Name: determining the toxicity of uv radiation and chemicals on primary and immortalized human corneal epithelial cells
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1. 生物用氯环油酸和生物异氯环氧乙烷的培养
<ol><li>使用以下补充剂在人眼上皮培养基 （HOEM） 中培养 pHCEC 和 iHCEC：6 mM L-谷氨酰胺、0.002% 细胞培养基补充剂 O（材料表）、1.0 μM 肾上腺素、0.4% 细胞培养基补充剂 P（材料表）、5 μg/mL rh 胰岛素、5 μg/mL 载脂转铁蛋白和 100 ng/mL 半琥珀酸氢化可的松半琥珀酸酯在胶原蛋白-1 包被培养瓶中（75 cm2 培养瓶中为 18 mL）。...

<ol>
	<li>McCanna, D. J., Harrington, K. L., Driot, J. Y., Ward, K. W., Tchao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+human+corneal+epithelial+cell+line+for+screening+the+safety+of+contact+lens+care+solutions+in+vitro.">Use of a human corneal epithelial cell line for screening the safety of contact lens care solutions in vitro.</a> Eye &amp; Contact Lens. 34 (1), 6-12 (2008).</li><li>Xu, M., Sivak, J. G., McCanna, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+effects+of+ophthalmic+solutions+on+human+corneal+epithelial+cells+using+fluorescent+dyes.">Comparison of the effects of ophthalmic solutions on human corneal epithelial cells using fluorescent dyes.</a> Journal of Ocular Pharmacology and Therapeutics. 29 (9), 794-802 (2013).</li><li>Youn, H. Y., McCanna, D. J., Sivak, J. G., Jones, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+ultraviolet-induced+damage+in+human+corneal,+lens,+and+retinal+pigment+epithelial+cells.">In vitro ultraviolet-induced damage in human corneal, lens, and retinal pigment epithelial cells.</a> Molecular Vision. 17, 237-246 (2011).</li><li>Hakkarainen, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+cytotoxic+effects+of+marketed+ophthalmic+formulations+on+human+corneal+epithelial+cells.">Acute cytotoxic effects of marketed ophthalmic formulations on human corneal epithelial cells.</a> International Journal of Pharmaceutics. 511 (1), 73-78 (2016).</li><li>Spurr, S. J., Gipson, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+corneal+epithelium+with+Dispase+II+or+EDTA.+Effects+on+the+basement+membrane+zone.">Isolation of corneal epithelium with Dispase II or EDTA. Effects on the basement membrane zone.</a> Investigative Ophthalmology &amp; Visual Science. 26 (6), 818-827 (1985).</li><li>Kahn, C. R., Young, E., Lee, I. H., Rhim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+epithelial+primary+cultures+and+cell+lines+with+extended+life+span:+in+vitro+model+for+ocular+studies.">Human corneal epithelial primary cultures and cell lines with extended life span: in vitro model for ocular studies.</a> Investigative Ophthalmology &amp; Visual Science. 34 (12), 3429-3441 (1993).</li><li>Araki-Sasaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+SV40-immortalized+human+corneal+epithelial+cell+line+and+its+characterization.">An SV40-immortalized human corneal epithelial cell line and its characterization.</a> Investigative Ophthalmology &amp; Visual Science. 36 (3), 614-621 (1995).</li><li>Griffith, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+human+corneal+equivalents+constructed+from+cell+lines.">Functional human corneal equivalents constructed from cell lines.</a> Science. 286 (5447), 2169-2172 (1999).</li><li>Toouli, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+mammary+epithelial+cells+immortalized+by+simian+virus+40+T-Antigen+or+by+the+telomerase+catalytic+subunit.">Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit.