• Title/Summary/Keyword: collagen assay

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Thrombospondins Mediate the Adhesion of Osteoblast to Extracelluar Matrix

  • Lim, Dong-Jin;Bae, In-Ho;Jeong, Byung-Chul;Kim, Sun-Hun;Park, Bae-Keun;Kang, In-Chul;Lee, Shee-Eun;Song, Sang-Hun;Koh, Jeong-Tae
    • International Journal of Oral Biology
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    • v.33 no.3
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    • pp.105-111
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    • 2008
  • Thrombospondins (TSP-1, TSP-2) are secretory extracellular glycoproteins that are involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. The present study was undertaken to elucidate the involvement of thrombospondins in the adhesion of osteoblast-like cells using the TSP-1 or TSP-2 antisense MG63 and MC3T3-E1 cell lines. For downregulation of TSPs expression, we prepared antisense constructs for TSP-1 and TSP-2 using the pREP4 an episomal mammalian expression vector, which be able to produce the specific antisense oligonucleotides around chromosome. MG63 and MC3T3-E1 osteoblast-like cells were transfected with the antisense constructs and nonliposomal Fugene 6, and then selected under hygromycin B (50 ${\mu}g/ml$) treatment for 2 weeks. Western blot analysis revealed that expression of the TSP proteins was downregulated in the antisense cell lines. The cell adhesion assay showed that adhesive properties of TSP-1 and TSP-2 antisense MG63 cells on the polystyrene culture plate were reduced to 17% and 21% of the control cells, respectively, and those of the TSP-1 and TSP-2 antisense MC3T3-E1 cells also decreased to 19% and 27% of control, respectively. Adhesion of TSP-1 and TSP-2 antisense MC3T3-E1 cells on Type I collagen-coated culture plate decreased to 27% and 76%, respectively. These results indicate that TSP-1 and TSP-2 proteins may have an important role in adhesion of osteoblast-like cells to extracellular matrix.

Interaction of Bone Marrow Stromal Stem Cells with Adhesive Protein and Polypeptide-adsorbed Poly(lactide-co-glycolide) Scaffolds (골수유래 간엽줄기세포와 점착성 단백질 및 폴리펩타이드가 흡착된(락티이드/글리콜라이드) 공중합체 지지체와의 상호작용)

  • Choi, Jin-San;Lee, Sang-Jin;Jang, Ji-Wook;Khang, Gil-Son;Lee, Young-Moo;Lee, Bong;Lee, Hai-Bang
    • Polymer(Korea)
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    • v.27 no.5
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    • pp.397-404
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    • 2003
  • The interaction of cell adhesive protein and polypeptide with bone marrow stromal stem cells (BMSCs) grown in tissue engineered films and scaffolds were examined. Several proteins or polypeptide known as cell-adhesive were coated adsorption on poly(lactide-co-glycolide) (PLGA) films and scaffolds and adhesion and proliferation behavior of BMSC on those surfaces were compared. The protein and polypeptide used include collagen IV, fibrinogen, laminin, gelatin, fibronectin, and poly(L-lysine). The protein and polypeptide were adsorbed on the PLGA film surfaces with almost monolayer coverage except poly(L-lysine). BMSCs were cultured for 1, 2, and 4 days on the protein- or polypeptide-adsorbed PLGA films and scaffolds. The cell adhesion and proliferation behaviors were assessed by sulforho damine B assay. It was observed that the protein- or polypeptide-adsorbed surfaces showed better cell adhesion and proliferation than the control.

Inhibiton of MMP-13 mRNA expression by Doxycycline combination with Mefenamic Acid in the rat Periodontal ligament cells (백서 치주인대세포에서 Doxycycline의 Mefenamic Acid 병용사용 시 MMP-13mRNA 발현 억제 효과)

