• 대한생식의학회:학술대회논문집
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• 대한불임학회 2006년도 The 5th Biannual Meeting of Pacific Rim Society for Fertility and Sterility
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• pp.170.1-170.1
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• 2006

• 한국발생생물학회지:발생과생식
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• 제12권1호
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• pp.1-8
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• 2008

배아줄기세포는 다양한 분화 유도 방법을 통해 신경계세포로 분화시킬 수 있을 뿐만 아니라, 보다 더 엄격한 선발조건을 적용함으로써 특정 종류의 신경세포만을 확보할 수도 있게 되었다. 세포사멸연구를 포함한 신경생리학적 연구의 대상으로써 중요한 요건은 이렇게 확보한 배아줄기세포 유래의 신경계세포들이 정상적인 신경생리학적 특성을 갖고 있어야 하며, 동시에 그런 신경생리학적 특성이 체외에서 일정기간 동안 이상 자연적인 세포사멸없이 유지되어야 한다는 것이다. 생쥐 배아줄기세포를 retinoic acid로 처리한 후, astrocytes monolayer 위에서 신경계세포로 분화시키면 장기간 생존이 가능한 다수의 신경계세포를 손쉽게 확보할 수 있을 뿐만 아니라, 면역세포화학적 방법을 통해 신경세포의 생사를 개별세포 수준에서 추적할 수 있다. 배아줄기세포 유래의 신경계세포는 glutamate agonist들에 대해 수용체 특이적 흥분성 신경독성 반응을 보이며, 이 반응은 신경계세포로의 분화가 진행될수록 더욱 뚜렷해지는 양상을 보인다. 신경계세포의 발생분화, 생존에 관여하는 Neurotrophin, GDNF 계열의 신경계 작용 성장인자들의 수용체가 배아줄기세포 유래의 신경계세포에서 발현되고 있으며, 이들에 의해 신경계세포로의 분화과정에서 세포의 생존능 및 신경독성처리에 대한 세포사멸반응이 조절될 수도 있다. 따라서 배아줄기세포 유래의 신경계세포는 신경세포의 생존과 사멸, 그리고 세포손상으로부터의 보호와 같은 신경약리학적 연구를 위한 중요한 특성을 나타내고 있기에 관련 연구를 위한 새로운 연구시스템이 될 수 있는 것이다. 특히 일반적인 신경세포는 유전적 변형이 어려워 다양한 신경약리학적 연구에 많은 제약을 받아 왔으나, 이제 유전자 변형 배아줄기세포로부터 얻은 신경세포를 활용하여 연구할 수 있게 됨으로써, 보다 복합적인 신경약리학적 기초연구도 가능하게 되었다. 특히 최근 인간 배아줄기세포 유래 신경계세포도 유사한 신경독성 반응을 보이고 있음이 확인됨으로써(Schrattenholz & Klemm, 2007), 이제 배아줄기세포는 신경약리학적 기초 연구만이 아닌, 나아가 대량의 약물 스크리닝과 같은 제약산업에도 활용될 수 있는 가능성을 제시하고 있다.

• Journal of Dental Anesthesia and Pain Medicine
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• 제17권3호
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• pp.191-198
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• 2017

Background: For peripheral nerve regeneration, recent attentions have been paid to the nerve conduits made by tissue-engineering technique. Three major elements of tissue-engineering are cells, molecules, and scaffolds. Method: In this study, the attachments of nerve cells, including Schwann cells, on the nerve conduit and the effects of both growth factor and adhesion molecule on these attachments were investigated. Results: The attachment of rapidly-proliferating cells, C6 cells and HS683 cells, on nerve conduit was better than that of slowly-proliferating cells, PC12 cells and Schwann cells, however, the treatment of nerve growth factor improved the attachment of slowly-proliferating cells. In addition, the attachment of Schwann cells on nerve conduit coated with fibronectin was as good as that of Schwann cells treated with glial cell line-derived neurotrophic factor (GDNF). Conclusion: Growth factor changes nerve cell morphology and affects cell cycle time. And nerve growth factor or fibronectin treatment is indispensable for Schwann cell to be used for implantation in artificial nerve conduits.