</a> Oncogene. 21 (1), 128-139 (2002).</li><li>Kaur, G., Dufour, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines:+Valuable+tools+or+useless+artifacts.">Cell lines: Valuable tools or useless artifacts.</a> Spermatogenesis. 2 (1), 1-5 (2012).</li><li>Zorn-Kruppa, M., Tykhonova, S., Belge, G., Diehl, H. A., Engelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+corneal+cell+cultures+in+cytotoxicity+testing.">Comparison of human corneal cell cultures in cytotoxicity testing.</a> Altex. 21 (3), 129-134 (2004).</li><li>Huhtala, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+an+immortalized+human+corneal+epithelial+cell+line+and+rabbit+corneal+epithelial+cell+culture+in+cytotoxicity+testing.">Comparison of an immortalized human corneal epithelial cell line and rabbit corneal epithelial cell culture in cytotoxicity testing.</a> Journal of Ocular Pharmacology and Therapeutics. 18 (2), 163-175 (2002).</li><li>Pisella, P. J., Fillacier, K., Elena, P. P., Debbasch, C., Baudouin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+effects+of+preserved+and+unpreserved+formulations+of+timolol+on+the+ocular+surface+of+albino+rabbits.">Comparison of the effects of preserved and unpreserved formulations of timolol on the ocular surface of albino rabbits.</a> Ophthalmic Research. 32 (1), 3-8 (2000).</li><li>Youn, H. Y., Moran, K. L., Oriowo, O. M., Bols, N. C., Sivak, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+and+UV-B-induced+damage+of+the+cultured+bovine+lens.">Surfactant and UV-B-induced damage of the cultured bovine lens.</a> Toxicology in vitro. 18 (6), 841-852 (2004).</li><li>Hughes, R., Kilvington, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+hydrogen+peroxide+contact+lens+disinfection+systems+and+solutions+against+Acanthamoeba+polyphaga.">Comparison of hydrogen peroxide contact lens disinfection systems and solutions against Acanthamoeba polyphaga.</a> Antimicrobial Agents and Chemotherapy. 45 (7), 2038-2043 (2001).</li><li>Delic, N. C., Lyons, J. G., Di Girolamo, N., Halliday, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damaging+effects+of+ultraviolet+radiation+on+the+cornea.">Damaging effects of ultraviolet radiation on the cornea.</a> Photochemistry and Photobiology. 93 (4), 920-929 (2017).</li><li>Ahuja, D., Saenz-Robles, M. T., Pipas, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SV40+large+T+antigen+targets+multiple+cellular+pathways+to+elicit+cellular+transformation.">SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation.</a> Oncogene. 24 (52), 7729-7745 (2005).</li><li>Tong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pin1+inhibits+PP2A-mediated+Rb+dephosphorylation+in+regulation+of+cell+cycle+and+S-phase+DNA+damage.">Pin1 inhibits PP2A-mediated Rb dephosphorylation in regulation of cell cycle and S-phase DNA damage.</a> Cell Death &amp; Disease. 6, 1640 (2015).</li><li>Muller, P. A., Vousden, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+mutations+in+cancer.">p53 mutations in cancer.</a> Nature Cell Biology. 15 (1), 2-8 (2013).</li><li>Seshacharyulu, P., Pandey, P., Datta, K., Batra, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatase:+PP2A+structural+importance,+regulation+and+its+aberrant+expression+in+cancer.">Phosphatase: PP2A structural importance, regulation and its aberrant expression in cancer.</a> Cancer Letters. 