• Title/Summary/Keyword: confocal microscopy

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Dexamethasone Facilitates NF-κB Signal Pathway in TNF-α Stimulated Rotator Cuff Tenocytes

  • Ji, Jong-Hun;Kim, Young-Yul;Patel, Kaushal;Cho, Namjoon;Park, Sang-Eun;Ko, Myung-Sup;Park, Suk-Jae;Kim, Jong Ok
    • Journal of Microbiology and Biotechnology
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    • v.29 no.2
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    • pp.297-303
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    • 2019
  • Corticosteroids are commonly used for pain control in rotator cuff tear. Deregulated $NF-{\kappa}B$ activation is a hallmark of chronic inflammatory diseases and has been responsible for the pathogenesis of rotator cuff tear. The Dexamethasone(DEXA) is a synthetic corticosteroid. The purpose of this study was to examine the exact effect of dexamethasone on $NF-{\kappa}B$ signaling in rotator cuff tear. We measured $NF-{\kappa}B$ expression in four groups: control, $TNF-{\alpha}$-treated, DEXA-treated, and combined treatment with $TNF-{\alpha}$ and DEXA. Tenocytes were isolated from patients with rotator cuff tears and pre-incubated with $TNF-{\alpha}$ (10 ng/ml), DEXA ($1{\mu}M$), or both of them for 10 min, 1 h, and 2 h. Expression of p65, p50, and p52 in the nuclei and cytosol was analyzed by western blotting and immunofluorescence imaging using confocal microscopy. We also evaluated nucleus/cytosol (N/C) ratios of p65, p50, and p52. In our study, the combined treatment with DEXA and $TNF-{\alpha}$ showed increased N/C ratios of p65, p50, and p52 compared with those in the $TNF-{\alpha}$ group at all time points. Additionally, in the DEXA group, N/C ratios of p65, p50, and p52 gradually increased from 10 min to 2 h. In conclusion, DEXA promoted the nuclear localization of p65, p50, and p52, but was not effective in inhibiting the inflammatory response of $TNF-{\alpha}$-stimulated rotator cuff tear.

Bonding efficacy of cured or uncured dentin adhesives in indirect resin (간접 레진수복시 상아질 접착제의 중합 여부에 따른 결합 효능)

  • Jang, Ji-Hyun;Lee, Bin-Na;Chang, Hoon-Sang;Hwang, Yun-Chan;Oh, Won-Mann;Hwang, In-Nam
    • Restorative Dentistry and Endodontics
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    • v.36 no.6
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    • pp.490-497
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    • 2011
  • Objectives: This study examined the effect of the uncured dentin adhesives on the bond interface between the resin inlay and dentin. Materials and Methods: Dentin surface was exposed in 24 extracted human molars and the teeth were assigned to indirect and direct resin restoration group. For indirect resin groups, exposed dentin surfaces were temporized with provisional resin. The provisional restoration was removed after 1 wk and the teeth were divided further into 4 groups which used dentin adhesives (OptiBond FL, Kerr; One-Step, Bisco) with or without light-curing, respectively (Group OB-C, OB-NC, OS-C and OS-NC). Pre-fabricated resin blocks were cemented on the entire surfaces with resin cement. For the direct resin restoration groups, the dentin surfaces were treated with dentin adhesives (Group OB-D and OS-D), followed by restoring composite resin. After 24 hr, the teeth were assigned to microtensile bond strength (${\mu}TBS$) and confocal laser scanning microscopy (CLSM), respectively. Results: The indirect resin restoration groups showed a lower ${\mu}TBS$ than the direct resin restoration groups. The ${\mu}TBS$ values of the light cured dentin adhesive groups were higher than those of the uncured dentin adhesive groups (p < 0.05). CLSM analysis of the light cured dentin adhesive groups revealed definite and homogenous hybrid layers. However, the uncured dentin adhesive groups showed uncertain or even no hybrid layer. Conclusions: Light-curing of the dentin adhesive prior to the application of the cementing material in luting a resin inlay to dentin resulted in definite, homogenous hybrid layer formation, which may improve the bond strength.

