• 한국발생생물학회지:발생과생식
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• 제12권1호
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• pp.87-95
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• 2008

만능성 인간 배아줄기세포로부터 확립된 신경줄기세포 또는 신경전구세포는 퇴행성 신경질환 세포치료제로 이용될 수 있는 다양한 종류의 신경세포로 분화 유도될 수 있다. 하지만, 인간 배아줄기세포로부터 신경세포를 생산하기 위한 기술은 아직 많은 장애를 가지고 있다. 인간 배아줄기세포 유래 신경전구세포에서 특징적으로 나타나는 신경관 유사로제트에 대한 이해는 인간 배아줄기세포 신경 분화의 효율을 높이는데 유용한 정보를 제공할 것으로 사료된다. 일반적으로 신경로제트(neural rosette)는 분화 중인 배아체를 부착 배양함으로써 유도하지만, 이 방법은 시간이 걸리고 복잡하다는 단점이 있다. 본 연구에서는 신경로제트가 부착배양을 하지 않고 부유배양으로 형성될 수 있는지 조사하였다. 우선적으로, 배아체 형성 및 신경분화에 인간 배아줄기세포 클럼프(clump) 크기가 영향을 주는지를 조사하였고, 사방 $500\;{\mu}m$ 크기의 인간 배아줄기세포 클럼프가 신경 분화 유도에 가장 효과적임을 확인하였다. 로제트 형성을 유도하기 위해, 사방 $500\;{\mu}m$ 크기의 인간 배아줄기세포 클럼프를 1주일 동안 EB 배양배지에 부유 배양함으로써 균일한 크기의 배아체를 얻은 후, NES 배양 배지에서 부가적으로 $1{\sim}2$주 동안 계속 부유 배양한 결과, $7{\sim}10$일 사이에 신경관 유사 로제트가 형성됨을 확인하였다. 로제트 형성 세포의 신경전구세포로서 특성은 RT-PCR과 면역형광염색법을 이용한 신경전구세포 특이적 마커(vimentivi, nestin, MSI1, MSI2, Sox1, Tuj1) 발현을 통해 확인하였다. 또한, 성장인자를 제외한 NES 배양 배지에서 신경로제트를 $2{\sim}6$주 동안 지속적으로 배양하면 성숙 신경세포로의 말단 분화가 유도됨을 확인하였다. 신경세포 특이적 마커(Tuj1, MAP2, GABA)와 신경아교 특이적 마커($S100{\beta}$, GFAP)는 $2{\sim}3$주 또는 4주 후에 각각 발현이 유도됨을 확인하였고, 희소 돌기아교 특이적 마커(O1과 CNPase)는 $5{\sim}6$주 후에 발현이 증가함을 확인하였다. 본 연구결과는 신경로제트가 부유 배양시스템에서 성공적으로 형성됨을 보여주고 있으며, 이는 인간 배아줄기세포의 신경 분화를 이해하고, 신경전구세포 유도 과정을 단순화하는데 효과적으로 이용될 수 있을 것으로 사료된다.

• 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.

• 대한후두음성언어의학회지
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• 제12권2호
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• pp.126-132
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• 2001

Background and Objectives : Signal traduction through phospholipase C(PLC) participate in the regulation of cell growth and differentiation. Growth factors bind to their receptors and thereby induce tyrosine phophorylation of the phospholipase C-${\gamma}$1(PLC-${\gamma}$1). PLC-${\gamma}$1 is a substrate for several receptor tyrosine kinases and its catalytic activity is increased by tyrosine phosphorylation. Tyrosine kinase phosphorylation of PLC-${\gamma}$1 stimulates PLC activation and cell proliferation. However the signal transduction pathway and the significance of PLC in injured recurrent laryngeal nerve regeneration is unknown. Therefore after we obtained fuctionally recovered rats using PEMF in this study, we attempt to provide some evidence that PLC plays a role in nerve regeneration itself and regeneration related to PEMF through the analysis of the difference between fucntional recovery group and non-recovery group in the recurrent laryngeal nerve. Materials and Method : Using 32 healthy male Sprague-Dawley rats, transections and primary anastomosis were performed on their left recurrent laryngeal nerves. Rats were then randomly assigned to 2 groups. The experimental group(n=16) received PEMS by placing them in custom cages equipped with Helm-holz coils(3hr/day, 5days/wk, for 12wk). The control group(n=16) were handled the same way as the experimental group, except that they did not receive PEMS. Laryngo-videoendoscopy was performed before and after surgery and followed up weekly. Laryngeal EMG was obtained in both PCA and TA muscles. Immunohistochemisty staining and Western blotting analysis using monoclonal antibody was performed to detect PLC-${\gamma}$1 in recurrent laryngeal nerve and nodose ganglion. Results : 10 rats(71%) in experimental group and 4 rats(38%) in the control group showed recovery of vocal fold motion. Functionally-recoverd rats show PLC-${\gamma}$1 positive cells in neuron and ganglion cells after 12 weeks from nerve injury. Conclusion : This study shows that PLC1-${\gamma}$ involved in singnal trasduction pathway in functinal recovery of injured recurrent laryngeal nerve and PEMF enhance the functional recovery by effect on this molecule.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제28권2호
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• pp.111-117
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• 2006