335 (1), 9-18 (2013).</li><li>Yang, D., Okamura, H., Morimoto, H., Teramachi, J., Haneji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+2A+Calpha+regulates+proliferation,+migration,+and+metastasis+of+osteosarcoma+cells.">Protein phosphatase 2A Calpha regulates proliferation, migration, and metastasis of osteosarcoma cells.</a> Laboratory nvestigation; A Journal of Technical Methods and Pathology. 96 (10), 1050-1062 (2016).</li><li>Xie, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+and+inactivation+of+the+PP2A+complex+promotes+the+proliferation+and+angiogenesis+of+hemangioma+endothelial+cells+through+activating+AKT+and+ERK.">Disruption and inactivation of the PP2A complex promotes the proliferation and angiogenesis of hemangioma endothelial cells through activating AKT and ERK.</a> Oncotarget. 6 (28), 25660-25676 (2015).</li><li>Mallet, J. D., Rochette, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelength-dependent+ultraviolet+induction+of+cyclobutane+pyrimidine+dimers+in+the+human+cornea.">Wavelength-dependent ultraviolet induction of cyclobutane pyrimidine dimers in the human cornea.</a> Photochemical &amp; Photobiological Sciences. 12 (8), 1310-1318 (2013).</li><li>Cejkova, J., Cejka, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxidative+stress+in+corneal+diseases+and+injuries.">The role of oxidative stress in corneal diseases and injuries.</a> Histology and Histopathology. 30 (8), 893-900 (2015).</li><li>Wang, S. Q., Balagula, Y., Osterwalder, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoprotection:+a+review+of+the+current+and+future+technologies.">Photoprotection: a review of the current and future technologies.</a> Dermatologic Therapy. 23 (1), 31-47 (2010).</li><li>Svobodova, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+after+acute+exposure+of+mice+skin+to+physiological+doses+of+UVB+and+UVA+light.">DNA damage after acute exposure of mice skin to physiological doses of UVB and UVA light.</a> Archives of Dermatological Research. 304 (5), 407-412 (2012).</li><li>Kennedy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+irradiation+induces+the+production+of+multiple+cytokines+by+human+corneal+cells.">Ultraviolet irradiation induces the production of multiple cytokines by human corneal cells.</a> Investigative Ophthalmology &amp; Visual Science. 38 (12), 2483-2491 (1997).</li><li>Kode, A., Yang, S. R., Rahman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+cigarette+smoke+on+oxidative+stress+and+proinflammatory+cytokine+release+in+primary+human+airway+epithelial+cells+and+in+a+variety+of+transformed+alveolar+epithelial+cells.">Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells.</a> Respiratory Research. 7, 132 (2006).</li><li>Epstein, S. P., Chen, D., Asbell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+biomarkers+of+inflammation+in+response+to+benzalkonium+chloride+on+corneal+and+conjunctival+epithelial+cells.">Evaluation of biomarkers of inflammation in response to benzalkonium chloride on corneal and conjunctival epithelial cells.</a> Journal of Ocular Pharmacology and Therapeutics. 25 (5), 415-424 (2009).</li></ol>

评估了使用两种类型的HCEC的潜在差异。将细胞置于相同浓度的细胞的相同培养基（HOEM）中，然后暴露于短时间和长期的紫外线辐射和三种眼部毒素中。根据它们的生理效应选择紫外线辐射和化学物质的剂量，这些效应对细胞的损害足以产生可以比较的中间反应。紫外线辐射的暴露时间为5和20分钟，所选剂量的BAK （0.001%），SDS（0.0025%）和H 2 O2（0.01%）的暴露时间为5和15分钟是理想...

