• Title, Summary, Keyword: Disc cells

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Different Expression of Extracellular Matrix Genes: Primary vs. Recurrent Disc Herniation

  • Kuh, Sung-Uk;Kwon, Young-Min;Chin, Dong-Kyu;Kim, Keun-Su;Jin, Byung-Ho;Cho, Yong-Eun
    • Journal of Korean Neurosurgical Society
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    • v.47 no.1
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    • pp.26-29
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    • 2010
  • Objective: Recurrent lumbar disc herniation has been reported to occur in 5% to 15% of surgically treated primary lumbar disc herniation cases. We investigated the molecular biologic characteristics of primary herniated discs and recurrent discs to see whether the recurrent discs has the similar biological features with primary herniated discs. Methods: Primary hemiated disc and recurrent disc cells were obtained by discectomy of lumbar disc patients and cells were isolated and then taken through monolayer cultures. We compared chondrogenic and osteogenic mRNA gene expression, and western blot between the two groups. Results: The mRNA gene expression of recurrent disc cells were increased 1.47* times for aggrecan, 1.38 times for type I collagen, 2.04 times for type II collagen, 1.22 times for both Sox-9 and osteocalcin, and 1.31 times for alkaline phosphatase, respectively, compared with the primary herniated lumbar disc cells (*indicates p < 0.05). Westem blot results for each aggrecan, type I collagen, type II collagen, Sox-9, osteocalcin, and alkaline phosphatase were similar between the primary herniated disc cells and recurrent disc cells. Conclusion: These results indicate that the recurrent disc cells have similar chondrogenic and osteogenic gene expression compared to primary herniated disc cells. Therefore, we assumed that the regeneration of remaining discs could fill the previous discectomy space and also it could be one of the factors for disc recurrence especially in the molecular biologic field.

The Relation Between Sox9, TGF-${\beta}1$, and Proteoglycan in Human Intervertebral Disc Cells

  • Lee, Yong-Jik;Kong, Min-Ho;Song, Kwan-Young;Lee, Kye-Heui;Heo, Su-Hak
    • Journal of Korean Neurosurgical Society
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    • v.43 no.3
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    • pp.149-154
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    • 2008
  • Objective: The aim of this study is to elucidate the effects of transforming growth factor-${\beta}$ (TGF-${\beta}$)1 and L-ascorbic acid on proteoglycan synthesis, and the relationship between Sox9, proteoglycan, and TGF-${\beta}1$ in intervertebral disc cells. Methods: Human intervertebral disc tissue was sequentially digested to 0.2% pronase and 0.025% collagenase in DMEM/F-12 media and extracted cells were cultured in $37^{\circ}C$, 5% $CO_2$ incubator. When intervertebral disc cells were cultured with TGF-${\beta}1$ or L-ascorbic acid, the production level of sulfated glycosaminoglycan (sGAG) was estimated by dimethyl methyleneblue (DMMB) assay. The changes of Sox9 mRNA and protein levels via TGF-${\beta}1$ were detected by RT-PCR and Western blot analysis in each. Results: The amount of sGAG was increased with the lapse of time during incubation, and sGAG content of pellet cultured cells was much larger than monolayer culture. When primary cultured intervertebral disc cells in monolayer and pellet cultures were treated by TGF-${\beta}1$ 20 ng, sGAG content of experimental group was increased significantly compared to control group in both cultures. L-Ascorbic acid of serial concentrations (50-300 ug/ml) increased sGAG content of mono layer cultured intervertebral disc cells significantly in statistics. The co-treatment of TGF-${\beta}1$ and L-ascorbic acid increased more sGAG production than respective treatment. After treating with TGF-${\beta}1$, Sox9 mRNA and protein expression rates were significantly increased in disc cells compared with the control group. Conclusion: This study suggests that TGF-${\beta}1$ would increase sulfated glycosaminoglycan (sGAG) and other proteoglycans such as versican by elevating Sox9 mRNA and protein expressions in order.

Small Interfering RNA-Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells

  • Park, Jong-Beom;Park, Chanjoo
    • Asian Spine Journal
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    • v.11 no.5
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    • pp.686-693
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    • 2017
  • Study Design: In vitro cell culture model. Purpose: To investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells. Overview of Literature: Synthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear. Methods: Rat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls. Results: Serum deprivation increased apoptosis by 40.3% (p<0.001), decreased proliferation by 45.3% (p<0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures (p<0.001). Finally, Fas siRNA-mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures (p<0.05 for both). Conclusions: The observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.

