• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.33-40
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• 2001

Objective: This study was to establish the human embryonic stem (ES) cells derived from frozen-thawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. Methods: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. Results: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and ${\beta}$-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. Conclusion: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.

• Journal of Oriental Physiology
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• v.14 no.2 s.20
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• pp.117-126
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• 1999

The purpose of this study is to examine the toxic effects caused by Glucose Oxidase(GO) and the effects of herbal extracts such as Acori Rhizoma(AR) on the treatment of the toxic effects. For this purpose, experiments with the cultured cell from the cerebrums of new born mice were done. The results of these experiments were as follows. 1. GO, a oxygen radical, decreased the survival rate of the cultured cells on NR assay and MTT assay. 2. GO, a oxygen radical, decreased the amount of neurofilaments and total protein. 3. AR have efficacy of increasing the amount of neurofilament. 4. AR have efficacy of increasing the amount of total protein. From the above results, It is concluded that AR has marked efficacy as a treatment for the damages caused in the GO-mediated oxidative process. And AR is thought to have certain pharmacological effects on controlling over aging. Further clinical study of this pharmacological effects of AR should be complemented.

• Journal of Korean Neurosurgical Society
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• v.64 no.5
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• pp.705-715
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• 2021

Objective : Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3-asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. Methods : Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9-10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×10⁵ cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. Results : Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. Conclusion : Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.51-51
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• 2001

Cocaine and amphetamine-regulated transcript (CART) is a satiety factor that is regulated by leptin. It was reported that the mice intracerebroventricularly injected with CART showed behavioral changes resembled with the typical behavioral alterations found in the mice carrying disorders in the brain serotonergic (5-HT) system. Hence, this study was conducted to find out the relationships between CART and 5-HT. We first examined the mRNA levels of CART after the injections of para-chlorophenylalanine (pCPA, 300 mg/kg i.p., single injection or daily for three consecutive days) in the rat brains by in situ hybridization using the mouse CART cDNA probe cloned in our laboratory. Systemic administrations of pCPA, a potent inhibitor of tryptophan hydroxylase, the rate limiting enzyme of 5-HT biosynthesis, acutely depletes the brain 5-HT transporter (5-HTT) in the dorsal raphe nucleus (DRN), which reuptakes terminal 5-HT. Results indicated that the mRNA level of CART significantly decreased in the arcuate nucleus, paraventricular nucleus, and lateral hypothalamic nucleus by three days of daily injection with pCPA with no noticeable change detected 24 hrs after the single injection. The message levels of 5-HTT in DRN decreased in both single and three days of injections. Secondly, to investigate whether CART affect to 5-HT, mouse genomic CART gene, which is consist of 3 exons and 2 introns and mouse neurofilament light (NF-L) chain promoter were cloned. Then, we constructed neuron specific expression vector, which was transfected into HeLa cell using lipid-mediated transfection system. Expression of GFP and CART linked to NF-L-chain promoter in the transfected HeLa cell were detected by using fluorescent microscope and RT-PCR. These results confirmed normal expression of DNA constructs in vitro. Then, to increase brain specific expression of CART in vivo transgenic mice carrying CART gene controlled the deleted NF-L-chain promoter were generated by the DNA microinjection into pronuclei of fertilized embryos. Transgenic mice were detected by Southern blot. Further study is necessary to examine CART expression and 5-HTT in these transgenic mice. Therefore, these results suggest that there maybe a positive molecular correlation between CART and 5-HT in responding to the stimuli.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.389-397
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• 2001

Cocaine-amphetamine regulated transcript (CART), a satiety factor regulated by leptin, is associated with food intake and motor behavior. In knock out studies, Leu34Phe mutation of human CART gene resulted in obese phenotype but mice carrying a targeted deletion of the CART gene exhibited no dramatic increase of body weight on normal fat diet. To establish a new transgenic mouse model for determining the function of CART on feeding behavior in vivo, we constructed the fusion gene, CART gene under the control of neurofilament light chain promoter, which regulates gene expression at the stage of neuronal differentiation. Transgenic mice were generated by microinjection method and screened by PCR and Southern blot analyses. In these transgenic mice, overexpression of CART was detected by in situ hybridization in spinal cords and brains at 13.5 days post-coitum embryos. At six weeks of age, RT-PCR analysis showed that exogenous CART mRNA was expressed strongly in brains and spinal cords, but not much in other tissues. Our results suggest that these transgenic mice provide a new model to investigate the function of CART gene in neuronal network associated with feeding behavior.

