• Title/Summary/Keyword: in-vitro

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Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells (렌티바이러스와 아데노바이러스를 통하여 쥐의 중간엽줄기세포에 사람 나트륨/옥소 공동수송체 유전자를 전달하였을 때의 발현성능 비교)

  • Park, So-Yeon;Kim, Sung-Jin;Lee, Won-Woo;Lee, Heui-Ran;Kim, Hyun-Joo;Chung, June-Key;Kim, Sang-Eun
    • Nuclear Medicine and Molecular Imaging
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    • v.42 no.5
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    • pp.394-400
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    • 2008
  • Purpose: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Materials and Methods: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, and $98.4{\pm}1.3%$ at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and 1-125 uptake. Results: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro 1-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC ($29,704{\pm}6,659\; picomole/10^6\;cells$) was greater than that in adeno-hNIS-rMSC at MOI 100 ($6,168{\pm}2,134\;picomole/10^6\;cells$). Conclusion: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

Effect of Resveratrol on the Induction of Cdk Inhibitor p21 and Pro-apoptotic Bax Expression by amyloid-β in Astroglioma C6 Cells (신경교 세포에서 resveratrol이 amyloid-β에 의해 유도되는 Cdk inhibitor p21 및 Bax 발현의 감소 효과)

  • Kim Young Ae;Lim Sun-Young;Ko Woo Shin;Choi Byung Tae;Lee Yong Tae;Rhee Sook-Hee;Park Kun-Young;Lee Won-Ho;Choi Yung Hyun
    • Journal of Life Science
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    • v.15 no.2 s.69
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    • pp.169-175
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    • 2005
  • Resveratrol (3,4',5-trihydroxy-trans-stilbene), a phytoalexin found in grape skins, peanuts, and red wine, has been reported to have a wide range of biological and pharmacological properties. $Amyloid-\beta$ deposition and senile plaque-associated astrocytes are common neuropathological features of Alzheimer's disease. In this study, we have explored the effects of resveratrol on $amyloid-\beta-peptide-mediated$ cytotoxicity in vitro and modulation of cell growth-regulatory gene products in astroglioma C6 cells to elucidate its possible mechanism for anti-cytotoxicity. Exposure of C6 cells to $Amyloid-\beta$ resulted in dose-dependent growth inhibition and morphological changes of C6 cells, which were recovered by pre-treatment with resveratrol. The anti-proliferative effect of $amyloid-\beta$ was associated with the induction of tumor suppressor p53 and cyclin-dependent kinase (Cdk) inhibitor p21 (WAF1/CIP1) expression assessed by RT-PCR and Western blot analysis in time-dependent manner in C6 cells. In addition, the pro-apoptotic Bax expression was also up-regulated in $amyloid-\beta-treated$ C6 cells without alteration of anti-apoptotic Bcl-2 and $Bcl-X_L$ expression. However, pre-treatment of resveratrol significantly inhibited $amyloid-\beta-induced$ p53, p21 and Bax levels, suggesting that the modulation of p53, p21 and Bax levels could be one of the possible pathways by which resveratrol functions as anti-cytotoxic agent. Our results demonstrate that resveratrol may enhance the protection against $amyloid-\beta-induced$ cytotoxicity by promoting the survival of glial cells.

Antioxidative Effects of Korean Bamboo Trees, Wang-dae, Som-dae, Maengjong-juk, Jolit-dae and O-juk (한국산 왕대, 솜대, 맹종죽, 조릿대 및 오죽의 항산화 효과)

  • Lee, Min-Ja;Moon, Gap-Soon
    • Korean Journal of Food Science and Technology
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    • v.35 no.6
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    • pp.1226-1232
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    • 2003
  • Bamboo trees have been used for a long time as folk remedies for treatment of hypertension and stroke symptoms in Oriental regions. These pharmaceutical effects of bamboos look like to be related to its antioxidant capacity and phytochemicals in bamboos. To evaluate the antioxidative effects of bamboo trees, five kinds of bamboo varieties dominant in Korean peninsular were chosen and determined its total antioxidaive activities, free radical scavenging activities and nitrite scavenging activities by TEAC (Trolox Equivalent Antioxidant Capacity) assay, DPPH and Griess reagent assay using in vitro system, respectively. To evaluate the correlation between antioxidative activities and Maillard reaction during hot water extraction, contents of reducing sugar and total nitrogen and brown color intensity at 420 nm were determined. When total antioxidative activities, free radical scavenging activities and nitrite scavenging activities of five kinds of bamboo trees were compared, wang-dae (Phyllostachys bambusoides S. et Z.) showed the strongest effect among samples, although all kinds of extracts showed relatively strong effects against oxidation. The bamboo culms extract showed stronger antioxidative effects than that of bamboo leaves. In each fraction obtained from 70% ethanol extract, antioxidative effect were increased in order of dichloromethane>ethyl acetate>butanol>water>hexane fraction. In reducing sugar analysis of extracts, reducing sugar contents of water extracts were higher than that of 70% ethanol extracts and wang-dae water extract showed the highest level which was 708.92 mg/g. Total nitrogen contents of the extracts were $1.785{\sim}2.605\;mg%$ and contents in water extracts were lower than that in 70% ethanol extracts. Brown color intensity at 420 nm showed similar tendency with results in reducing sugar contents.