Effects of High Glucose and Advanced Glycosylation Endproducts (AGE) on ZO-1 Expression in cultured Glomerular Epithelial Cells (GEpC) (당과 후기당화합물에 의한 사구체 상피세포 ZO-1 발현의 변화)

  • Lee Jin-Seok;Lee Hae- Soo;Yoon Ok-Ja;Ha Tae-Sun
    • Childhood Kidney Diseases
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    • v.8 no.2
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    • pp.138-148
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    • 2004
  • Purpose: Regardless of the underlying diseases, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the highly specialized interdigitating foot processes. We examined the molecular basis for this alteration of the podocyte phenotypes, including quantitative and distributional changes of ZO-1 protein as a candidate contributing to the pathogenic changes in the barrier to protein filtration. Methods: To investigate whether high glucose and advanced glycosylation endproduct(AGE) induce podocyte cytoskeletal changes, we cultured rat GEpC under 1) normal glucose(5 mM=control) or 2) high glucose(30 mM) or 3) AGE-added or 4) high glucose plus AGE-added conditions. The distribution of ZO-1 was observed by confocal microscope and the change of ZO-1 expression was measured by Western blotting and RT-PCR. Results: By confocal microscopy, we observed that ZO-1 moves from peripheral cytoplasm to inner actin filaments complexes in both AGE-added and high glucose condition. In Western blotting, high glucose or AGE-added condition decreased the ZO-1 protein expression by 11.1%(P>0.05) and 2.3%(P>0.05), respectively compared to the normal glucose condition. High glucose plus AGE-added condition further decreased ZO-1 protein expression to statistically significant level(12%, P<0.05). No significant change was seen in the osmotic control. In RT-PCR, high glucose plus AGE-added condition significantly decreased the expression of ZO-1 mRNA by 12% compared to normal glucose condition. Conclusion: We suggest that both high glucose and AGE-added condition induce the cytoplasmic translocation and suppresses the production of ZO-1 at transcriptional level and these changes may explain the functional changes of podocytes in diabetic conditions.

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Mechanism Underlying a Proteasome Inhibitor, Lactacystin-Induced Apoptosis on SCC25 Human Tongue Squamous Cell Carcinoma Cells (사람혀편평상피세포암종세포에서 proteasome 억제제인 lactacystin에 의해 유도된 세포자멸사의 기전에 대한 연구)

  • Baek, Chul-Jung;Kim, Gyoo-Cheon;Kim, In-Ryoung;Lee, Seung-Eun;Kwak, Hyun-Ho;Park, Bong-Soo;Tae, Il-Ho;Ko, Myung-Yun;Ahn, Yong-Woo
    • Journal of Oral Medicine and Pain
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    • v.34 no.3
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    • pp.261-276
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    • 2009
  • Lactacystin, a microbial natural product synthesized by Streptomyces, has been commonly used as a selective proteasome inhibitor in many studies. Proteasome inhibitors is known to be preventing the proliferation of cancer cells in vivo as well as in vitro. Furthermore, proteasome inhibitors, as single or combined with other anticancer agents, are suggested as a new class of potential anticancer agents. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in SCC25 human tongue sqaumous cell carcinoma cell line treated with lactacystin. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingiva fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay respectively. The hoechst staining, hemacolor staining and TUNEL staining were conducted to observe SCC25 cells undergoing apoptosis. SCC25 cells were treated with lactacystin, and Western blotting, immunocytochemistry, confocal microscopy, FAScan flow cytometry, MMP activity, and proteasome activity were performed. Lactacystin treatment of SCC25 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Interestingly, lactacytin remarkably revealed cytotoxicity in SCC25 cells but not normal cells. And tested SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, the up-regulation of Bax, and the activation of caspase-7, caspase-3, PARP, lamin A/C and DFF45 (ICAD). Flow cytometric analysis revealed that lactacystin resulted in G1 arrest in cell cycle progression which was associated with up-regulation in the protein expression of CDK inhibitors, $p21^{WAF1/CIP1}$ and $p27^{KIP1}$. We presented data indicating that lactacystin induces G1 cell cycle arrest and apoptois via proteasome, mitochondria and caspase pathway in SCC25 cells. Therefore our data provide the possibility that lactacystin could be as a novel therapeutic strategy for human tongue squamous cell carcinoma.