  • Seo, Jin-Hee;Ciu, De-Zhe;Kim, Young-Joon
    • Journal of Periodontal and Implant Science
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    • v.35 no.1
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    • pp.99-109
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    • 2005
  • It has been focused on the importance of the host inflammatory response in periodontal pathogenesis and progression, treatment has been introduced to control the host response and the method, which diminishes production and activity of MMP by doxycycline, has been used in periodontal field. MMP is a proteolytic enzyme which plays a major role in tissue destruction and MMP-1 is secreted in the periodontally healthy tissue, while MMP-8, 9, 13, etc in the inflammatory state. Among these, MMP-13 has been discovered lately and reported to degrade primarily type II collagen. Periodontal ligament (PDL) cell plays a role in destruction of periodontal tissue. This study was to evaluate the effect of doxycycline and mefenamic acid, non-steroidal antiinflammatory drug on MMP-13 mRNA expression in the rat PDL cell. Doxycycline concentration of $1{\sim}100\;{\mu}g/ml$ was added rat PDL cell and cell activity was measured by MIT assay at day 1 and 3. MMP-13 gene expression was evaluated by RT-PCR after PDL cells were pre-treated for 1hour with doxycycline (50 ${\mu}g/ml$) alone or with mefenamic acid ($10^{-6}M$), then added $IL-1{\beta}$(1.0 ng/ml) and incubated for 16-18 hours. The results are as follows: 1. Cell activity decreased Significantly at 24 and 72 hours in 100 ${\mu}g/ml$ (p<0.05). 2. Level of MMP-13 mRNA was in 20.2% increase by $IL-1{\beta}$ and in pre-treating doxycycline group, expression of $IL-1{\beta}$ induced MMP-13 mRNA was inhibited by 31% than $IL-1{\beta}$ treated only. 3. Mefenamic acid did not inhibit on the expression of $IL-1{\beta}$ induced MMP-13 mRNA, while mefenamic acid in combination with doxycycline inhibited the expression by 41% compared to only $IL-1{\beta}$ stimulation. These results suggest that doxycycline synergistically inhibit the expression of $IL-1{\beta}$ induced MMP-13 mRNA in combination with mefenamic acid.

Construction and Production of Concatameric Human TNF Receptor-Immunoglobulin Fusion Proteins

  • Yim, Su-Bin;Chung, Yong-Hoon
    • Journal of Microbiology and Biotechnology
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    • v.14 no.1
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    • pp.81-89
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    • 2004
  • Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and lymphotoxin-$\alpha$ (LT-$\alpha$, TNF-$\beta$) can initiate and perpetuate human diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and insulin-dependent diabetes mellitus (IDDM). TNFs can be blocked by the use of soluble TNF receptors. However, since monomeric soluble receptors generally exhibit low affinity or function as agonists, the use of monomeric soluble receptors has been limited in the case of cytokines such as TNF-$\alpha$, TNF-$\alpha$, interleukin (IL)-1, IL-4, IL-6, and IL-13, which have adapted to a multi component receptor system. For these reasons, very high-affinity inhibitors were created for the purpose of a TNFs antagonist to bind the TNFR and trigger cellular signal by using the multistep polymerase chain reaction method. First, recombinant simple TNFR-Ig fusion proteins were constructed from the cDNA sequences encoding the extracellular domain of the human p55 TNFR (CD120a) and the human p75 TNFR (CD120b), which were linked to hinge and constant regions of human $IgG_1$ heavy chain, respectively using complementary primers (CP) encoding the complementary sequences. Then, concatameric TNFR-Ig fusion proteins were constructed using recombinant PCR and a complementary primer base of recombinant simple TNFR-Ig fusion proteins. For high level expression of recombinant fusion proteins, Chinese hamster ovary (CHO) cells were used with a retroviral expression system. The transfected cells produced the simple concatameric TNFR-Ig fusion proteins capable of binding TNF and inactivating it. These soluble versions of simple concantameric TNFR-Ig fusion proteins gave rise to multiple forms such as simple dimers and concatameric homodimers. Simple TNFR-1g fusion proteins were shown to have much more reduced TNF inhibitory activity than concatameric TNFR-Ig fusion proteins. Concatameric TNFR-Ig fusion proteins showed higher affinity than simple TNFR-Ig fusion proteins in a receptor inhibitor binding assay (RIBA). Additionally, concatameric TNFR-Ig fusion proteins were shown to have a progressive effect as a TNF inhibitor compared to the simple TNFR-Ig fusion proteins and conventional TNFR-Fc in cytotoxicity assays, and showed the same results for collagen induced arthritis (CIA) in mice in vivo.

