• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.95-95
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• 2002

Human embryonic stem (hES) cells can be induced to differentiate into tyrosine hydroxylase expressing (TH+) cells that may serve as an alternative for cell replacement therapy for Parkinson's disease (PD). To examine in vitro differentiation of hES (MB03, registered in NIH) cells into TH+ cells, hES cells were induced to differentiate according to the 4-/4+ protocol using retinoic acid (RA), ascorbic acid (AA), and/or lithium chloride (LiCl) followed by culture in N2 medium for 14 days, during which time the differentiation occurs. Immunocytochemical stainings of the cells revealed that approximately 21.1% of cells treated with RA plus AA expressed TH protein that is higher than the ratio of TH+ cells seen in any other treatment groups (RA, RA+LiCl or RA+AA+LiCl). In order to see the differentiation pattern in vivo and the ability of in vitro differentiation-induced cells in easing symptomatic motor function of PD animal model, cells (2 $\times$ 10$^{5}$ cells/2${mu}ell$) undergone 4-/4+ protocol using RA plus AA without any further treatment were transplanted into unilateral striatum of MPTP-lesioned PD animal model (C57BL/6). Following the surgery, motor behavior of the animals was examined by measuring the retention time on an accelerating rotar-rod far next 10 weeks. No significant differences in retention time of the animals were noticed until 2 weeks post-graft; however, it increased markedly at 6 weeks and 10 weeks time point after the surgery. Immunohistochemical studies confirmed that a reasonable number of TH+ cells were found at the graft site as well as other remote sites, showing the migrating nature of embryonic stem cells. These results suggest that in viかo differentiated hES cells relieve symptomatic motor behavior of PD animal model and should be considered as a promising alternative for the treatment of PD.

• The Korean Journal of Physiology and Pharmacology
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• 제13권3호
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• pp.251-256
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• 2009

Previous observations suggest that Bis, a Bcl-2-binding protein, may playa role the neuronal and glial differentiation in vivo. To examine this further, we investigated Bis expression during the in vitro differentiation of P19 embryonic carcinoma cells induced by retinoic acid (RA). Western blotting and RT-PCR assays showed that Bis expression was temporarily decreased during the free floating stage and then began to increase on day 6 after the induction of differentiation. Double immunostaining indicated that Bis-expressing cells do not express several markers of differentiation, including NeuN, MAP-2 and Tuj-1. However, some of the Bis-expressing cells also were stained with GFAP-antibodies, indicating that Bis is involved glial differentiation. Using an shRNA strategy, we developed bis-knock down P19 cells and compared them with control P19 cells for the expression of NeuroD, Mash-1 and GFAP during RA-induced differentiation. Among these, only GFAP induction was significantly attenuated in Pl9-dnbis cells and the population showing GFAP immunoreactivity was also decreased. It is noteworthy that distribution of mature neurons and migrating neurons was disorganized, and the close association of migrating neuroblasts with astrocytes was not observed in P19-dnbis cells. These results suggest that Bis is involved in the migration-inducing activity of glial cells.

• Journal of Acupuncture Research
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• 제27권6호
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• pp.31-42
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• 2010

Objectives : The aim of this study was to investigate the anti-obesity potential and mechanisms of action of Rhizoma Atractylodis(RA) herbal acupuncture in high fat diet- induced obese ICR mice. Methods : Sample solutions for herbal acupuncture were prepared from the Rhizoma Atractylodis water extract powder at concentration of 150mg/kg and 300mg/kg with distilled water. Five week-old ICR mice acclimatized to the laboratory environment for 1 week were allocated into four groups: regular diet group (RD), high fat diet group(HFD), groups fed HFD with 150mg/kg RA herbal acupuncture treatment (RAE 150) and with 300mg/kg RA herbal acupuncture treatment(RAE 300). Herbal acupuncture groups were injected with either 150mg/kg or 300mg/kg of Rhizoma Atractylodis(RA) subcutaneously onto both Sinsu($BL_{23}$) alternately on the same time everyday for 30days. Body weight, gross appearance of epididymal fat area, blood glucose, insulin, insulin resistance(HOMA-IR), non-esterified fatty acid, cholesterol, triglyceride, AST, ALT, histological analysis of white adipose tissue, gene expression responsible for adipocyte differentiation and AMPK activation were analyzed. Results : RA herbal acupuncture inhibited the development of weight gain, hyperglycemia, hyperinsulinemia, hyperlipidemia, increases of AST and ALT, and the enlargement of fat cell size induced by HFD. Also, RA herbal acupuncture inhibited the expression of PPAR-${\gamma}$, C/$EBP{\alpha}$, aP2, LPL, FAS, SCD-1 and enhanced the activation of AMP-activated protein kinase. Conclusions : The results of this study demonstrate that RA herbal acupuncture can exert the anti-obesity effect and it is partially mediated by activation of AMPK and inhibition of the gene expressions responsible for adipocyte differentiation. Further studies will be required to ascertain the nti-obesity effect and mechanisms of action of RA herbal acupuncture in animal models and human for aclinical application.