CHANGE OF TASTE PREFERENCE AND TASTE BUD AFTER UNILATERAL LINGUAL NERVE TRANSECTION IN RAT (백서 편측 설신경 손상 후 미각 및 설유두의 변화에 대한 연구)

  • Kim, Yoon-Tae;Jeon, Seung-Ho;Yeom, Hak-Ryol;Kang, Jin-Han;Ahn, Kang-Min;Kim, Sung-Min;Jahng, Jeong-Won;Park, Kyung-Pyo;Lee, Jong-Ho
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.31 no.6
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    • pp.515-525
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    • 2005
  • Purpose of study: Lingual nerve damage can be caused by surgery or trauma such as physical irriatation, radiation, chemotherapy, infection and viral infection. Once nerve damage occurred, patients sometimes complain taste change and loss of taste along with serious disturbance of tongue. The purpose of this study was to evaluate the effects of unilateral lingual nerve transection on taste as well as on the maintenance of taste buds. Materials & Methods: Male Sprague-Dawley rats weighing 220-250g received unilateral transection of lingual nerve, subjected to the preference test for various taste solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) with two bottle test paradigm at 2, 4, 6, or 8 weeks after the operation. Tongue was fixed with 8% paraformaldehyde. After fixation, they were observed with scanning electron microscope(JSM-$840A^{(R)}$, JEOL, JAPAN) and counted the number of the dorsal surface of the fungiform papilla for changes of fungiform papilla. And, Fungiform papilla were obtained from coronal sections of the anterior tongue(cryosection). After cryosection, immunostaining with $G{\alpha}gust$(I-20)(Santa Cruz Biotechnology, USA), $PLC{\beta}2$(Q-15)(Santa Cruz Biotechnology, USA), and $T_1R_1$(Alpha Diagnostic International, USA) were done. Immunofluorescence of labeled taste bud cells was examined by confocal microscopy(F92-$300^{(R)}$, Olympus, JAPAN). Results: The preference score for salty and sweet tended to be higher in the operated rats with statistical significance, compared to the sham rats. Fungiform papilla counting were decreased after lingual nerve transaction. In 2 weeks, maximum differences occurred. Gustducin and $T_1R_1$ expressions of taste receptor in 2 and 4 weeks were decreased. $PLC{\beta}2$ were not expressed in both experimental and control group. Conclusion: This study demonstrated that the taste recognition for sweet and salty taste changed by week 2 and 4 after unilateral lingual nerve transection. However, regeneration related taste was occurred in the presence of preserving mesoneurial tissue and the time was 6 weeks. Our results demonstrated that unilateral lingual nerve damage caused morphological and numerical change of fungiform papilla. It should be noted in our study that lingual nerve transection resulted in not only morphological and numerical change but also functional change of fungiform papillae.

Photodynamic Therapy with Photofrin Reduces Invasiveness of U87 Malignant Human Glioma Cells (교모세포종 세포주 U87에서 Photofrin을 사용한 광역학 치료가 종양 침습성에 미치는 영향)

  • Woo, Hye Kyung;Cho, Kyung-Keun;Rha, Hyung Kyun;Lee, Kyung Jin;Park, Sung Chan;Cho, Jung Ki;Park, Hea Kwan;Kang, Joon Ki;Choi, Chang Rak
    • Journal of Korean Neurosurgical Society
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    • v.30 no.sup2
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    • pp.189-196
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    • 2001
  • Objective : We tested the hypothesis that photodynamic therapy(PDT) with Photofrin inhibits tumor invasion of U87 human glioma cells using several in vitro assay to measure tumor invasiveness. The effects of PDT on cell growth, directional migration and cell invasion were investigated. Material and Method : Tumor cells were treated with Photofrin at various doses and at a fixed optical(632nm) dose of $100mJ/cm^2$. Cytotoxicity was tested using the MTT method. Invasion assays including the matrigelartificial basement membrane barrier migration and spheroid confrontation with confocal microscopic analysis were used to study the relationship between PDT and invasiveness. Result : U87 cells showed a dose dependent cytotoxic response to increasing Photofrin dose. Data from the matrigel artificial basement membrane assay indicate that PDT inhibits the U87 cell migration dose dependently. Low doses of subcytotoxic PDT treatment, such as 2.5ug/ml Photofrin dose, also appeared to significantly inhibit migration of U87 cells(p<0.05). In co-cultures between U87 cell spheroids and brain aggregates, progressive invasion with destruction of the brain aggregate occurs. The extent of tumor cell infiltration and proportion or intact brain aggregate remaining after 24h differs in Photofrin PDT treated versus Photofrin only control, with changes suggestive of a dose-response effect. Conclusion : our data indicate that PDT with Photofrin significantly inhibits the invasiveness of U87 cells, and this inhibition is dose dependent.

