OBJECTIVETo purpose of study aimed at investigating the technique of culturing oral epithelia in vitro and to set up an experimental model for further reconstructing oral mucosa in vitro.

METHODSThe oral mucosa was taken from young New Zealand rabbits, and the mucosa was digested with enzyme and suspended in liquid to form cellular suspension. Being seeded, the cells were cultured motionlessly. The medium was changed regularly and the cells were subcultured.

RESULTSThe cultured cells were all epithelial cells without fibroblasts, and they were proved to be diploid cells. The cells were subcultured in 1-13 generations which survived for 50-60 days.

CONCLUSIONThe oral epithelium of young New Zealand rabbit can be cultured in vitro, maintaining the ability to proliferate in a certain period. It is a pilot study to reconstruct oral tissue in vitro.

Animals ; Animals, Newborn ; Cells, Cultured ; Coculture Techniques ; Epithelial Cells ; cytology ; Female ; L Cells (Cell Line) ; Male ; Mice ; Mouth Mucosa ; cytology ; Rabbits ; Tissue Engineering

The aim of this study was to investigate the effect of hypoxia-reoxygenation on the proliferation of fibroblast, and to elucidate the role of oxygen free radicals in this process. Malme-3 fibroblast, derived from human skin fibroblast, was used for this study. The hypoxia or reoxygenation condition was made by exposing cultured cells to the environment of 95% N2, 5% CO2 or 95% room air, 5% CO2, respectively. Cell proliferation was estimated by the cell numbed, and DNA synthesis was measured by the [3H]-thymidine uptake. Release of oxygen free radicals was measured by the means of Ohkawa's method of lipid peroxidation. The effect of oxygen free radicals was confirmed by using dimethylthiourea(DMTU) and alpha-tocopherol, two known oxygen free radical scavengers. The results are as follows: 1. The dissolved oxygen of the culture medium was 8.97+/-1.23 ppm in the normal condition. When the culture dish was exposed to the hypoxic condition for 3 or 6 hours, the dissolved oxygen of the culture medium decreased markedly to the level of 3.10+/-0.46 ppm or 2.37+/-0.47 ppm, respectively 2. The number of cultured cells increased in a hypoxia duration-dependent manner up to 6 hours when the cells were cultured for 24 hours after hypoxia. The same pattern was observed in the cells cultured for 48 hours after hypoxia. Lipid peroxidation in the culture increased after the exposure to hypoxia-reoxygenation. DMTU or alpha-tocopherol blocked the increase in lipid peroxidation induced by the exposure to hypoxia-reoxygenation. 3. [3H]-thymidine uptake of the cultured cells increased after the exposure to hypoxia-reoxygenation. 4. DMTU or alpha-tocopherol blocked the proliferation of fibroblasts induced by the exposure to hypoxia-reoxygenation. The increase in lactate dehydrogenase (LDH) activity was also noted after the exposure to hypoxia-reoxygenation, and this increase was blocked by DMTU or alpha-tocopherol. These results indicate that the hypoxia-reoxygenation induces the proliferation of fibroblasts, and that oxygen free radicals play an important role in this process. Moreover, oxygen free radical scavengers may be of potential therapeutic value in preventing fibrosis.
alpha-Tocopherol ; Anoxia ; Cell Line* ; Cell Proliferation ; Cells, Cultured ; DNA ; Fibroblasts* ; Fibrosis ; Free Radical Scavengers* ; Free Radicals ; Humans* ; L-Lactate Dehydrogenase ; Lipid Peroxidation ; Oxygen* ; Skin

Human epithelial cell lines were utilized to examine the effects of anoxia on cellular growth and metabolism. Three normal human epithelial cells lines (A549, NHBE, and BEAS-2B) as well as a cystic fibrosis cell line (IB3-1) and its mutation corrected cell line (C38) were grown in the presence and absence of oxygen for varying periods of time. Interleukin-8 (IL-8) levels were measured by enzyme-linked immunosorbent assay technique. Cellular metabolism and proliferation were assayed by determining mitochondrial oxidative burst activity by tetrazolium compound reduction. The viability of cells was indirectly measured by lactate dehydrogenase release. A549, NHBE, and BEAS-2B cells cultured in the absence of oxygen showed a progressive decrease in metabolic activity and cell proliferation after one to three days. There was a concomitant increase in IL-8 production. Cell lines from cystic fibrosis (CF) patients did not show a similar detrimental effect of anoxia. However, the IL-8 level was significantly increased only in IB3-1 cells exposed to anoxia after two days. Anoxia appears to affect certain airway epithelial cell lines uniquely with decreased cellular proliferation and a concomitant increased production of a cytokine with neutrophilic chemotactic activity. The increased ability of the CF cell line to respond to anoxia with increased secretion of inflammatory cytokines may contribute to the inflammatory damage seen in CF bronchial airway. This study indicates the need to use different cell lines in in vitro studies investigating the role of epithelial cells in airway inflammation and the effects of environmental influences.
Anoxia ; Cell Line ; Cell Proliferation ; Cystic Fibrosis ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; Humans ; Inflammation ; Interleukin-8 ; L-Lactate Dehydrogenase ; Neutrophils ; Oxygen ; Respiratory Burst