Inhibitory Activity of Blueberries on UVB-induced Oxidative Stress and Matrix Metalloproteinase Expression in Human Skin Fibroblasts (인간피부 섬유아세포에서 UVB 유도된 산화 스트레스와 기질금속단백질가수분해효소 발현에 블루베리의 저해능)

  • Jang, Young Ah;Kim, Se Gie
    • Journal of Life Science
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    • v.29 no.12
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    • pp.1321-1328
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    • 2019
  • Intermediate-wavelength solar radiation, also known as ultraviolet B (UVB: 290-320 nm) radiation, may cause premature aging and oxidative damage-dependent skin cancer in humans. UVB-induced formation of reactive oxygen species (ROS)-often a consequence of excessive exposure to these rays-could activate matrix metalloproteinases (MMPs) such as MMP-1 and MMP-3. These enzymes break down type I collagen in human fibroblasts. In this study, we assessed the antioxidant and anti-aging effects of ethyl acetate extract of blueberry (EEB). An antioxidant test in blueberries evaluated ROS production using CCD-986sk cells and DPPH assay. In order to evaluate the anti-wrinkle efficacy of blueberries, the MMP-1 production and type 1 procollagen synthesis evaluated and the expression of MMP 1, 3 were tested through Western blot and RT- PCR. EEB exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and reduced the production of UVB-induced ROS. Also, EEB inhibited UVB-induced processes associated with photoaging and skin cancer, such as reduction in procollagen production and increase in MMP-1 production. More precisely, EEB (50 ㎍/ml) markedly suppressed mRNA and protein levels of MMP-1 and -3. The anti-aging effects are attributable to the antioxidant activity of EEB. These findings indicate that EEB has a protective effect against UVB-induced aging in human fibroblast cells by regulating the levels of type-1 procollagen, MMP-1, and MMP-3.

The Physicochemical Characteristics of Marinated Beef Galbi under Different Cooking Conditions (양념 소갈비의 조리과정에서의 물리화학적 특성 평가)

  • Hong, Sang-Pil;Kim, Young-Ho;Lee, Nam-Hyouck;Heo, Yeong-Uk
    • Journal of the Korean Society of Food Culture
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    • v.28 no.1
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    • pp.78-88
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    • 2013
  • Marinated beef galbi is a traditional Korean dish cooked with soy sauce, pear juice, onion, sesame oil, and sugar. However, there are many differences in beef galbi, including flavor and physicochemical aspects, depending on cooking conditions. Therefore, the physicochemical characteristics of marinated beef galbi prepared through various recipes was evaluated for its effects on pH, texture, aging, proteolysis, heating conditions, cooking time, and flavor compounds (pyrazines, IMPs, or FAAs). There were significant differences in salt concentration (0.8~3.03%), pH (4.89~6.22), and solid soluble contents (1.34-6.31 Brix) between recipes in this study. In the Pearson assay for sensory evaluation, overall preference correlated well with texture (a well-known sensory attribute in meat evaluation). Controlling the pH of meat through soaking in lemon solution, alkali water, phosphate, and baking powder solution, improved water holding capacity as much as 9 to 15% compared with the control. The myofibril index (MFI) of marinated meat stored at $4^{\circ}C$ increased 32% with 24 hours of aging and reached 39% at 48 hours of aging, and its fragmentation was observed through microscopy. SDS-PAGE showed hydrolysis of acid-soluble collagen by the pear juice, possibly related to meat tenderness. On the basis of surface temperature, the cooking time was estimated to be 8 minutes with pan heating at $170^{\circ}C$, 6 minutes at $270{\sim}300^{\circ}C$, and 4 minutes with charcoal at $700{\sim}900^{\circ}C$. Different pyrazine compounds, such as 2-methyl-3-phenylpyrrol(2,3-b) pyrazine (the typical product of the browning reaction) was mainly detected, and IMP (one of the main taste compounds in beef) was in higher amounts with the charcoal treatment, potentially related to its flavor preference among treatments. Our results demonstrate an effective case study and cooking system for beef galbi.

Anti-wrinkle Activity of Low Molecular Weight Peptides Derived from the Collagen Isolated from Asterias amurensis (불가사리(Asterias amurensis) 콜라겐 유래 저분자 펩타이드의 피부주름 억제활성)

  • Kwon, Min-Chul;Kim, Cheol-Hee;Kim, Hyo-Sung;Syed, Abdul Qadir;Hwang, Bo-Yong;Lee, Hyeon-Yong
    • Korean Journal of Food Science and Technology
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    • v.39 no.6
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    • pp.625-629
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    • 2007
  • This study was carried out to investigate the anti-wrinkle effects of peptides derived from collagens isolated from Asterias amurensis, which was collected in the East Sea. The molecular weights of the peptides were between 10-50 kDa, as determined through sephadek G-75 gel. The cytotoxicities against CCD-986sk cells and HEL-299 cells were measured using the MTT assay. The cytotoxicity of all the fractions(F1: Fraction No. 4-13, 116 kDa; F2: Fraction No. 25-30, 100 kDa; F3: Fraction No. 45-55, 58 kDa; F4: Fraction No. 59-63, 43 kDa; F5: Fraction No. 79-90, 24 kDa) was less than 25%, by the addition of 1.0 mg/mL. These peptides did not show any adverse effects on human skin cells. In the presence of F1 at 1.0 mg/mL, matrix metalloproteinase-1 (MMP-1) expression of UVA-induced human normal fibroblasts was reduced to 34.8%. Overall, the results seem to suggest that peptides of approximately 20 kDa have superior anti-wrinkle effects.