• Journal of Acupuncture Research
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• 제24권6호
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• pp.1-14
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• 2007

Backgrounds : Rheumatoid Arthritis(RA) is known as the chronic inflammatory diseasethat induces persistent inflammation in the joint cavity. The destruction of cartilage occurs as the result of bones destoyed by pannus, several influential cytokines induced by the synovial capsulitis, varieties of proteinases, $O_2$ radicals, and the secondary degenerative changes of articular cartilage. The type 2 collagen-induced arthritis model is used in recent experimental research on rheumatoid arthritis. Cervus elaphus sibiricus (Nockyong) has the effect of relieving pain by nourishing the muscles, joints, and bones. It is also known to be efficacious in promoting and enhancing the immune system. The objective of this study was to investigate the effect of Cervus elaphus sibiricus herbal acupuncture to inhibit the generation of proinflammatory enzyme on type 2 collagen-induced arthritis. I investigated the inhibition of mRNA transcription of MIF(macrophage migration inhibitory factor), $TNF-{\alpha}$(Tumor necrosis $factor-{\alpha}$) and MMP-9 (matrix metalloproteinase-9) of Cervus elaphus sibiricus herbal acupuncture using an in vitro test. Also investigated was the inhibition of differentiation of Th 1 cells and activation of cytokines(MIF, $TNF-{\alpha}$, IL-6, MMP-9), which are known to cause initial RA ,and are also related to the morphology of the synovial membranes of the joint capsule, by an in vivo test, using CIA(collagen induced arthritis) model mice. Materials & methods : The laboratory animals used in this experiment were 4 week-old DBA female mice, weighing approximately 20 grams, and adjusted to the laboratory environment. The experiment was divided into the normal group(NOR)-no treated group, control group(CON)-CIA induced group, and sample group(SAM)-Cervus elaphus sibiricus herbal acupuncture treated group. RA was induced in the mice via injection of $50{\mu}{\ell}$ C II mixed CFA. The Cervus elaphus sibiricus herbal acupuncture solution was applied on $GB_{35}$(陽陵泉) for 26 days from the 3rd day of RA inducement. The concentration of the solution was determined via a MTT assay. To research the effect on the expression of MIF, $TNF-{\alpha}$ and MMP-9 mRNA, RT-PCR was performed on synovial membrane cells from the knee joint of CIA mice. C II induced RA knee joint's histo-chemical synovial membrane was observed using a specimen model via the Hematoxilin and Eosin dying technique. Results : The expression of mRNA of RA-related cytokines such as MIF, $TNF-{\alpha}$, and MMP-9 dosedependently decreased in the cell from the synovial membranes of the joint, which is treated with Cervus elaphus sibiricus herbal acupuncture solution. In mice treated with Cervus elaphus sibiricusherbal acupuncture, the damage of synovial membranes of the joint was lessened, and differentiation of Th 1 cells was suppressed. The activation of RA-related cytokines such as MIF was suppressed, and the generation of $TNF-{\alpha}$ and MMP-9 showed a statistically significant decreas. Conclusions : It is speculated that Cervus elaphus sibiricus herbal acupuncture has the therapeutic effect of palliating the damage of the tissue impaired by RA by inhibition of the initial RA progression and by regulating excessive differentiation of Th 1 cell as it suppresses the generation of RA-related cytokines during the highest stage of RA by acting on pro-inflammatory enzymes.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.104-104
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• 2003