An area of current research is investigating the app1ication of human mesenchymal stem cells or hMSCs as a cell-based regenerative therapy. In order to achieve effective bone regeneration, appropriate matrices functioning as cell-carriers must be identified and optimized in terms of function, efficacy and biocompatibility. Two methods of approaching optimization of matrices are to facilitate adhesion of the donor hMSCs and furthermore to facilitate recruitment of host progenitor cells to osteoblastic differentiation. Pleiotrophin is an extracellular matrix protein that was first identified in developing rat brains and believed to be associated with developing neuronal pathways. A recent publication by Imai and colleagues demonstrated that transgenic mice with upregulated pleiotrophin expression developed a greater volume of cortical as well as cancellous bone. The proposed mechanism of action of pleiotrophin is demonstrated here. Through either environmental stresses and/or intracellular regulation, there is an increase in pleiotrophin production. The pleiotrophin is released extracellularly into areas requiring bone deposition. A receptor-mediated process recruits host osteoprogenitor cells into these areas. Therefore, the aim of our study was to investigate the osteoconductive properties of pleiotrophin. We wanted to determine if pleiotrophin coating facilitates cellular adhesion and furthermore if this has any effect on hMSCs derived bone formation in an animal model. The results showed a dose dependent response of cellular adhesion in fibronectin samples, and cellular adhesion was facilitated with increasing pleiotrophin concentrations. Histologic findings taken after 5 weeks implantation in SCID mouse showed no presence of bone formation with only a dense fibrous connective tissue. Possible explanations for the results of the osteogenesis assay include inappropriate cell loading.

• Molecules and Cells
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• 제38권7호
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• pp.663-668
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• 2015

hBMSCs are multipotent cells that are useful for tissue regeneration to treat degenerative diseases and others for their differentiation ability into chondrocytes, osteoblasts, adipocytes, hepatocytes and neuronal cells. In this study, biodegradable elastic hydrogels consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(${\varepsilon}$-caprolactone) (PCL) scaffolds were evaluated for tissue engineering because of its biocompatibility and the ability to control the release of bioactive peptides. The primary cultured cells from human bone marrow are confirmed as hBMSC by immunohistochemical analysis. Mesenchymal stem cell markers (collagen type I, fibronectin, CD54, $integrin1{\beta}$, and Hu protein) were shown to be positive, while hematopoietic stem cell markers (CD14 and CD45) were shown to be negative. Three different hydrogel scaffolds with different block compositions (PEG:PCL=6:14 and 14:6 by weight) were fabricated using the salt leaching method. The hBMSCs were expanded, seeded on the scaffolds, and cultured up to 8 days under static conditions in Iscove's Modified Dulbecco's Media (IMDM). The growth of MSCs cultured on the hydrogel with PEG/PCL= 6/14 was faster than that of the others. In addition, the morphology of MSCs seemed to be normal and no cytotoxicity was found. The coating of the vascular endothelial growth factor (VEGF) containing scaffold with Matrigel slowed down the release of VEGF in vitro and promoted the angiogenesis when transplanted into BALB/c nude mice. These results suggest that hBMSCs can be supported by a biode gradable hydrogel scaffold for effective cell growth, and enhance the angiogenesis by Matrigel coating.