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.
Biocompatible Materials/*toxicity ; Bone Substitutes/*toxicity ; Cell Survival/drug effects ; *DNA Damage ; Hydroxyapatites/*toxicity ; L Cells (Cell Line) ; *Mutagenicity Tests ; *Prostheses and Implants

Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.
Apoptosis/drug effects ; Biocompatible Materials/*toxicity ; Bone Substitutes/*toxicity ; Cell Death/*drug effects ; Durapatite/*toxicity ; L Cells (Cell Line) ; *Prostheses and Implants

BACKGROUND: Chronic pain is often associated with changes in the immune responses, which highlights the need for the aggressive pain control to obtain a better prognosis. This study examined splenic NK cell cytotoxicity in an attempt to assess the possible changes in the immune function under chronic neuropathic pain after a partial transsection of the sciatic nerve. METHODS: After confirming tactile allodynia in response to the von Frey filament, a modified lactate dehydrogenase (LDH) release assay was used to determine the cytotoxic activity of splenic NK cells on the YAC-1 cell line in C3H/HeN (H-2k) mice (n = 6). NK cells as effector cells were mixed with YAC-1 cells as target cells (1 x 10(4)/100microliter), resulting in an effector-target ratio of 1 : 25, 1 : 50, 1 : 100 in the culture medium. RESULTS: At 1 and 2 weeks after the nerve injury, all the subjects showed significant mechanical sensitivity compared with those observed before surgery. The percentage of NK cell cytotoxicity of the neuropathic mice increased significantly 1 week after the nerve injury but decreased within 2 weeks compared with the normal mice. CONCLUSIONS: In terms of the altered NK cell cytotoxicity, neuropathic pain can cause changes in the normal performance of the immune function.
Animals ; Cell Line ; Chronic Pain ; Cytotoxicity Tests, Immunologic ; Hyperalgesia ; Immune System ; Killer Cells, Natural* ; L-Lactate Dehydrogenase ; Mice* ; Neuralgia ; Peripheral Nerve Injuries* ; Peripheral Nerves* ; Prognosis ; Sciatic Nerve

OBJECTIVETo explore whether coal tar pitch smoke extract (CTP) induced pyroptosis in human bronchial epithelial cells (BEAS-2B).

METHODSBEAS-2B cells were treated with different concentrations of CTP (1, 3 µg/ml) for 8h and 24 h, respectively. Lactic dehydrogenase (LDH) activity and interleukin-1 beta (IL-1β) levels in the supernatants of cell culture media were measured with LDH activity or human IL-1β ELISA kit, respectively. The activity of Caspase-1 was measured with Caspase-1 colorimetric assay kit.

RESULTSThe activity of caspase-1 in 1 and 3 µg/ml CTP groups were (9.29 ± 0.30) and (8.67 ± 0.59) µmol/ml respectively which were both significantly increased compared to that (7.42 ± 0.59) µmol/ml in the control group (P < 0.05) after 8 h exposure, but there was no significant difference in the activity of LDH and levels of IL-1β in the cell culture media among the CTP and control groups. 24 h after exposure, the activity of LDH in the CTP (1, 3 µg/ml) groups were (1323.03 ± 28.53) and (1148.45 ± 16.42) U/dl respectively which were significantly higher than that (1091.93 ± 26.64) U/dl in the control group (P < 0.05), and the levels of IL-1β in the CTP (1 and 3 µg/ml) groups were (125.37 ± 25.00) pg/ml and (92.04 ± 19.09) pg/ml respectively which were significantly higher than that (46.20 ± 14.43) pg/ml in the control group (P < 0.05), but there was no significant difference in the activity of Caspase-1 among CTP and control groups (P < 0.05).

CONCLUSIONCTP treatment induced early increase in caspase-1 activity followed by the increase in LDH activity and IL-1 levels, indicative of pyroptosis in human bronchial epithelial cells.

Apoptosis ; Bronchi ; cytology ; Caspase 1 ; metabolism ; Cell Line ; Coal Tar ; adverse effects ; Epithelial Cells ; cytology ; Humans ; Interleukin-1beta ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Smoke ; adverse effects