Immunochemical Localization of Tetrahydrocannabinol (THC) in Chemically Fixed Glandular Thrichomes of Cannabis (Cannabaceae)

  • Eun Soo Kim;Paul G. Mahlberg
    • Animal cells and systems
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    • v.3 no.2
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    • pp.215-219
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    • 1999
  • Monoclonal antibody for delta-9-tetrahydrocannabiol (THC Ab), conjugated with protein A-gold, was employed as a probe to detect THC localization in the gland and subjacent cells of chemically fixed bracts of Cannabis. THC was detected in the outer wall of the disc cells, fibrillar matrix, the surface feature of secretory vesicles, and sheath throughout development of the secretory cavity. The probe was absent from vesicles. Label was also present in anticlinal walls of disc cells and walls of dermal and mesophyll cells. Little or no THC Ab was present in disc cells and none were detected in control tissues. This distribution pattern of THC Ab was similar to that in tissues prepared by high pressure cryofixation-cryosubstitution. Consistent association of THC with wall and wall-derived materials suggests that cannnabinoids are synthesized outside the plasma membrane and bound to a wall component, where-upon they are transported to the cavity with wall materials released from the disc cell wall during development of the secretory cavity.

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CHANGES IN THE SHAPE AND ULTRASTRUCTURE OF THE ARTICULAR DISC OF THE RAT MANDIBULAR JOINT WITH AGING (가령에 따른 백서 악관절 원판의 형태 및 미세구조적 변화)

  • Suh, Hye-Kyung;Kyung, Hee-Moon;Sung, Jae-Hyun;Bae, Yong-Chul
    • The korean journal of orthodontics
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    • v.24 no.2
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    • pp.331-348
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    • 1994
  • The purpose of this study was to investigate changes in the shape and ultrastructure of the articular disc of the rat mandibular joint with aging. Mechanical stress applied to the articular disc changes during neonatal, suckling, juvenile, adult and senile stages. Mandibular joints of 6 groups of rats(1-, 7-, 17-, 27-, 55-day and over-1-year groups) were removed en bloc and processed for light and electro microscopic study. The changes in the shape of articular disc were examined by light microscope in each group. Structural and ultrastructural changes in the articular disc were examined by light and electron microscope in each group. The results were as follows : In the 1-day and 7-day groups, the articular disc was long and slender in shape and the articular disc was not fitted with the shape of the mandibular fossa and condyle. However' after that time, the anterior and posterior portions of the articular disc were more bulged and the middle portion was shorter and biconcave. Thus the articular disc was well fitted with the shape of the mandibular fossa and condyle. The cell density decreased with aging. In the l -day and 7-day groups, the Golgi apparatus, rough endoplasmic reticulum and free ribosome, which are involved in the synthesis of intracellular and extracellular matrix, were developed. In the 17-day, 27-day and 55-day groups, not only the cell organelles involved in the synthesis of the intracellular and extracellular matrix but also the cell organelles involved in the remodeling of the extracellular matrix(i.e., finger-like cell process, lysosome and mitochondria)were well developed. With advancing age, intracytoplasmic microfilaments were more accumulated and condroid cells increased. In the over-1-year group, the majority of cells of the articular disc were chondroid cells. The majority of cytoplasmic compartment were filled with intracytoplasmic microfilaments and cell organelles were not developed. Therefore, metabolic activities of the cell was markedly reduced and cells contained structures enduring mechanical stress, and cells which were in the process of degeneration were observed occasionally.

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Gene Therapy Using GM-CSF Gene Transferred by a Defective Infectious Single-cycle Herpes Virus in Micro-residual Organotropic Head and Neck Squamous Cell Cancer Model (향장기성 두경부 편평세포암종의 미세잔존암 모델에서 GM-CSF 유전자를 이입시킨 제한복제성 헤르페스바이러스 벡터를 이용한 종양백신의 유전자 치료)