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Enhancement of Immune Activities of Peptides from Asterias amurensis Using a Nano-encapsulation Process (나노 입자 불가사리 펩타이드의 면역 활성 증진)

  • Jeong, Hyang-Suk;Oh, Sung-Ho;Kim, Seoung-Seop;Jeong, Myoung-Hoon;Choi, Woon-Yong;Seo, Yong-Chang;Choi, Geun-Pyo;Kim, Jin-Chul;Lee, Hyeon-Yong
    • Korean Journal of Food Science and Technology
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    • v.42 no.4
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    • pp.424-430
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    • 2010
  • Immuno-modulatory activities of peptides from Asterias amurensis were investigated using a nano-encapsulation process. The molecular weights of the peptides in the range of 5-7 kDa were separated using Sephadex G-75 gel filtration. Eighty-five percent of the nano-particles were in the 300 nm range using dynamic light scattering. The cytotoxicity of the A. amurensis nano-particles against CCD-986sk human dermal fibroblast cells was 11.64% after adding 1.0 mg/mL of the samples, which was lower than that from the control (13.28% collagen). The secretion of $NO^-$ from macrophages was estimated as $40\;{\mu}M$ after adding 1.0 mg/mL of gelatin nano-particles, which was higher than the others. Prostaglandin $E_2$ production from UV-induced human skin cells decreased greatly to 860 pg/mL after adding 1.0 mg/mL of the samples. Confocal microscopy revealed that nano-particles effectively penetrated the cells within 1 hour. From these results, we consider that nano-encapsulation of the peptides from A. amurensis can improve their biological functions.

17 beta-Estradiol Increases Peak of $\textrm{Ca}^{2+}$ Current in Mouse Early Embryo (에스트로겐이 생쥐 초기배의 $\textrm{Ca}^{2+}$ 전류에 미치는 영향)

  • 강다원;신용원;김은심;홍성근;한재희
    • Journal of Embryo Transfer
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    • v.16 no.2
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    • pp.79-89
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    • 2001
  • Steroid hormones control the expression of many cellular regulators, and a role thor estrogen in mouse oocytes has been well documented. The preovulatory $E_2$increment is generally accepted as the endocrine process regulating induction of in vivo oocyte maturation To address whether the activity of the T-type $Ca^{2+}$ channel is altered by 17 beta-estradiol ( $E_2$), we examined the actions of $E_2$on the calcium channel of mouse oocytes and early embryos. Oocrtes were collected from the oviduct of mice treated with pregnant mare's serum gonadotropin (PMSG) and human choronic gonadotropin (hCG). Whole cell voltage clamp technique and confocal microscopy were used to examine that $E_2$increase intracellular $Ca^{2+}$ concentration ([C $a^{2+}$]$_{i}$ ) via voltage dependent $Ca^{2+}$ channel (VDC) and estrogen receptor (FSR), and $E_2$concentration by the use of radioimmunoassay (RIA) were examined in mouse. The results obtained were as follows: The peak of $Ca^{2+}$ current induced by $E_2$increased 122% to 1.50$\pm$0.03 nA from 1.23$\pm$0.21 nA (n=15) in the presence of 5 mM extracellular $Ca^{2+}$ concentration ([C $a^{2+}$]$_{o}$ ). The increased $Ca^{2+}$ current was temporally associated with $Ca^{2+}$ transients. The intracellular $Ca^{2+}$ level increased 207%~30 s following the addition of 1${\mu}{\textrm}{m}$ $E_2$(relative fluorescence intensity: 836.4$\pm$131.2 for control, n=10, 1736.4$\pm$192.0 in the presence of $E_2$, n=10). $E_2$increased amplitude of $Ca^{2+}$ current and [C $a^{2+}$]$_{i}$ . $E_2$-induced $Ca^{2+}$ current and $E_2$concentration in blood were showed difference on the stage of embryo. These results suggest that $E_2$modulate $Ca^{2+}$ channel to increase $Ca^{2+}$ influx.$Ca^{2+}$ influx.