• The Korean Journal of Physiology and Pharmacology
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• 제8권5호
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• pp.245-251
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• 2004

The effects of fetal mesencephalic cell grafts on the restoration of nigrostriatal dopaminergic function were studied in the intrastriatal 6-hydroxydopamine-lesioned rats. Four weeks after lesioning, transplantation of ventral mesencephalic cells from embryonic day 14 fetuses showed the number of tyrosine hydroxylase (TH) positive cells and fiber outgrowth in the grafted striatum, and significantly ameliorated symptomatic motor behavior of the animals, as determined by apomorphine-induced rotation. Furthermore, in substantia nigra pars compacta (SNc), the numbers of TH + cells and fibers were markedly restored. Dopamine content of ipsilateral SNc was close to that of contralateral SNc $(91.9{\pm}9.8%)$ in the transplanted animals, while the ratio was approximately 32% in sham-grafted animals. These results indicate that grafted cells restored the activity for the dopaminergic neurons located in SNc, although they were transplanted into striatum. In addition, we showed that the implanted fetal cells expressed high level of glial cell line-derived neurotrophic factor (GDNF), suggesting that the transplanted fetal cells might serve as a dopamine producer and a reservoir of neurotrophic factors. These results may be helpful in consideration of the therapeutic transplantation at early stage of PD.

• BMB Reports
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• 제51권11호
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• pp.590-595
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• 2018

Parkinson's disease (PD) is a common chronic neurodegenerative disease mainly caused by the death of dopaminergic neurons. However, no complete pharmacotherapeutic approaches are currently available for PD therapies. 1-methyl-4-phenylpyridinium $(MPP^+)$-induced SH-SY5Y neurotoxicity has been broadly utilized to create cellular models and study the mechanisms and critical aspects of PD. In the present study, we examined the role of a novel azetidine derivative, 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792), against $MPP^+$-induced neurotoxicity in SH-SY5Y cells. Treatment of KHG26792 significantly attenuated $MPP^+$-induced changes in the protein levels of Bcl-2 and Bax together with efficient suppression of $MPP^+$-induced activation of caspase-3 activity. KHG26792 also attenuated mitochondrial potential and levels of ROS, $Ca^{2+}$, and ATP in $MPP^+$-treated SH-SY5Y cells. Additionally, KHG26792 inhibited the induced production of nitric oxide and malondialdehyde. Moreover, the protective effect of KHG26792 is mediated through regulation of glutathione peroxidase and GDNF levels. Our results suggest a possibility that KHG26792 treatment significantly protects against $MPP^+$-induced neurotoxicity in SH-SY5Y cells and KHG26792 may be a valuable therapeutic agent for the treatment of PD induced by an environmental toxin.

• The Korean Journal of Physiology and Pharmacology
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• 제12권2호
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• pp.37-41
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• 2008

Melatonin has been reported to protect neurons from a variety of neurotoxicity. However, the underlying mechanism by which melatonin exerts its neuroprotective property has not yet been clearly understood. We previously demonstrated that melatonin protected kainic acid-induced neuronal cell death in mouse hippocampus, accompanied by sustained activation of Akt, a critical mediator of neuronal survival. To further elucidate the neuroprotective action of melatonin, we examined in the present study the causal mechanism how Akt signaling pathway is regulated by melatonin in a rat primary astrocyte culture model. Melatonin resulted in increased astrocytic Akt phosphorylation, which was significantly decreased with wortmannin, a specific inhibitor of PI3K, suggesting that activation of Akt by melatonin is mediated through the PI3K-Akt signaling pathway. Furthermore, increased Akt activation was also significantly decreased with luzindole, a non-selective melatonin receptor antagonist. As downstream signaling pathway of Akt activation, increased levels of CREB phoshorylation and GDNF expression were observed, which were also attenuated with wortmannin and luzindole. These results strongly suggest that melatonin exerts its neuroprotective property in astrocytes through the activation of plasma membrane receptors and then PI3K-Akt signaling pathway.