Bakanae Disease Reduction Effect by Use of Silicate Coated Seed in Wet Direct-Seeded Rice (규산코팅 벼 종자를 이용한 담수직파재배 시 벼 키다리병 경감효과)

  • Kang, Yang-Soon;Kim, Wan Joong;Kim, Yeon Ju;Jung, Ki-Hong;Choi, Ul-Su
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.61 no.1
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    • pp.9-16
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    • 2016
  • To investigate the effect of soluble silicate zeolite dressing of the rice against bakanae disease, field trial in reclaimed land and in vitro were carried out. The coated rice seeds (SCS) which were dressed with the mixture of 25% silicic acids (binder), and the zeolite (coating powder). In wet direct seeding, uniform scattering of rice seeds on the soil surface and the better seedling establishment were shown in SCS treatment plots. The incidence of bakanae disease began from the mid tillering stage toward the heading stage. Around heading stage, the ratio of infected tillers reached its highest point by 9.9% in non-SCS treatment plots. While, in SCS treatment plots, the ratio of infected tillers was no more than 0.01%. The vitality of the pathogenic fungi of bakanae disease in the SCS and non-SCS samples were assessed. Samples were incubated for one week keeping proper humidity at $30^{\circ}C$ after inoculated with panicles of infected rice plants from experimental field plots. In non-SCS treatment, pinkish colonies were formed on the grain surface of panicle of infected plants, and mycelium, macro-conidia and micro-conidia were developed actively inside part of infected grain inoculated. While in SCS treatment, micro-conidia and mycelium were not survived and the growth of macro-conidia, mycelia were greatly inhibited and withered. Based on the results, it is concluded that the environmental friendly control of bakanae disease by use of SCS is possible and soluble silicate can be applied as agents for replacement of seed disinfection.

In Vitro Plant Regeneration for Mass Propagation of Epimedium koreanum Nakai (삼지구엽초의 다량번식을 위한 기내 식물체 분화)

  • Han, Young-Hee;Choi, Byoung-Ryourl;Soh, Ho-Seob;Lee, Seong-Jae;Choi, Young-Jin;Kim, Se-Young
    • Horticultural Science & Technology
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    • v.18 no.6
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    • pp.834-838
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    • 2000
  • As an endeavor to establish a micropropagation system for Epimedium koreanum Nakai., this study was carried out to define methods to disinfect its explants and media for callus induction, proliferation and plant regeneration. The lowest infection rates by fungi or bacteria on apical and axillary bud explants of rhizome were observed when they were immerged in 0.3% NaOCl solution for 20 min after soaked in 0.1% $AgNO_3$ solution for 30 min, but leaf explants were seldom infected with fungi or bacteria by this disinfectant method. The highest rate of plantlet formation was obtained from the explants disinfected in 0.3% NaOCl solution for 20 min after soaked in 0.1% $AgNO_3$ solution for 60 min for tip buds and in 0.1 % $AgNO_3$ solution for 30 min for axillary buds of rhizome. Induction rate of callus was the highest from the explants disinfectd in 0.3% NaOCl solution for 20 min after soaked in 0.2% $AgNO_3$ solution for 15 min. Callus growth was proper in a modified 1/2 MS medium including half strength of $NH_4NO_3$ with $0.02-0.2mg{\cdot}L^{-1}$ BA and $2.0mg{\cdot}L^{-1}$ NAA. Low rate of plantlet regeneration was obtained in 1/2 UM or 1/2 White medium with $2.0mg{\cdot}L^{-1}$ BA and $0.2mg{\cdot}L^{-1}$ AA.

