• Restorative Dentistry and Endodontics
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• 제25권1호
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• pp.63-70
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• 2000

The properties of ideal retrograde filling materials include the ability to seal the root canal system in three dimensions and well tolerated by periradicular tissues. Biocompatibility testing has been done mainly with cytotoxicity tests using cell culture. Little attention has been paid to the potential adverse influence on the inflammatory and immune reaction in the periapical tissue. The purpose of this study was to investigate the effects of retrograde filling materials on human mononuclear cells in vitro. Freshly mixed and set specimens from six materials (Z100, Tetric Ceram, Fuji II, Fuji II LC, F2000, Compoglass Flow, and ZOE) were eluated with cell culture medium for 24 hours. Cytotoxic effects of these extracts were evaluated by determining cell viability and enzyme activity using MTT and lactate dehydrogenase (LD). The production of inflammatoy bone resorptive cytokine, TNF-${\alpha}$ was measured from human peripheral blood mononuclear cells (PBMC) exposed to the extracts by means of Endogen Human TNF-${\alpha}$ ELISA kit (Wobrun, MA, U.S.A.). Eluates and diluted (1 : 10) eluates with cell culture medium from freshly mixed Fuji IT had cytotoxic effects on mononuclear cells using MTT and LD. However, eluates from set Fuji II were not cytotoxic. Eluates form set ZOE exhibited cytotoxicity with LD test. TNF-${\alpha}$ levels were high in eluates from freshly mixed Fuji II and Z100. Diluted eluates from freshly mixed Z100 and F2000 stimulated the production of TNF-${\alpha}$. However, there were no significant difference in TNF-${\alpha}$ levels compared to controls. These results indicate that some materials could possibly stimulate bone resorption in the periapical tissue by means of the production of bone resorptive cytokine.

• 한국임상수의학회지
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• 제14권2호
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• pp.230-234
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• 1997

In vitro effect of cortisol on the proliferation of canine peripheral blood mononuclear cells (MNC) was examined. The MNC was isolated from peripheral blood by a gradient centrifugation with Picoll-Hypaque. The cell proliferation assayed using a noneradioactive 5-Bromo-2'-deoxy-uridine (BrdU) kit. The MNC proliferated well in response to either phrtobeRagg$]$utinin-p (PHA-P) or culture supernatant from MNC stimulated with PHA-p. However, these proliferative responses of MNC were not affected by addition of coitisol of 1 to 1,OOfl ng/ml. The addition of cortisol in MNC culture with either PHA-P or corture supernatBnt from MNC stimulated with PHA-P far 4 days wag not also influenced on the viabilities of cultured MNC. In conclusions it was able to assay the cell proliferation with BrdU instead of radioactive isotope e.g. tritiated thymidine (3H-TdR). These results suggested that cortisol does not at least influence on MNC proliferation in vitro.

• Journal of Genetic Medicine
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• 제5권1호
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• pp.55-60
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• 2008

목 적 : 염색체 검사는 의학의 많은 영역과 혈액종양학 분야에서 중요한 역할을 하고 있다. 세포의 기능을 잘 유지하면서 장기간 보관할 수 있는 가장 효과적인 방법은 냉동보존(cryopreservation)으로 알려져 있으며, 이는 미래에 신기술을 적용할 수 있고 회귀연구를 가능하게 한다. 이에 본 연구에서는 제대혈에서 분리된 단핵세포의 냉동전과 해동후의 염색체 검사 결과를 비교하고자 한다. 방 법 : 실험에 대한 동의서를 획득한 제대혈 1례가 검사되었다. Ficoll-Hypaue로 단핵세포를 분리하였고, DMSO 등 냉동보존을 위한 전처리 후 프로그램 냉동기(Cryomed 1010, 미국)로 냉동하여 질소탱크($-196^{\circ}C$)에 3일간 보관하였고, 급속냉동 후 염색체 검사를 시행하였다. 냉동전과 해동후의 염색체 핵형을 분석하였다. 결 과 : 1례의 제대혈은 3군으로 나누어, 냉동 전 배양과정 없던 CB-1군은 염색체 핵형 검사가 판독 불가능하였으며, 냉동 전 3일간 배양 후 냉동보관을 하였던 CB-2와 CB-3군은 냉동전과 해동후의 염색체 핵형이 판독 가능하였고, 서로 일치하였다. 결 론 : 검체의 분포 불균형과 검체 수가 적다는 제한점이 있으나, 제대혈에서 냉동전과 해동후의 염색체 핵형이 일치하는 결과에서 제대혈 단핵세포의 유전적 안정성과 장기간 보관의 가능성을 예측할 수 있을 것으로 사료된다.