• 한국수정란이식학회지
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• 제29권3호
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• pp.221-228
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• 2014

Despite that porcine spermatogonial stem cells (pSSCs) have been regarded as a practical tool for preserving eternally genetic backgrounds derived from pigs with high performance in the economic traits or phenotypes of specific human diseases, there were no reports about precise definition of niche conditions promoting proliferation and maintenance of pSSCs. Accordingly, we tried to determine niche conditions supporting proliferation and maintenance of undifferentiated pSSCs for short-term. For these, undifferentiated pSSCs were progressively cultured in different composition of culture medium, seeding density of pSSCs, type of feeder cells and concentration of growth factors, and then total number of and alkaline phosphatase (AP) activity of pSSCs were investigated at post-6 day culture. As the results, the culture of $4{\times}10^5$ pSSCs on mitotically in activated $2{\times}10^5$ STO cells in the mouse embryonic stem cell culture medium (mESCCM) supplemented with 30 ng/ml glial cell line-derived neurotrophic factor (GDNF) was identified as the best niche condition supporting effectively the short-term maintenance of undifferentiated pSSCs. Moreover, the optimized short-term culture system will be a basis for developing long-term culture system of pSSCs in the following researches.

• 생명과학회지
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• 제19권4호
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• pp.449-456
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• 2009

신경아세포종은 미분화된 신경외배엽 세포로부터 유래한 신경능세포에 의해 형성된 소아기에 보는 가장 많이 발생하는 악성 종양 중 하나이다. 신경아세포종인 Neuro-2a 세포는 신경세포의 분화, 세포사 억제 효능, 세포독성 검정 등에 활용되고 있다. Neuro-2a 역시 다른 신경아세종과 같이 염색체 변이를 가지고 있지만, 이에 대해 고밀도의 게놈수준에서 염색체 변이에 대해 보고된 바가 없다. 본 연구에서는 고집적 마이크로어레이(최소 43,000 개의 코딩, non-코딩 유전자 서열이 집적된 마이크로어레이)기반의 비교유전체보합법을 활용하여, 고해상도의 Neuro-2a 유전체 이상을 분석하였다. 마이크로 어레이 데이터는 Hidden Markov Model을 활용하여, 유전체 변이를 double loss, single loss, normal, single gain 그리고 amplification으로 나누어 분석하였다. Neuro2a는 MYCN 유전자의 증폭은 관찰되지 않았고, GDNF, BDNF, NENF등의 neurotrophic factor 가운데 NENF의 gain 현상이 관찰 되었다. 염색체의 이상은 4,8,10,11,15번에서 발견되었으며, 염색체 3,17,18,19에서는 전부 20개 미만의 염색체 이상이 발견되었다. 염색체 이상이 연속적으로 일어난 부위 중 gain으로서 가장 긴 부분은 Chr8:8,427,841-35,162,415의 약 26.7 Mb이며, single loss로서 가장 긴 곳은 Chr4:73,265,785-88,374,165의 약 15.1 Mb였다. 염색체의 위치는 UCSC 데이터베이스 (UCSC mm8, NCBI Build 36)에 근거하였다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제31권3호
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• pp.199-218
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• 2005

Purpose of Study: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats. Materials and Methods: Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with ${\beta}$-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells$(1{\times}10^6)$ or BDNF-Ad infected Schwann cells$(1{\times}10^6)$ were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis. Results: 1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were $1.54{\pm}4.0{\times}10^6$ and $9.66{\pm}9.6{\times}10^6$. 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell $0.69\;{\mu}g/{\mu}l$ of DNA was detected and in BDNF-Adenovirus transfected Schwann cell $0.795\;{\mu}g/{\mu}l$ of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal. Conclusion: BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.