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INFLUENCE OF LIGHT IRRADIATION OVER SELF-PRIMING ADHESIVE ON DENTIN BONDING (상아질접착제에 대한 광조사가 접착에 미치는 영향)

  • 류현욱;김기옥;김성교
    • Restorative Dentistry and Endodontics
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    • v.26 no.5
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    • pp.409-417
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    • 2001
  • The purpose of this study was to investigate the influence of light irradiation over self-priming adhesive on dentin bonding. After acid etching the exposed dentin, a self-priming adhesive (Prime&Bond$^{\circledR}$NT dental adhesive system Dentsply DeTrey, GmbH, Konstanz, Germany) was applied and light irradiation was done for 20 sec with regular intensity (600 mW/$\textrm{cm}^2$) in group I and for 3 sec with ultra-high intensity (1930 mW/$\textrm{cm}^2$) in group III. No light irradiation was done over self-priming adhesive in groups II and IV. Composite resin was added on the self-priming adhesive and irradiated for 40 sec with regular intensity (600 mW/$\textrm{cm}^2$) in groups I and II and for 3 sec with ultra-high intensity (1930 mW/$\textrm{cm}^2$) in groups III and IV. To see the effect of light curing time on dentin bonding, another 3 group specimens were prepared. Without light-irradiation over self-priming adhesive, added composite resin was irradiated for 3, 6, or 12 sec with ultra-high intensity light. After bonded specimens were stored in 37$^{\circ}C$ distilled water for 24 hours, shear bond strength were measured using a universal testing machine (4202, Instron, Instron Co., U.S.A.) and fractured surfaces were examined under a stereomicroscope (SZ-PT Olympus, Japan). Statistical analysis were done with one-way, two-way ANOVA and chi-square test. The results were as follows : 1. The shear bond strengths from the groups irradiated over self-priming adhesive were significantly higher than those from the groups without irradiation (p<0.05). 2. There was no significant shear bond strength difference between regular intensity light irradiation groups and ultra-high intensity ones (p>0.05). 3. There was no significant shear bond strength difference among various irradiation time groups with ultra-high intensity ones (p>0.05). 4. In stereomicroscopic examination of fractured surfaces, adhesive-cohesive mixed failure mode was mostly seen in all groups, and there was no significant difference in failure mode among groups (p>0.05).

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Bioactive Utility of the Extracts from Rhus verniciflua Stokes (RVS) : Biological Function of the Extracts from RVS (옻나무 추출물의 생리활성 이용에 대한 연구 : 옻나무 추출물의 생물학적 기능)

  • Lim, Kye-Taek;Lee, Jeong-Chae
    • Korean Journal of Food Science and Technology
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    • v.31 no.1
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    • pp.238-245
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    • 1999
  • Antioxidative effects of the water or ethanol extracts from Rhus verniciflua Stokes (RVS) were measured by protection against hydroxyl radicals in mouse brain tissue culture. In the water extracts from RVS, cell viabilities were estimated 60.0, 66.0, 72.0, 84.0 and 90.0% at addition of 1, 2, 4, 7 and $10{\mu}L$, respectively, compared with GO (20 mU/mL) alone. The cell viability in the ethanol extracts was similarly with water extracts. In the antitumor effects, the results showed that percentages of the HeLa cell death were approximately 24% for 12 hrs, 57% for 48 hrs at addition of 10%/well ethanol extracts respectively. To know inhibition of tumor growth, in vivo, mice (BALB/c) were inoculated with 0.25 mL CT-26 $(1{\times}10^6\;cells/mL)$ subcutaneously. After the generation of tumor, the results of RVS extracts (ethanol, water) injection showed generally that the tumor size in BALB/c was reduced. For physicochemical characterization of the RVS extracts, purified substances of water or ethanol extracts were analized with SDS-PAGE and ICP spectrometer. In electrophoresis, gel showed 2 bands (210, 230 KDa). The results of ICP verified that RVS extracts contain $Cu^{2+}$ in both samples. Conclusively, this substance might be a laccase which has a biological effective function, as a natural bioactive substance.

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Tensile Bond Strength of Composite Resin Treated with Er:YAG Laser (Er:YAG 레이저를 활용한 와동형성시 컴포짓 결합강도)

  • Shin, Min;Ji, Young-Duk;Rhu, Sung-Ho;Cho, Jin-Hyoung
    • Journal of Oral Medicine and Pain
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    • v.30 no.2
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    • pp.269-276
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    • 2005
  • This in vitro study evaluated the influence of a flowable composite resin on the tensile bond strength of resin to enamel and dentin treated with Er:YAG laser and diamond bur. 96 Buccal enamel and mid-coronal dentin were laser-irradiated using an Er:YAG laser and treated with diamond bur. Each groups(48) were divided two small groups depends on acid-etching procedure. Light-cure flowable resin(Metafil Flo) and self-cure resin(Clearfil FII New Bond) were used in this study. After surface etching with 37% phosphoric acid and the application of an adhesive system, specimens were prepared with a hybrid composite resin. After 24hours storage in distilled water at 37$^{\circ}C$, all samples were submitted to the tensile bond strength evaluation, using a universal testing machine(Z020, Zwick, Germany). The obtained results were as follows: 1. TBS of acid-etching group were higher than those of non-etching group in both enamel and dentin treated with Er:YAG laser and diamond bur. Laser 'conditioning' was clearly less effective than acid-etching. Moreover, acid etching lased enamel and dentin significantly improved the microTBS of M-Flo. 2. In enamel, TBS of laser-irradiated group were lower than those of bur-prepared group. However, in flowable resin subgroup, there were not differed those between two groups in dentin. 3. In laser-treated group, TBS of flowable composite resin were higher than those of self-curing resin in dentin, however, there was no difference in enamel. From this study, we can conclude that the self- and light-cure composite resin bonded significantly less effective to lased than to bur-cut enamel and dentin, and that acid-etch procedure remains mandatory even after laser ablation. We suggest that Er:YAG laser was useful for preparing dentin cavity with flowable resin filling.