• Restorative Dentistry and Endodontics
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• 제17권2호
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• pp.263-286
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• 1992

This study was performed to evaluate and compare the cytotoxic effects of five root canal sealers to several different cell lines. Five root canal sealers were AH-26, N2, Sealapex, Tubliseal, and Vitapex. Each sealers were mixed according to the manufacturer's instructions, and culture media were added to each sealers immediately after mixing (the immediate group) and after three days (the third day group) and seven days (the seventh day group) respectively. And every sealer solutions were diluted to 1:1, 1:2, 1:3 and 1:4. Three different permanent cell lines (HEp-2, McCoy, MRC-S) and human gingival fibroblasts and mononuclear cells were challenged by each sealer solution and the cytopathic effects were evaluated using MTT-ELISA, MTT-microscopy, and lactate dehydrogenase (LD) activity. The results were as follows: 1. In HEp-2 and MRC-5 cells, Vitapex was the least cytotoxic sealers. 2. AH-26 showed mild cytotoxic effects to HEp-2, gingival fibroblast and mononuclear cells. 3. N2 was the most toxic sealer to gingival fibroblast and it showed relatively strong cytotoxicity to HEp-2, McCoy and MRC-S cells. 4. Tubliseal showed strong cytotoxic effects to HEp-2, McCoy, MRC-S, and mononuclear cells. 5. Sealapex showed strong cytotoxic effect to HEp-2, McCoy, and gingival fibroblasts.

• 대한수의학회지
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• 제42권2호
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• pp.209-217
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• 2002

This study was performed to produce monoclonal antibodies (mAb) specifically reacting with chicken leukocyte surface antigens. Popliteal lymph node cells of BALB/c mice previously immunized through foot-pad with peripheral blood mononuclear cells (PBMC) of chickens separated by Ficoll-Histopaque method. They were fused with P3X63Ag14 mouse myeloma cells. A total of 34 hybridomas secreted antibodies specifically binding to the PBMC. According to the reactivity patterns with PBMC, the mAbs were divided into 4 groups. Group 1 mAbs (IIB3, IIB10, IIE10) specifically reacted with non-adherent lymphocytes but not with adherent cells which were mainly composed of thrombocytes and monocytes in PBMC culture. These mAbs were reactive with 25-59% of thymus cells and 42-64% of spleen cells of chickens. They did not show any significant reactivity with cells in the bursa of Fabricius, T-cell (MDCC-MSB1) and B-cell (LSCC-1104B1) lines. These results indicate that Group I mAbs specifically reacted with T-lymphocyte subpopulation. Monoclonal antibodies in Group II (IC6, IG2-2 and IID9) showed specific reactivity with monocytes but not with thrombocytes or non-adherent cells in PBMC culture. These mAbs, though not reacted with the chicken macrophage cell line, HD11, also bound to macrophages of the spleen and lung in immunohistochemical staining. Five mAbs in Group III showed characteristics of binding to lymphocytes and monocytes, but not to thrombocytes. Twenty-three mAbs in Group IV showed specific reactivity to lymphocytes, monocytes, and thrombocytes. Two mAbs (IC3 and IE9) in Group IV reacted with most of PBMC.