Development of Scar Improving Materials using Enkephalin Derivatives (엔케팔린 유도체를 이용한 흉터 개선 소재 개발)

  • Kim, Yang Woo;Kim, Hyoung Shik;Kim, Soo-Yun;Choi, Yun-Hee;Moh, Sang Hyun;Cheon, Young Woo
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.16 no.8
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    • pp.5336-5342
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    • 2015
  • Although demand for scar treatment has been rising as our quality of life is improved, most scar treatment products rely on importation. Enkephalin is one of the neuropeptides secreted from neuronal ends. As both skin and neuron are derived from the exoderm during the development process, skin cells express opioid receptors as neuronal cells do. Opioid receptors are categorized into three types, mu(m)-, delta(d)-, and kappa(k)- opioid receptors, all of which are directly involved in the wound healing process. In this study, enkephalin derivatives are synthesized by Alanin Scan and their efficacy was evaluated and compared. In vitro wound healing effects, stimulatory effects of collagen synthesis, and skin hydration effects were also evaluated and confirmed. Among Enkephalin derivatives, AS13 showed highest wound healing effect.

Efficacy of callus induced from Ullengdo niche plants for skin protection (식물세포배양기술을 이용한 울릉도 자생식물의 세포주 개발 및 피부세포 효능)

  • Choi, Yun Hui;Jung, Hae Soo;Cho, Moon Jin;Song, Mi Young;Seo, Hyo Hyun;Moh, Sang Hyun
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.15 no.8
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    • pp.5070-5077
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    • 2014
  • Many countries in the world have protected their native plants and utilized them as industrial materials in each country. In this aspect, it is increasingly important to develop cosmetics materials using native plants in Korea. Cosmetic materials have been developed with niche plants, such as Campanula takesimana Nakai, Dianthus superbus, Aster spathulifolius in Ullengdo, in which a specific plant distribution by distinct climate and environment was present. Water and ethanol extractions were performed from the calluses of Campanula takesimana Nakai, Dianthus superbus, Aster spathulifolius. HPLC analysis revealed different compositions and functions of effective elements in each ethanol extract. For example, all types of ethanol extracts showed an ability to heal wounds. In particular, the expression of the inflammation-related gene, COX-2, was decreased in response to the ethanol extracts of Dianthus superbus. These results indicate that the ethanol extracts from niche plants' calluses in Ullengdo are natural and environmentally-friendly compounds, and can be used as medical supplies associated with anti-inflammation and wound healing.

Effects of PLGA/Fibrin Scaffolds on Attachment and Proliferation of Costal Cartilage Cells (PLGA/피브린 지지체가 늑연골 세포의 부착과 성장에 미치는 영향)

  • Song, Jeong Eun;Lee, Yujung;Lee, Yun Me;Cho, Sun Ah;Jang, Ji Eun;Lee, Dongwon;Khang, Gilson
    • Polymer(Korea)
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    • v.37 no.2
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    • pp.141-147
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    • 2013
  • Poly(lactide-co-glycolic acid) (PLGA) has been widely used in the drug delivery and tissue engineering applications because of its good mechanical strength and biodegradation profile. However, cell attachment to the scaffold is low compared with that on fibrin although cells can be attached to the polymer surface. In this study, PLGA scaffolds were soaked in cells-fibrin suspension and polymerized with dropping fibrinogen-thrombin solution. Cellular proliferation activity was observed in PLGA/fibrin-seeded costal cartilage cells (CC) on 1, 3, and 7 days using the MTT assay and SEM. The effects of fibrin on the extracellular matrix (ECM) formation were evaluated using CC cell-seeded PLGA/fibrin scaffolds. The PLGA/fibrin scaffolds elicited more production of glycosaminoglycan (GAG) and collagen than the PLGA scaffold. In this study, fibrin incorporated PLGA scaffolds were prepared to evaluate the effects of fibrin on the cell attachment and proliferation in vitro and in vivo. In this result, we confirmed that proliferation of cells in PLGA/fibrin scaffolds were better than in PLGA scaffolds. The PLGA/fibrin scaffolds provide suitable environment for growth and proliferation of costal cartilage cells.