myelin basic protein and the activity of 2',3'-cyclic nucleotide phosphohydrolase(CNPase) were measured in oligodendrocyte and the amounts of peripheral myelin in Schwann cell. When $TGF\;{\beta}_1$, was treated for 2 days with cultured glial cell, $TGF\;{\beta}_1$, decreased the $^3H-thymidine$ uptake and proliferation index of double immunostain of astrocytes, which indicates the inhibition of astroglial DNA synthesis, but stimulated the growth of Schwann cell. Also, $TGF\;{\beta}_1$, decrease the GS activity and increased the amounts of GFAP in astrocyte. In the case of Schwann cells the amounts of peripheral myelin was increased when treated with $TGF\;{\beta}_1$. However, $TGF\;{\beta}_1$, didn't show any effect on the proliferation and biochemical changes in oligodendrocyte. These results suggest that $TGF\;{\beta}_1$, might have a critical action in the regulation of proliferation and biochemical changes in glial cells, especially astrocyte.

• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2019년도 춘계학술대회
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• pp.528-532
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• 2019

본 연구는 뉴런 세포와 슈반 세포의 공동 배양에 의한 수초화 발생 과정과 herpes simplex virus-1 감염에 의한 탈수초화 발생과정을 전자 현미경과 분자생물학적 분석에 의하여 확인하고자 하였다. 쥐의 배아로부터 후근신경절(dorsal root ganglion, DRG)을 분리하여 슈반(Schwann) 세포와 뉴런 세포(neuronal cell)를 in vitro에서 각각 배양하였다. 유사 분열 억제인자로 처리한 뉴런세포와 정제된 슈반 세포를 함께 공동 배양을 하여 수초화를 발생시켰다. 이렇게 수초화된 공동 배양 세포에 herpes simplex virus-1를 감염시켜 탈수초화를 진행시켰다. 수초 형성의 존재를 의미하는 myelin protein zero(MPZ) 항체를 사용하고 전자 현미경을 이용하여 수초 발생 및 탈수초화 과정을 관찰하였다. 이 연구는 과학 기술부, ICT 및 미래 계획 (NRF-2016R1A2B4016552 및 2017R1A2B3005753)이 자금을 지원하는 국립 연구 재단 (NRF)을 통한 기초 연구 프로그램의 지원을 받았다.

• Applied Microscopy
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• 제18권1호
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• pp.92-102
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• 1988

살리실산이 백서 와우의 나선신경절의 미세구조에 미치는 영향을 전자현미경으로 관찰하였다. 생후 $4{\sim}5$주의 Sprague-Dawley계 백서에 $500{\sim}600mg/kg$의 용량으로 살리실산 나트륨을 7일간 피하주사한 후에 각각 24시간(1군), 6주(2군), 10주(3군) 경과 후에 와우조직을 얻어 나선신경절에 나타나는 미세구조의 변화를 관찰하였다. 1. 1군에서는 신경절세포와 위성세포 그리고 Schwann cell의 세포질내에 다양한 크기의 막소구의 팽창이 나타났으며 Schwann cell 내에서는 미토콘드리아가 팽윤되거나 다낭성 세포소체를 형성하였다. 2. 2군에서는 신경절세포나 위성세포내의 막소구들이 더욱 팽창하고 그 일부는 융합하여 세포질이 감소하였다. Type I 세포의 신경 세포체나 신경섬유 주위를 싸고 있는 수초는 각층 사이의 거리가 멀어지거나 일부는 소실되었고, Type II 세포내에는 신경미섬유가 증가하였다. 3. 3군에서는 수초의 변형 및 파괴로 신경절세포의 세포질 이탈이 관찰되었고 Corti기 및 골성 나선판에 위치하는 신경섬유들의 소실이 관찰되었다. 이상의 결과를 종합하면 나선신경절세포와 그 주위를 둘러싸는 위성세포 및 Schwann cell은 과량의 살리실산 나트륨 투여후에 미세구조의 변화를 보였으며 1군에서 2군, 3군으로 갈수록 변화가 더욱 현저하였다.

• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2017년도 춘계학술대회
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• pp.714-717
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• 2017

슈반세포와 뉴런 세포가 쥐의 배아의 척수신경절로 부터 각각 in vitro에서 분리되었다. 배양된 슈반세포와 뉴런 세포는 동일한 평판접시에서 공동배양 되었다. 이들 세포들은 때 수초화가 진행되었다. 이렇게 수초화된 공동 배양은 Semliki forest virus에 의해 감염되었고 그 때 탈수초화 과정을 유발시켰다. 우리는 수초화된 뉴런에 존재하는 peripheral myelin protein 22의 항체를 이용하여 수초화 과정과 탈수초화 과정을 확인하였다.

• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2016년도 추계학술대회
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• pp.919-922
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• 2016

쥐의 배아의 척수신경절로 부터 슈반세포와 뉴런 세포가 각각 분리되어 배양되었다. in vitro 시스템에서 배양되었다. 정제된 뉴런 세포는 항 유사분열제로 처리하였고 정제한 슈반세포들과 함께 공동배양 하였다. 이 때 수초화 과정이 진행되었다. 이렇게 수초화된 공동 배양에 Herpes simplex virus-1을 감염시켜 탈수초화를 진행 시켰다. 우리는 수초화의 존재를 의미하는 neuropeptide Y의 항체를 이용하여 수초화와 탈수초화를 확인하였다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제28권5호
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• pp.375-395
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• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2015년도 춘계학술대회
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• pp.943-946
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• 2015

탈수초화를 위해 쥐의 배아(임신 16일)의 슈반세포와 뉴런 세포가 척수신경절로 부터 in vitro 시스템에서 배양되었다. 항 유사분열제로 첨가된 분리 정제된 척수신경절 세포와 분리 정제된 슈반세포가 공동배양 배양되었고 배양되었고 수초화 과정이 구축되었다. 이렇게 형성된 공동 배양에 M. leprae-specific phenolic glycolipid-1 (PGL-1)을 처리하고 myelin basic protein (MBP)의 항체를 이용하여 탈수초화가 형성되었음을 확인하였다.

• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2016년도 춘계학술대회
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• pp.718-721
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• 2016

쥐의 배아의 척수신경절로 부터 슈반세포와 뉴런 세포가 각각 배양되었다. in vitro 시스템에서 배양되었다. 항 유사분열제로 처리한 정제 뉴런 세포와 정제 슈반세포들이 공동배양 되었고 그 때 수초화 과정이 진행되었다. 수초화된 공동 배양에 Semliki forest virus를 감염시켜 탈수초화를 진행 시켰다. 우리는 수초화의 형성을 의미하는 neuropeptide Y의 항체를 이용하여 수초화와 탈수초화를 확인하였다.

• 대한한방내과학회지
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• 제33권1호
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• pp.83-101
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• 2012

Objectives : This study was aimed to investigate the therapeutic effect of Bogijetongtanggammi-bang (BJTG) on injury of the peripheral nerve tissues. Methods : Rats were divided into 2 groups. The rats of the first group were injected with Taxol (1.25 mg/kg) to their sciatic nerves, once each. The sciatic nerves of the rats of the second group were crushed by forcept for 30 seconds. Rats were administered with BJTG (400 mg/kg) or 0.9% saline for 5 days. Changes of DRG neurons, Schwann cells, Cdc2, caspase 3. phospho-p44/42 Erk1/2, phospho-vimentin and ${\beta}1$ integrin were observed by fluorescent microscope and analysed in western blot. Results : In Taxol-treated SD rat models, BJTG up-regulated neurite outgrowth, Schwann cells, Cdc2 and phospho-Erk1/2, and down-regulated caspase 3. In pressure-injured rat models, BJTG up-regulated axons of sciatic nerve, Schwann cells, Cdc2, phospho-vimentin, ${\beta}1$ integrin, and down-regulated caspase 3. Conclusions : Taken together, BJTG was promotive of nerve regeneration on SNI as well as Taxol-induced nerve injury. BJTG had a pharmaceutical property enhancing recovery of injured peripheral nerves and could be a candidate for drug development after further research.