Inhibitory Effect of Camp Antagonist and Pka Inhibitors, and Stimulatory Effect of Adenylate Cyclase Agonist on Cathepsin K Processing in Cultured Mouse Osteoclasts (cAMP 길항제와 PKA 억제제 및 Adenylate Cyclase 촉진제의 백서 파골세포에서 Cathepsin K 생성에 대한 효과)

  • Shim, Youn-Soo
    • Journal of dental hygiene science
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    • v.6 no.1
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    • pp.1-9
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    • 2006
  • Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and was thought to play a key role in matrix degradation during bone resorption. It was shown that the intracellular maturation of cat K was prevented by the cAMP antagonist, Rp-cAMP, and the protein kinase A (PKA) inhibitors of KT5720 and H89. In contrast, forskolin, a adenylate cyclase agonist, rather induced Cat K processing and maturation in osteoclasts. Furthermore, to determine whether cat K processing and maturation signaling involves protein kinase C (PKC), mouse total bone cells were treated with calphostin C, a specific inhibitor of PKC, however, no effect was observed, indicating that calphostin C did not affect to osteoclast-mediated cat K processing and maturation. Thus, it is indicated that the cAMP-PKA signaling pathway regulates cat K maturation in osteoclasts. Since secreted proenzymes have the potential to reenter the cell via M6P receptor, to prevent this possibility, it was tested cAMP antagonist Rp-cAMP and the PKA inhibitors KT5720 and H89 in the absence or presence of M6P. Inhibition of cat K processing by Rp-cAMP, KT5720, or H89 was observed in a dose-dependent manner. Furthermore, the addition of M6P resulted in enhanced potency of Rp-cAMP, KT5720 and H89. These dose-dependently inhibited in vitro bone resorption with a potency similar to that observed for inhibition of cat K processing.

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Anti-aging Effects of L-Carnitine on Human Skin (L-카르니틴의 사람피부에 대한 항노화 효과)

  • Lee Bum-Chun;Choe Tae-Boo;Sim Gwan-Sub;Lee Geun-Soo;Park Sung-Min;Lee Chun-Il;Pyo Hyeong-Bae
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.30 no.3 s.47
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    • pp.393-397
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    • 2004
  • L-Carnitine $({\beta}-hydroxy-{\gamma}-trimethyl-ammoniumbutyric{\;}acid)$ is a small water-soluble molecule important in mammalian fat metabolism. It is essential for the normal oxidation of fatty acids by the mitochondria, and is involved in the trans-esterification and excretion of acyl-CoA esters. In this paper, to investigate the relationship between aging and L-carnitine, we investigated the effects of in vitro matrix-metalloproteinase (MMP) inhibition and activity and expression of UYA-induced MMPs in human skin fibroblasts. Also, we studied to develop as anti-aging cosmetics with L-carnitine. Fluorometric assays of the proteolytic activities of MMP-1 (collagenase) were performed using fluorescent collagen substrates. ELISA (enzyme linked immune sorbent assay), gelatin-substrate zymography, RT-PCR ELISA techniques were used for the effects of L-carnitine on MMP expression, activity, and MMP mRNA expression in UVA irradiated fibroblast $(5\;J/cm^2)$, respectively. In addition, we performed clinical study with L-carnitine cream. L-carnitine inhibited the activities of MMP-1 in a dose-dependent manner and the $IC_{50}$ values calculated from semi-log plots were 2.45 mM, and L-carnitine showed strong inhibition on MMP-2 (gelatinase) activity in UVA irradiated fibroblast by zymography. Also, UVA induced MMP-1, 2 expression was reduced $43\%,\;53\%$ by treated with L-carnitine at 1.25 mM, and MMP-1 mRNA expression was reduced dose-dependent manner. Therefore L-carnitine was able to significantly inhibit the MMP activity, and regulate MMP expression in protein and mRNA level. The results of clinical study showed that $1.0\%$ L-carnitine treated group reduced wrinkle significantly compared with placebo treated group (P<0.05). All these results suggest that L-carnitine may be useful as new anti-aging cosmetics for protection against UVA induced Mm expression and activity.