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Effect of the Flavonoid Luteolin for Dextran Sodium Sulfate-induced Colitis in NF-${\kappa}B^{EGFP}$ Transgenic Mice (Dextran Sodium Sulfate 유발 장염 모델에서 루테올린의 치료효과)

  • Jang, Byung-Ik
    • Journal of Yeungnam Medical Science
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    • v.23 no.1
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    • pp.26-35
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    • 2006
  • Background: Luteolin, a flavone found in various Chinese herbal medicines is known to possess anti-inflammatory properties through its ability to inhibit various proinflammatory signaling pathways including NF-${\kappa}B$ and p38 MAPK. In this study, we investigated the potential therapeutic effect of luteolin on dextran sodium sulfate (DSS)-induced colitis. Materials and Methods: We used a transgenic mouse model expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-${\kappa}B$ $cis$-elements. C57BL/6 NF-${\kappa}B^{EGFP}$ mice received 2.5% DSS in their drinking water for six days in combination with daily luteolin administration (1mg/kg body weight, 0.1ml vol, intragastric) or vehicle. NF-${\kappa}B$ activity was assessed macroscopically with a Charge-Coupled Device (CCD) camera and microscopically by confocal analysis. Results: A significant increase in the Disease Activity Index (DAI), histological score (p<0.05), IL-12 p40 secretion in colonic stripe culture (p<0.05) and EGFP expression was observed in luteolin and/or DSS-treated mice compared to water-treated mice. Interestingly, a trend toward a worse colitis (DAI, IL-12p40) was observed in luteolin-treated mice compared to non-treated DSS-exposed mice. In addition, EGFP expression (NF-${\kappa}B$ activity) strongly increased in the luteolin-treated mice compared to control mice. Confocal microscopy showed that EGFP positive cells were primarily lamina propria immune cells. Conclusions: These results suggest that luteolin is not a therapeutic alternative for intestinal inflammatory disorders derived for primary defects in barrier function. Thus, therapeutic intervention targeting these signaling pathways should be viewed with caution.

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Comparative evaluation of marginal and internal fit of metal copings fabricated by various CAD/CAM methods (다양한 CAD/CAM 방식으로 제작한 금속하부구조물 간의 변연 및 내면 적합도 비교 연구)

  • Jeong, Seung-Jin;Cho, Hye-Won;Jung, Ji-Hye;Kim, Jeong-Mi;Kim, Yu-Lee
    • The Journal of Korean Academy of Prosthodontics
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    • v.57 no.3
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    • pp.211-218
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    • 2019
  • Purpose: The purpose of the present study was to compare the accuracy of four different metal copings fabricated by CAD/CAM technology and to evaluate clinical effectiveness. Materials and methods: Composite resin tooth of the maxillary central incisor was prepared for a metal ceramic crown and duplicated metal die was fabricated. Then scan the metal die for 12 times to obtain STL files using a confocal microscopy type oral scanner. Metal copings with a thickness of 0.5 mm and a cement space of $50{\mu}m$ were designed on a CAD program. The Co-Cr metal copings were fabricated by the following four methods: Wax pattern milling & Casting (WM), Resin pattern 3D Printing & casting (RP), Milling & Sintering (MS), Selective laser melting (SLM). Silicone replica technique was used to measure marginal and internal discrepancies. The data was statistically analyzed with One-way analysis of variance and appropriate post hoc test (Scheffe test) (${\alpha}=.05$). Results: Mean marginal discrepancy was significantly smaller in the Group WM ($27.66{\pm}9.85{\mu}m$) and Group MS ($28.88{\pm}10.13{\mu}m$) than in the Group RP ($38.09{\pm}11.14{\mu}m$). Mean cervical discrepancy was significantly smaller in the Group MS than in the Group RP. Mean axial discrepancy was significantly smaller in the Group WM and Group MS then in the Group RP and Group SLM. Mean incisal discrepancies was significantly smaller in the Group RP than in all other groups. Conclusion: The marginal and axial discrepancies of the Co-Cr coping fabricated by the Wax pattern milling and Milling/Sintering method were better than those of the other groups. The marginal, cervical and axial fit of Co-Cr copings in all groups are within a clinically acceptable range.