• Biomolecules & Therapeutics
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• 제18권1호
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• pp.39-47
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• 2010

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor involved in neuronal differentiation, plasticity, survival and regeneration. BDNF draws massive attention mainly due to the potential as a therapeutic target in neurological diseases such as depression and Alzheimer's disease. In a primary screening for the natural compounds enhancing BDNF release from cultured rat primary cortical neuron, we found that compounds such as baicalein, tanshinone IIa, cinnamic acid, epiberberine, genistein and wogonin among many others increased BDNF release. All the compounds at $0.1{\mu}M$ of concentration barely showed stimulatory effect on BDNF induction, however, their combination (mixture 1; baicalein, tanshinone IIa and cinnamic acid, mixture 2; epiberberine, genistein and wogonin) showed synergistic increase in BDNF release as well as mRNA and protein expression. The level of BDNF expression was comparable to the maximum BDNF stimulation attainable by a positive control oroxylin A ($20{\mu}M$) without cell toxicity as determined by MTT analysis. Both mixtures synergistically increased the phosphorylation of extracellular signal-regulated kinase (ERK) as well as cAMP response element binding protein (CREB), an immediate and essential regulator of BDNF expression. Similar to these results, mixture of these compounds synergistically inhibited the up-regulation of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide treatments in rat primary astrocytes. These results suggest that the combinatorial treatment of natural compounds in lower concentration might be a useful strategy to obtain sufficient BDNF stimulation in neurological disease condition such as depression, while minimizing potential side effects and toxicity of higher concentration of a single compound.

• Journal of Periodontal and Implant Science
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• 제27권2호
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• pp.425-441
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• 1997

The neuropeptide substance P(SP) has been implicated in the mediation of inflammation and immune-mediated disease such as arthritis. Recently, it was reported that SP was markedly increased around the blood vessels in inflamed gingiva as well as in close association with the inflammatory cell infiltrate. These results support that SP may contribute to the pathophysiology of neuronal inflammation in human periodontal tissues. SP may regulate inflammatory/immune responses by stimulating the proliferation of human T cells, differentiation and antibody-secreting potential of B cells, macrophage respiratory burst, connective tissue proliferation, and the secretion of cytokines from monocytes and T cells. Here, I studied potential role of SP as a costimulatory chemical signal in inflammatory/immune responses, by determining the released proinflammatory cytokines such as $MIP-1{\alpha}$, $IL-1{\beta}$, and IL-6 from culture supernatants of homogeneous immune cell lines. Serum free cell supernatants were concentrated with TCA precipitation, fractionated with SDS-PAGE, and subjected into western blot analysis. Among 15 cell lines tested, macrophage/monocyte cell line RAW264.7 and WRl9m.1 showed the highest level of induction of $MIP-1{\alpha}$ when stimulated with LPS. Discrete IL-6 bands with multiple forms of molecular mass were detected from supernatants of B cell lines A20(32kDa), Daudi(32, 35kDa), and SKW6.4(29kDa), which were expressed constitutively. $IL-1{\beta}$ could not be detected by the method of western blot analysis from supernatants of all cell lines tested except RAW264.7, WRl9m.1, and erythroid cell line K562 which showed the least amount of $IL-{\beta}$ secretion. SP $10^{-9}M$ with suboptimal dose of LPS treatment showed synergistic induction of $MIP-1{\alpha}$ release from RAW264.7 or WR19m.1, and also IL-6 release from A20, but this synergism is not the case in costimulation of RAW264.7 or WRl9m.1 with SP $10^{-9}M$ and TPA. Although treatment of T cell line CTLL-R8 with SP $10^{-7}M$ or PHA+TPA induced modest level of $MIP-1{\alpha}$ secretion, synergism was not observed when they are applied together. These findings all together suggest the possibility of a regulatory role of SP in inflammatory/immune reaction through differential modulation of bioactivities of other chemical cosignals.