As an effort to direct differentiation of human embryonic stem (hES, MB03) cells to dopamine-producing neuronal cells, Nurr1 was transfected using conventional transfection protocol into MB03 and examined the expression of tyrosine hydroylase (TH) after differentiation induced by retinoic acid (RA) and ascorbic acid (AA). Experimentally, cells were transfected with linearized Nurr1 cDNA in pcDNA3.1 (+)-hygovernight followed by selection in medium containing hygromycin-B (150 $\mu$/ml). Expression of Nurr1 mRNA was confirmed by RT-PCR and protein by immunocytochemistry in the drug resistant clones. In order to study the effect of Nurr1 protein on the differentiation pattern of ES cells, one of the positive clones (MBNr24) was allowed to form embryoid body (EB) for 2 days and were induced to differentiate for another 4 days using RA (1 $\mu M$) and AA (50 mM) (2-/4+ protocol) followed by selection in N2 medium for 10 or 20 days. After 10 days in N2 medium, cells immunoreactive to anti-GFAP, anti-TH, or anti-NF200 antibodies were 38.8%, 11%, and 20.5%, respectively. After 20 days in N2 medium, cells expressing GFAP, TH, or NF200 were 28%, 15% and 44.8%, respectively but approximately 9% of MB03 expressed TH protein when the cells were induced to differentiate using a similar prorocol, These results suggest that ectopic expression of Nurr1 enhances generation of TH+ cells as well as neuronal cells when hES cells were differentiated by 2-/4+ protocol.

• Molecules and Cells
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• 제39권4호
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• pp.352-357
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• 2016

Vertebrate neurogenesis requires inhibition of endogenous bone morphogenetic protein (BMP) signals in the ectoderm. Blocking of BMPs in animal cap explants causes the formation of anterior neural tissues as a default fate. To identify genes involved in the anterior neural specification, we analyzed gene expression profiles using a Xenopus Affymetrix Gene Chip after BMP-4 inhibition in animal cap explants. We found that the xCyp26c gene, encoding a retinoic acid (RA) degradation enzyme, was upregulated following inhibition of BMP signaling in early neuroectodermal cells. Whole-mount in situ hybridization analysis showed that xCyp26c expression started in the anterior region during the early neurula stage. Overexpression of xCyp26c weakly induced neural genes in animal cap explants. xCyp26c abolished the expression of all trans-/cis-RA-induced posterior genes, but not basic FGF-induced posterior genes. Depletion of xCyp26c by morpholino-oligonucleotides suppressed the normal formation of the axis and head, indicating that xCyp26c plays a critical role in the specification of anterior neural tissue in whole embryos. In animal cap explants, however, xCyp26c morpholinos did not alter anterior-to-posterior neural tissue formation. Together, these results suggest that xCyp26c plays a specific role in anterior-posterior (A-P) neural patterning of Xenopus embryos.

• BMB Reports
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• 제37권2호
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• pp.185-191
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• 2004

Tissue transglutaminase (tTGase) regulates various biological processes, including extracellular matrix organization, cellular differentiation, and apoptosis. Here we report the protective role of tTGase in the cell death that is induced by the tumor necrosis factor $\alpha$ (TNF-$\alpha$) and ceramide, a product of the TNF-$\alpha$ signaling pathway, in human neuroblastoma SH-SY5Y cells. Treatment with retinoic acid (RA) induced the differentiation of the neuroblastoma cells with the formation of extended neurites. Immunostaining and Western blot analysis showed the tTGase expression by RA treatment. TNF-$\alpha$ or $C_2$ ceramide, a cell permeable ceramide analog, induced cell death in normal cells, but cell death was largely inhibited by the RA treatment. The inhibition of tTGase by the tTGase inhibitors, monodansylcadaverine and cystamine, eliminated the protective role of RA-treatment in the cell death that is caused by TNF-$\alpha$ or $C_2$-ceramide. In addition, the co-treatment of TNF-$\alpha$ and cycloheximide ecreased the protein level of tTGase and cell viability in the RA-treated cells, supporting the role of tTGase in the protection of cell death. DNA fragmentation was also induced by the co-treatment of TNF-$\alpha$ and cycloheximide. These results suggest that tTGase expressed by RA treatment plays an important role in the protection of cell death caused by TNF-$\alpha$ and ceramide.