  • Kim Se-Heon;Choi Eun-Chang;Kim Han-Su;Chang Jung-Hyun;Kim Ji-Hoon;Kim Kwang-Moon
    • Korean Journal of Head & Neck Oncology
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    • v.19 no.1
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    • pp.25-33
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    • 2003
  • Background and Objectives: The Herpes Simplex type 2 Defective Infectious Single Cycle virus (DISC virus) is attenuated virus originally produced as viral vaccines but are also efficient gene transfer vehicle. The main goals of this study were to examine the efficiencies of the gene transfer using DISC vectors for various head and neck squamous cell carcinoma cell lines and to evaluate the efficacy of vaccination with DISC virus carrying a immunomodulatory genes (GM-CSF) as cancer therapy in a organotopic oral cavity squamous cell cancer model. Materials and Methods : We determinated the gene transfer efficiency of DISC virus by x-gal stain method and proved gene and protein expression of DISC-GMCSF transfected SCCVII cells by RT-PCR and ELISA method. Also we evaluated the ex vivo vaccination effects of SCCVII/GMCSF (DISC-GMCSF transfected SCCVII vaccine) vaccine on preventing the recurrence of micro-residual tumor. After the vaccination of SCCVII/GMCSF, specific cytotoxic T-cell responses was evaluated by CTL assay. Results: At an MOI of 10 DISC virus showed 64-88% of transfection rates in various head and neck squamous cancer cell lines. SCCVII cells transduced by DISC virus vector (MOI=10) carrying the GM-CSF gene, produced 4.5 nanogram quantities of GM-CSF per $10^6$ cells. In vivo vaccination using tumor cells transduced ex vivo with DISC-GMCSF resulted in better protection rate against subsequent tumor recurrence in organotopic oral cavity cancer model. Although tumor free survival rate was not statistically significantly increased in vaccination group (p=0.078), tumor specific cytotocic T-cell responses were significantly increased in SCCVII/GMCSF vaccination group. Conclusion: These data demonstrate that; 1) The DISC virus vector is capable of efficient gene transfer to various head and neck squamous cancer cell lines, 2) GM-CSF secreting genetically modified tumor vaccine (SCCVII/GMCSF) efficiently protected against tumor recurrence in organotopic micro-residual oral cavity cancer model and produced tumor specific cytotoxic T-cell response. DISC virus-mediated, cytokine gene transfer may prove to be useful as a clinical therapy for head and neck cancers.

Detection of O-Linked-N-Acetylglucosamine Modification and Its Associated Enzymes in Human Degenerated Intervertebral Discs

  • Nikolaou, Georgios;Zibis, Aristeidis Hristos;Fyllos, Apostolos H.;Katsioulis, Antonios;Sotiriou, Sotirios;Kotrotsios, Anastasios;Sgantzos, Markos;Vassiou, Aikaterini;Arvanitis, Dimitrios L.
    • Asian Spine Journal
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    • v.11 no.6
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    • pp.863-869
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    • 2017
  • Study Design: Human herniated discs were obtained from discectomy specimens for the immunohistochemical detection of O-GlcNAc and O-GlcNAcase (OGA)/O-GlcNAc transferase (OGT). Purpose: This study aimed to quantify the extent of O-GlcNAcylation and its associated enzymes (OGT/OGA) in human degenerated intervertebral discs. Overview of Literature: The O-GlcNAcylation of nuclear, cytoplasmic, and mitochondrial proteins as well as the effects of such post-translational modifications are currently the focus of extensive research. O-GlcNAcylation is believed to contribute to the etiology of chronic illnesses by acting as a nutrient and stress sensor in the cellular environment. Mature intervertebral disc cells are chondrocyte-like cells, and O-GlcNAc has been shown to promote chondrocyte apoptosis in vitro. We believe that O-GlcNAcylation is a key regulator of disc degeneration. Methods: Fifty-six specimens were fixed for 24 hours in a 10% solution of neutral-buffered formaldehyde, dehydrated, and embedded in paraffin. Tissue slices ($4-{\mu}m-thick$) were used for hematoxylin-eosin staining and immunohistochemistry. Results: We found that O-GlcNAcylation of cytoplasmic proteins was less than that of nuclear proteins in both single cells and cell clusters. Cytoplasmic O-GlcNAcylation occurs subsequent to nuclear O-GlcNAcylation and is directly proportional to disc degeneration. OGT and O-GlcNAc expression levels were identical in all specimens examined. Conclusions: O-GlcNAc and OGA/OGT expression is shown to correlate for the first time with intervertebral disc cell degeneration. Increasing disc degeneration is associated with increasing O-GlcNAcylation in both nuclear and cytoplasmic proteins in human disc cells.

Notochordal Cells Influence Gene Expression of Inflammatory Mediators of Annulus Fibrosus Cells in Proinflammatory Cytokines Stimulation