• Investigative Magnetic Resonance Imaging
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• 제16권2호
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• pp.124-135
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• 2012

목적: 본 연구에서는 숙달된 택시운전기사들을 대상으로 내비게이션 시스템을 이용하여 운전하는 한국인 대조군에 비교하여 해마의 부피와 모양 그리고 기능적 변화를 알아보고자 하였다. 대상과 방법: 총 8명의 대상군(4명의 숙달된 택시운전기사들과 4명의 같은 나이대 일반인 대조군)에 대해 삼차원 T1강조영상과 혈류산소 수준에 따른 기능성 자기공명영상을 촬영하였다. 해마의 용적과 모양, 기능성 자기공명영상 자료를 분석하여 두 군간에 신경 활성도 차이를 비교하였다. 결과: 해마의 용적은 두 군간에 통계적으로 유의한 차이를 보이지 않았다(p > 0.05). 해마의 모양을 보면, 택시운전기사군의 왼쪽 해마가 대조군에 비해 전체 길이가 약간 길고 머리와 꼬리부분이 약간 더 컸다(p < 0.05, uncorrected). 기능성 자기공명영상에서는 택시운전기사군에서 대조군에 비해 방추상회가 특히 활성화되어 있었다. 결론: 택시운전기사군의 해마가 보이는 이러한 구조적, 기능적 변화는 그들의 직업과 관련하여 공간적 탐색능력을 지속적으로 사용하면서 해마와 관련 뇌영역이 기능적으로 분화했음을 나타내는 소견으로 추측할 수 있겠다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제27권5호
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• pp.446-456
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• 2005

Nerve growth factor(NGF) has a critical role in peripheral nerve regeneration. The aim of this study is to construct a well-functioning hNGF-$\beta$ recombinat adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with adenovirus mediated hNGF-$\beta$ gene transfection into Schwann cells. First PCR associated cloning of GFP-tagged hNGF-$\beta$ which was ligated into E1/E3 deleted adenoviral vector was performed and tranfected into E. coli to construct hNGF-$\beta$ recombinant adenovirus. After production of recombinat adenovirus in a large scale, its transfection efficiency, expression, and function were evaluated using cell lines or primarily cultured cells of HEK293 cells, Schwann cells, fibroblast(NIH3T3) and myocyte(CRH cells). GFP expression was observed in 90% of infected cells compared to uninfected cells. Total mRNA isolated from hNGF-$\beta$ recombinat adenoviru infected cells showed strong RT-PCR band, however, LacZ recombinant adenovirus infected or uninfected cells did not. NGF quantification by ELISA showed a maximal release of 18.865 +/- 0.31ng/mL at 4th day. PC-12 cells exposed to media with hNGF-$\beta$ recombinant adenovirus infected Schwann cell demonstrated higher levels of differentiation compared with controls. We generated hNGF-$\beta$ recombinant adenovirus and induced over expression of NGF successfully in nonneuronal and neuronal cells. Following these result, it is expected to develop an improved treatment strategy peripheral nerve regeneration using the hNGF-$\beta$ gene transfected cells.

• Biomolecules & Therapeutics
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• 제20권1호
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• pp.27-35
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• 2012

Oroxylin A is a flavone isolated from a medicinal herb reported to be effective in reducing the inflammatory and oxidative stresses. It also modulates the production of brain derived neurotrophic factor (BDNF) in cortical neurons by the transactivation of cAMP response element-binding protein (CREB). As a neurotrophin, BDNF plays roles in neuronal development, differentiation, synaptogenesis, and neural protection from the harmful stimuli. Adenosine $A2_A$ receptor colocalized with BDNF in brain and the functional interaction between $A2_A$ receptor stimulation and BDNF action has been suggested. In this study, we investigated the possibility that oroxylin A modulates BDNF production in cortical neuron through the regulation of $A2_A$ receptor system. As expected, CGS21680 ($A2_A$ receptor agonist) induced BDNF expression and release, however, an antagonist, ZM241385, prevented oroxylin A-induced increase in BDNF production. Oroxylin A activated the PI3K-Akt-GSK-$3{\beta}$ signaling pathway, which is inhibited by ZM241385 and the blockade of the signaling pathway abolished the increase in BDNF production. The physiological roles of oroxylin A-induced BDNF production were demonstrated by the increased neurite extension as well as synapse formation from neurons. Overall, oroxylin A might regulate BDNF production in cortical neuron through $A2_A$ receptor stimulation, which promotes cellular survival, synapse formation and neurite extension.