• IMMUNE NETWORK
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• 제1권3호
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• pp.230-235
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• 2001

Background: Finding of the regulation of various gene expression by cytokine including $IFN-{\gamma}$ in hematopoietic stem cell will light up the understanding of pathogenesis of aplastic anemia in various aspects. To study on aplastic anemia, however, we have to circumvent the difficulty of directly obtaining bone marrow stem cells from the patient. Therefore, we tried to find out a cell can replace the bone marrow stem cells for study on cell signaling pathway and regulation of gene expression by $IFN-{\gamma}$. Materials and Methods: HL-60 cells, of 20 ng/mL of $IFN-{\gamma}$. Total RNA was isolated from the cells and RT-PCR of the indoleamine 2,3-dioxygenase (IDO), $IFN-{\gamma}$, TNF-${\alpha}$, $MIP-1{\alpha}$, and $TGF-{\beta}2$ was carried out for the estimation of the gene expression. Results: $IFN-{\gamma}$ induced IDO gene expression of mononuclear cells from umbilical cord blood showed similar pattern as compared to that of bone marrow. Whether $INF-{\gamma}$ was treated or not, $TNF-{\alpha}$ was expressed in both mononuclear cells from umbilical cord blood and bone marrow. However, HL-60 cells showed different expression patterns. HL-60 cells would express neither IDO nor $TNF-{\alpha}$ even under the culture with 20ng/mL of $IFN-{\gamma}$. Conclusion: Our results showed bone marrow can be replaced with mononuclear cells from umbilical cord blood in the study on the relation between aplastic anemia and $IFN-{\gamma}$ including $IFN-{\gamma}$ cell signaling pathway.

• 대한핵의학회지
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• 제29권1호
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• pp.125-132
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• 1995

The purpose of this study was to establish mononuclear cell cultures such as lymphocytes or buffy coat for the biological dosimetry of in vitro Irradiation of the radionuclide Tc-99m in order to exclude the effect of residual doses seen in the cultures of whole blood. Biological do simetry of Tc-99m on cultured mononuclear cells at doses ranging from 0.05 to 6.00 Gy, by scoring unstable chromosomal aberrations(Ydr) observed in cultured lymphocytes, were performed using peripheral venous blood of healthy normal person. The results showed that; (1) In vitro irradiation of radioisotope in separated lymphocyte or buffy coat showed trace amount of residual doses of isotope after washing. Residual doses of isotopes are increased in proportion to exposed time and irradiated dose without difference between I-131 and Tc-99m. (2) We obtained these linear-quadratic dose response equations in lymphocyte and buffy coat culture after in vitro irradiation of Tc-99m, respectively (Ydr = 0.001949 $D^2$ +0.006279D + 0.000185; Ydr= 0.002531 $D^2$-0.003274 D+0.003488). In conclusion, the linear quadratic dose-response equation from in vitro irradiation of Tc-99m with lymphocyte and buffy coat culture was thought to be useful for assessing Tc-99m induced biological effects. And mono-nuclear cell cultures seem to be the most appropriate experimental model for the assessment of biological dosimetry of internal irradiation of radionuclides.

• Clinical and Experimental Vaccine Research
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• 제12권4호
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• pp.328-336
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• 2023

Purpose: Human peripheral blood mononuclear cell (PBMC)-based in vitro systems can be of great value in the development and assessment of vaccines but require the right medium for optimal performance of the different cell types present. Here, we compare three commonly used media for their capacity to support innate and adaptive immune responses evoked in PBMCs by Toll-like receptor (TLR) ligands and whole inactivated virus (WIV) influenza vaccine. Materials and Methods: Human PBMCs were cultured for different periods of time in Roswell Park Memorial Institute (RPMI), Dulbecco's minimal essential medium (DMEM), or Iscove's modified DMEM (IMDM) supplemented with 10% fetal calf serum. The viability of the cells was monitored and their responses to TLR ligands and WIV were assessed. Results: With increasing days of incubation, the viability of PBMCs cultured in RPMI or IMDM was slightly higher than that of cells cultured in DMEM. Upon exposure of the PBMCs to TLR ligands and WIV, RPMI was superior to the other two media in terms of supporting the expression of genes related to innate immunity, such as the TLR adaptor protein gene MyD88 (myeloid differentiation factor 88), the interferon (IFN)-stimulated genes MxA (myxovirus resistance protein 1) and ISG56 (interferon-stimulated gene 56), and the leukocyte recruitment chemokine gene MCP1 (monocyte chemoattractant protein-1). RPMI also performed best with regard to the activation of antigen-presenting cells. As for adaptive immunity, when stimulated with WIV, PBMCs cultured in RPMI or IMDM contained higher numbers of IFNγ-producing T cells and secreted more immunoglobulin G than PBMCs cultured in DMEM. Conclusion: Taken together, among the different media assessed, RPMI was identified as the optimal medium for a human PBMC-based in vitro vaccine evaluation system.