OBJECTIVE: To investigate the role and mechanism of bombesin receptor subtype 3 (BRS-3) in the proliferation and protection against injury of human brochial epithelial cells (HBECs). METHODS: Effect of P3513 (a specific agonist of BRS-3) on the proliferation of HBECs was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method; the release rate of 3H-Udr, and LDH activity, catalase activity, and the expression of cadherin and integrin beta1 were also analyzed under O3 stress with or without P3513 treatment. RESULTS: The proliferation of HBECs was accelerated by P3513 in a concentration-dependent manner (10(-9) approximately 10(-7) mol/L). Ozone stress could promote the release rate of 3H-Udr, and LDH activity, which could be inhibited by P3513. P3513 could promote the activity of catalase. The effect of proliferation and protection against injury caused by P3513 could be inhibited by W7 (calmodulin inhibitor), PD98059 (tyrosin kinase inhibitor) and H89 (PKA inhibitor). P3513 could stimulate the expression of caderin and integrinbeta1 of ozone-stressed HBECs. CONCLUSION Activation of BRS-3 caused by P3513 may play an important role in protecting HBECs from oxidant injury, and the signal pathway is possibly relevant to Ca2+, MEK and PKA.
Bronchi ; cytology ; Cadherins ; metabolism ; Catalase ; metabolism ; Cell Line ; Cell Proliferation ; Epithelial Cells ; cytology ; Humans ; Integrin beta1 ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Protective Agents ; Reactive Oxygen Species ; adverse effects ; Receptors, Bombesin ; physiology

BACKGROUND AND OBJECTIVECytokine-induced killer (CIK) cells and autologous dendritic cells-CIK (DC-CIK) cells co-cultured with autologous dendritic cells (DCs) and CIK cells are commonly used for immunotherapy recently. We compared the anti-tumor immune response of CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells to explore a more effective anti-tumor adoptive immunotherapy approach.

METHODSPeripheral monocytes were isolated from patients with renal carcinoma, lung cancer, or maxillary squamous cell carcinoma and their healthy adult children. Isolated cells were cultured and induced as DCs and CIK cells in vitro. CIK cells from patients were co-cultured with autologous DCs and DCs from their children respectively, generating DC-CIK cells and semi-allogeneic DC-CIK cells. The anti-tumor activities of autologous CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells were measured by LDH assay. Intracellular staining was used to test the secretion of cytokines. Flow cytometry was applied for detecting the phonotype changes of these three types of cells. Cell proliferation and cell apoptosis were detected by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and Annexin V/PI respectively.

RESULTSCompared with autologous CIK cells and DC-CIK cells, semi-allogeneic DC-CIK cells significantly enhanced the anti-tumor activity and IFN-gamma secretion, reduced IL-4 secretion, increased the ratio of CD3(+)CD56(+) cells and CD3(+)CD8(+) cells, decreased the number of CD4(+)CD25(+) cells, promoted cell proliferation, and lessened cell apoptosis.

CONCLUSIONSSemi-allogeneic DC-CIK cells had a stronger anti-tumor effect than did autologous CIK cells and DC-CIK cells. Our results provided experimental evidence for clinical application of DC-CIK cells.

Apoptosis ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Cytokine-Induced Killer Cells ; cytology ; immunology ; metabolism ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Hep G2 Cells ; Humans ; Immunotherapy, Adoptive ; Interferon-gamma ; secretion ; Interleukin-4 ; secretion ; K562 Cells ; Kidney Neoplasms ; metabolism ; pathology ; L-Lactate Dehydrogenase ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Maxillary Neoplasms ; metabolism ; pathology

BACKGROUND: Chronic myelogenous leukemia is a chronic myeloproliferative disorder characterized by leukocytosis with myeloid elements at all stages of differentiation, t(9;22)(q34;q11) and bcr/abl rearrangement. We studied hydroxyurea induced apoptotic changes such as externalization of phosphatidylserine, caspase activities on human chronic myelogenous leukemic cell line, K562 cells. METHODS: K562 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and treated hydroxyurea. Viability was examined by MTT assay. Apoptosis were examined by annexin V stain, caspase (such as caspase-, caspase-, caspase-, caspase-, and caspase-) activities, and DNA fragmentation. RESULTS: The viability of K562 cells were markedly decreased in a dose dependent manner of hydroxyurea. Phosphatidylserine externalization was detected by annexin V stain after 3 hours in hydroxyurea treated K562 cells and the value of lactate dehydrogenase was not significantly changed in their culture media. The upstream effector of caspase- was slightly increased and had influenced on caspase-. And downstream acting caspase protease of caspase- was markedly increased in a time dependent manner at hydroxyurea treated K562 cells. In addition, however the activities of caspase- and caspase- were not increased. We also found DNA fragmentation at hydroxyurea treated K562 cells between 48 hours and 72 hours on agarose gel electrophoresis. CONCLUSIONS: Hydroxyurea induces apoptotic change in K562 cells via externalization of phosphatidylserine, activations of caspase-, caspase-, caspase- proteases, and DNA fragmentation.
Annexin A5 ; Apoptosis ; Cell Line* ; Culture Media ; DNA Fragmentation ; Electrophoresis, Agar Gel ; Humans ; Hydroxyurea* ; K562 Cells* ; L-Lactate Dehydrogenase ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukocytosis ; Myeloproliferative Disorders ; Peptide Hydrolases