  • Moon, Hong-Joo;Joe, Hoon;Kwon, Taek-Hyun;Choi, Hye-Kyoung;Park, Youn-Kwan;Kim, Joo-Han
    • Journal of Korean Neurosurgical Society
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    • v.48 no.1
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    • pp.1-7
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    • 2010
  • Objective : Notochordal cells in the intervertebral disc interact with nucleus pulposus (NP) cells and support the maintenance of disc homeostasis by regulation of matrix production. However, the influence of notochordal cells has not been evaluated in the annulus fibrosus (AF), which is the primary pain generator in the disc. We hypothesized that the notochordal cell has the capacity to modulate inflammatory mediators secreted by AF cells secondary to stimulation. Methods : Notochordal and AF cells were isolated from adult New Zealand white rabbits. AF pellets were cultured with notochordal cell clusters or in notochordal cell-conditioned media (NCCM) for 24 or 48 hours with proinflammatory cytokines at varying concentrations. Gene expression in AF pellets were assayed for nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, and interleukin (IL)-6 by real time reverse transcriptase polymerase chain reaction (RT-PCR). Results : AF pellet in NCCM significantly decreased the iNOS and COX-2 messenger ribonucleic acid (mRNA) levels compared to AF pellets alone and AF pellets with notochordal cells (p < 0.05). AF pellet resulted in dose-dependent iNOS and COX-2 expression in response to IL-$1{\beta}$, stimulation, demonstrating that 1 ng/ml for 24 hours yielded a maximal response. AF pellet in NCCM significantly decreased the expression of iNOS and COX-2 in response to 1ng/ml IL-$1{\beta}$, stimulation at 24 hours (p < 0.05). There was no difference in IL-6 expression compared to AF pellets alone or AF pellets with notochordal cell clusters. Conclusion : We conclude that soluble factors from notochordal cells mitigate the gene expression of inflammatory mediators in stimulated AF, as expected after annular injury, suggesting that notochordal cells could serve as a novel therapeutic approach in symptomatic disc development.

Matrix Degradative Enzymes and Their Inhibitors during Annular Inflammation : Initial Step of Symptomatic Intervertebral Disc Degeneration

  • Kim, Joo Han;Park, Jin Hyun;Moon, Hong Joo;Kwon, Taek Hyun;Park, Youn Kwan
    • Journal of Korean Neurosurgical Society
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    • v.55 no.5
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    • pp.237-243
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    • 2014
  • Objective : Symptomatic disc degeneration develops from inflammatory reactions in the annulus fibrosus (AF). Although inflammatory mediators during annular inflammation have been studied, the roles of matrix metalloproteinases (MMPs) and their inhibitors have not been fully elucidated. In this study, we evaluated the production of MMPs and tissue inhibitors of metalloproteinase (TIMPs) during annular inflammation using an in vitro co-culture system. We also examined the effect of notochordal cells on annular inflammation. Methods : Human AF (hAF) pellet was co-cultured for 48 hours with phorbol myristate acetate-stimulated macrophage-like THP-1 cells. hAF pellet and conditioned media (CM) from co-cultured cells were assayed for MMPs, TIMPs, and insulin-like growth factor (IGF)-1 levels using real-time reverse-transcriptase polymerase chain reaction and enzyem-linked immunosorbent assay. To evaluate whether notochordal cells affected MMPs or TIMPs production on annular inflammation, hAF co-cultured with notochordal cells from adult New Zealand White rabbits, were assayed. Results : MMP-1, -3, -9; and TIMP-1 levels were significantly increased in CM of hAF co-cultured with macrophage-like cells compared with hAF alone, whereas TIMP-2 and IGF-1 levels were significantly decreased (p<0.05). After macrophage exposure, hAF produced significantly more MMP-1 and -3 and less TIMP-1 and -2. Interleukin-$1{\beta}$ stimulation enhanced MMP-1 and -3 levels, and significantly diminished TIMP-2 levels. Co-culturing with rabbit notochordal cells did not significantly influence MMPs and TIMPs production or COL1A2 gene expression. Conclusion : Our results indicate that macrophage-like cells evoke annular degeneration through the regulation of major degradative enzymes and their inhibitors, produced by hAF, suggesting that the selective regulation of these enzymes provides future targets for symptomatic disc degeneration therapy.

Histology and Morphometries of the Epidermis of the Fins and Sucking Disc of the Mudskipper, Periophthalmus modestus (Pisces, Gobiidae)

  • Park, Jong-Young;Kim, Ik-Soo;Lee, Yong-Joo;Kim, So-Young
    • Animal cells and systems
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    • v.8 no.2
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    • pp.111-115
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    • 2004
  • The epidermis of the mudskipper, Periophthalmus modestus, consists of three layers- the outermost layer, middle layer and stratum germinativum. Extensive fine blood capillaries are present near the superficial layer of epidermis and outermost layer in five fins and a sucking disc. The diffusion distance between the vascular capillaries and the surface of epidermis ranged from 3.6 to 10.9${\mu}$m: 3.6 ${\mu}$m in the sucking disc, 10.9 ${\mu}$m in the anal fin and 4.6 to 5.0 ${\mu}$m in the two dorsal fins. Rate of the surface area of respiratory epithelium, the surface area of the fine blood capillaries occupied per surface area of epidermis in 0.1mm, is 3.7 to 4.4% in two dorsal fins and 1.1% in the anal fin. The middle layer is simpler in structure consisting of small or voluminous cells swollen by epidermal cells, and this layer appeared web-like. Well-developed lymphatic spaces containing lymphocytes existed in the stratum germinativum. The five fins and sucking disc had no epidermal glands.