• Nutrition Research and Practice
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• 제3권2호
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• pp.77-83
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• 2009

Retinoic acids (RAs) modulate growth, differentiation, and apoptosis in normal, pre-malignant & malignant cells. In the present study, the effects of RA isomers (all-trans RA, 13-cis RA, and 9-cis RA) on the cell signal transduction of human breast cancer cells have been studied. The relationship between RAs and an enzymatic antioxidant system was also determined. Estrogen-receptor (ER) positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells were treated with different doses of each RA isomers, all-trans RA, 13-cis RA, or 9-cis RA. Treatment of RA isomers inhibited cell viability and induced apoptosis of MCF-7 cells as a result of increased caspase activity in cytoplasm and cytochrome C released from mitochondria. All-trans RA was the most effective RA isomer in both cell growth inhibition and induction of apoptosis in MCF-7 cells. However, no significant effect of RA isomers was observed on the cell growth or apoptosis in ER-negative MDA-MB-231 cells. In addition, activities of antioxidant enzymes such as catalase and glutathione peroxidase were decreased effectively after treatment of RA in MCF-7 cells, whereas SOD activity was rarely affected. Thus, the present data suggest that all-trans RA is the most potential inducer of apoptosis and modulator of antioxidant enzymes among RA isomers in MCF-7 human breast cancer cells.

• IMMUNE NETWORK
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• 제12권3호
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• pp.89-95
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• 2012

Immune cells express toll-like receptors (TLRs) and respond to molecular patterns of various pathogens. CpG motif in bacterial DNA activates innate and acquired immune systems through binding to TLR9 of immune cells. Several studies reported that CpG can directly regulate B cell activation, differentiation, and Ig production. However, the role of CpG in B cell growth and Ig production is not fully understood. In this study, we analyzed the effect of CpG on the kinetics of mouse B cell viability, proliferation, and Igs production. Overall, CpG enhanced mouse B cell growth and production of Igs in a dose-dependent manner. Unlike LPS, 100 nM CpG (high dose) did not support TGF-${\beta}1$-induced IgA and IgG2b production. Moreover, 100 nM CpG treatment abrogated either LPS-induced IgM or LPS/TGF-${\beta}1$-induced IgA and IgG2b production, although B cell growth was enhanced by CpG under the same culture conditions. We subsequently found that 10 nM CpG (low dose) is sufficient for B cell growth. Again, 10 nM CpG did not support TGF-${\beta}1$-induced IgA production but, interestingly enough, supported RA-induced IgA production. Further, 10 nM CpG, unlike 100 nM, neither abrogated the LPS/TGF-${\beta}1$- nor the LPS/RA-induced IgA production. Taken together, these results suggest that dose of CpG is critical in B cell growth and Igs production and the optimal dose of CpG cooperates with LPS in B cell activation and differentiation toward Igs production.

• Toxicological Research
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• 제26권1호
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• pp.61-66
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• 2010

Retinoids have many beneficial effects on dermatological applications. But, retinoids cause skin irritation. In this study, the safety of retinoids was clarified via both primary skin irritation test in rabbits and sensitization study using an integrated model for the differentiation of chemical-induced allergic and irritant skin reaction (IMDS), an alternative method to sensitization test. The effects of retinoids on the change of ultraviolet A (UVA)-induced matrix metalloproteinase-1 (MMP-1) in human skin fibroblasts and the modulation of type-1 pN collagen synthesis in hairless mice were examined to clarify the anti-wrinkle effects. Alltrans retinol (t-ROL) and its derivative, all-trans retinoic acid (t-RA), showed mild skin irritation but did not induce the sensitization. t-ROL and t-RA exerted anti-wrinkle effects by inhibiting the UVA-induced MMP-1 in human skin fibroblasts and increasing the type-1 pN collagen synthesis in hairless mice. These findings suggest that retinoids do not induce the allergy, and show anti-wrinkle effects by decreasing MMP-1 activation and increasing collagen synthesis.