• Biomolecules & Therapeutics
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• v.32 no.1
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• pp.154-161
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• 2024

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a nonessential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [¹⁴C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1^G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [¹⁴C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [¹⁴C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [¹⁴C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.15 no.4
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• pp.281-301
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• 1993

It was revealed that the morphology and projection pattern of terminal arbors from single primary afferent are different among distinct fiber types, functional types and the different subdivision of trigeminal sensory nucleus complex(TSNC). But it was not identified the ultrastructural morphology and synaptic connections of terminal arbors from each primary afferent within TSNC. So we employed the intra-axonal horseradish peroxidase(HRP) injection technique to define the terminal arbors of primary afferent fiber from slowly adapting mechanoreceptors in the periodontal ligament of the cat, and examined 66 labeled terminal arbors within the rostrodorsomedial part(Vo.r) of the trigeminal nucleus oralis, electromicroscopically with 90nm serial sections. All the boutons labelled with HRP contained clear, spherical and uniform sized synaptic vesicles(diameter : $47.66{\pm}3.58nm$ ). Most of the labelled boutons were boutons en passant type and they were connected by unmyelinated axonal strand. In which neurofilament and microtubule was not developed but occasionally contained synaptic vesicle in contrast to the myelinated axon. The size of the labelled bouton was relatively small(long diameter : $1.46{\pm}0.24{\mu}m$, short diameter $0.85{\pm}0.26{\mu}m$, average diameter $1.15{\pm}0.24{\mu}m$) and the shape of which varied from dome to elongated shape, but scalloped glomerulus shape was not developed. Each primary ending in Vo.r made synapse with one or two neuronal propiles(average : $1.11{\pm}0.31$), of which, 89.4% of labelled boutons made synapse with only one neuronal pro pile, the remainder, 10.6% of labelled boutons, made synapse with two neuronal propile. So characteristically they made very simple synapse. Most of labelled boutons(80.03%) made asymmetrical synapse only with dendritic shaft or spine, and 6.1% of labelled boutons received symmetrical synapse from pleomorphic vesicle containing axonal ending(p-ending). So presynaptic inhibiton was relatively scarce. Synaptic triad, in which a p-ending is presynaptic both pre-and post-synaptic element of the axo-dendritic contact from the labelled primary ending was not observed.

• Applied Microscopy
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• v.32 no.1
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• pp.67-80
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• 2002

This study was carried out to investigate the effects of deer antler extract on the regeneration of peripheral nerves. Sprague-Dawley male rats weighing about 300 gm were fed deer antler extract for 1, 2, and 3 weeks per oral (1.5 ml/100 gm B.W.), respectively, once a day and transected both sides of sciatic nerve of each leg. After keeping for 6 hours, sciatic nerves taken from proximal part of transected region were treated with conventional transmission electron microscopical method and then observed with electron microscope. The results obtained were summarized as follows; 1. Sciatic nerves of normal control group were not showing any sprouts and electron dense axolemmal projections were frequently observed. 2. Sciatic nerves of saline treated groups were showing axonal sprouts at the nodes of Ranvier. The length of them was usually short, and numerous vesicles, vacuoles and organelles including neurofilament were contained. The number of nodes of Ranvier containing sprouts from 100 longitudinal sectioned nerve fibers was 29 (29%) in 1 week treated group, 32 (32%) in 2 weeks treated group, and 30 (30%) in 3 weeks treated group, respectively. 3. Sciatic nerves of deer antler treated groups were showing axonal sprouts at the node of Ranvier as well. Although most of the sprouts were short, some sprouts of 2 weeks and 3 weeks treated groups were quite long. Sprouts usually contained numerous vesicles, vacuoles and cell organelles such as neurofilaments and mitochondria. The number of nodes of Ranvier containing sprouts from 100 longitudinal sectioned nerve fibers was 38 (38%) in 1 week treated group, 46 (46%) in 2 weeks treated group, and 48 (48%) in 3 weeks treated group respectively. The results described above explain pretreatment of deer antler extract improves the sprout formation of transected sciatic nerves, and then it suggests deer antler may be effective for the regeneration of peripheral nerves.