• 대한치과교정학회지
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• 제25권6호
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• pp.697-704
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• 1995

Chicken calvarial bone is known to contain various cell types, but their exact composition is unknown. By characterizing the chicken calvarial bone biochemically, it can be used to study biochemical, histochemical actions of bone cells in general. Calvaria of 18-day-old white leg horn embryo was aseptically dissected and bone cell populations were isolated by sequential enzymatic digestion. Histochemical study for osteoclast-like bone cell. population was performed with tartrate resistant acid phosphatase(TRAP) stain and for osteoblast-like bone cell population, alkaline phosphatase(ALP) stain was performed. Biochemical study for osteoblast-like bone cell population was performed using alkaline phosphatase(ALP) assay. Following conclusions were obtained from this study. 1. TRAP positive multi and mononuclear cells were mostly observed in group I and II, indicating that osteoclast-like bone cell population is mostly found in these groups. 2. All the cultured groups showed almost equal ALP activities and were positive for ALP stain, indicating that osteoblast-like bone cell population is evenly dispersed in all culture groups. 3. Experimental group treated with $1,25(OH)_{2}D_3$ showed increase in ALP activity in contrast to the control group, confirming previous studies that $1,25(OH)_{2}D_3$ increases ALP activities in in vitro bone cultures. 4. Results from von Kossa's stain indicated that in vitro bone formation had occured after 3 weeks of culture with beta-glycero phosphate.

• 한국임상수의학회지
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• 제37권2호
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• pp.61-66
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• 2020

Nickel is a nutritionally essential trace element that plays an important role in the immune system of several animal species. The aim of this study was to examine the effect of nickel chloride on chemotactic activity of peripheral blood polymorphonuclear cells (PMNs) and whether this effect is associated with interleukin (IL)-8 and a nuclear factor-kappa B (NF-κB)-dependent pathway. Peripheral blood mononuclear cells (PBMCs) and PMNs were isolated by Percoll solution (Specific gravity; 1.080) and 1.5% dextran treatment, respectively. A modified Boyden chamber assay was used to measure the chemotactic activity of PMNs. The level of IL-8 in culture supernatant from PBMCs was measured by enzyme-linked immunosorbent assay (ELISA). Both of PBMCs and PMNs exhibited a low viability when cultured with concentration of greater than 1,000 μM of nickel chloride for 24 h. Thus, nickel chloride was used at concentration of 500 μM, which preserved cell viability. Treatment with nickel did not directly affect the chemotactic activity of PMNs. However, the chemotactic activity of PMNs was remarkably increased by culture supernatant from PBMCs treated with nickel chloride (500 μM) for 24 h. Recombinant porcine IL-8 polyclonal antibody (pAb) neutralized the enhancing effect on the chemotactic activity of PMNs by culture supernatant from PBMCs treated with nickel and this culture supernatant had higher IL-8 levels than the culture supernatant from untreated PBMCs. In addition, n-tosyll-phenylalanine chloromethyl ketone (TPCK), a NF-κB inhibitor, antagonized the enhancing effect on the chemotactic activity of PMNs by the culture supernatant from PBMCs treated with nickel. These results suggested that nickel stimulates porcine PBMCs to produce IL-8, which increases the chemotaxis of PMNs via NF-κB-dependent pathway.