进行体外毒理学研究以预测可能对细胞造成损害的化学物质和其他试剂的毒性作用。在评估对角膜的毒性时，人角膜上皮细胞（HCEC）已被用于评估这些影响的模型中1，2，3，4。这些模型通常评估生理效应，例如细胞代谢活动的变化，细胞增殖和其他细胞功能，例如炎性细胞因子的产生和释放。对于这些毒理学研究，已经选择了来自不同来源的细胞来评估化学物质和紫外线辐射对HCEC的破坏作用2，3。pHCEC系可从提供来自成人供体组织的这些细胞的公司获得。原代细胞可以用分散酶处理，并轻轻刮掉角膜进行培养5。然后对细胞进行病毒和污染测试，然后用10%二甲基亚砜冷冻保存。
原代细胞系的优点是细胞在遗传上与供体的细胞相同。这是理想的，因为 体外 模型应尽可能模仿 体内 组织。原代细胞系的缺点是它们的细胞分裂或传代次数有限6。可用的细胞数量有限，限制了使用单一原代培养物可以进行的实验数量，增加了实验的成本。
永生化细胞系也已用于细胞培养毒性模型。然而，与从体内组织获得的原代细胞系不同，永生化的细胞系已经过遗传改变。永生化细胞是通过将病毒的基因结合到原代细胞6，7，8的DNA中而创建的。为永生化细胞系选择具有成功病毒基因掺入的细胞。永生化的优点是它允许无限期的快速增殖，提供无限数量的细胞来使用相同的细胞系进行多次实验。这允许实验之间的一致性并降低成本。
除了限制细胞增殖的基因变化外，关键功能基因表达的变化也可能发生9。因此，使用永生化细胞的缺点是，就对各种外部刺激的反应而言，它们可能不再 代表原始体内 细胞10。比较涉及观察化学物质对原代和永生化的人角膜角质细胞的毒性作用11，以及永生化的HCEC和兔角膜上皮原代细胞12。毒素对原代人角质细胞和永生化角质细胞的影响之间的比较显示没有显着差异11。使用本文中详述的方法，将确定这些测定法评估紫外线辐射和眼部毒素对pHCEC和iHCEC的毒性的有效性。
选择了体外测定中常用的三种眼部毒素：BAK，H 2 O2和SDS。BAK是一种阳离子防腐剂，常用于眼用溶液13，14，H2O2通常用于消毒隐形眼镜15，SDS是一种阴离子表面活性剂，存在于洗涤剂和洗发水中14。与眼部毒素类似，紫外线辐射也会对HCEC造成重大损害3。此外，过度暴露于紫外线会导致一种称为光角膜炎的眼部疾病，其特征是流泪、对光敏感和砂砾感的症状16.
可以使用无限数量的原代细胞培养物和已开发的各种永生化细胞系。因此，进行了一项研究，将原代HCEC与永生化的HCEC系进行比较，以确定包含这些类型细胞的模型之间的异同。
这项研究使用显微镜来评估pHCEC和iHCEC之间对紫外线和毒素的细胞生理反应的可能差异。还评估了紫外线辐射和化学物质对两种细胞系的细胞代谢活性和炎性细胞因子释放的影响。确定两种细胞系之间的差异的重要性在于了解这些细胞系的最佳用途，以评估：1）紫外线辐射对细胞的影响，2）毒素对细胞的影响，以及3）由此产生的代谢，细胞活力和细胞因子释放的变化，以供未来的研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>75 cm2 Vented Flask</td>
			<td>Corning</td>
			<td>354485</td>
			<td>This is the BioCoat brand, collagen-coated</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>costar</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>Fisher Scientific</td>
			<td>dal 1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin Staining buffer solution</td>
			<td>Invitrogen, Burlington, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V</td>
			<td>Invitrogen, Burlington, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 100 microscope with a Zeiss confocal laser scanning microscope 510 system</td>
			<td>Carl Zeiss Inc., Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 48 Well plates</td>
			<td>Corning</td>
			<td>354505</td>
			<td>This is the BioCoat brand, collagen-coated</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td>CYT5MPV</td>
			<td>Can read fluorescence from 280 - 700 nm (for assay 540/590)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hycone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>glass bottom coverslips</td>
			<td>MatTek Corporation, Ashland, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human Corneal Epithelial Cells</td>
			<td>University of Ottawa</td>
			<td>N/A</td>
			<td>SV40-immortalized human corneal epithelial cells from Dr. M. Griffith (Ottawa Eye Research Institute, Ottawa, Canada) and have been characterized Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science. 1999;286:2169-72.</td>
		</tr>
		<tr>
			<td>Human Ocular Epithelia Media (HOEM) with the following supplements:</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>SCMC001</td>
			<td>Epigro base media supplemented with 6 mM L-Glutamine, 0.002% EpiFactor O (cell media supplement O in article) , 1.0 &#956;M Epinephrine, 0.4% EpiFactor P (cell media supplement P in article), 5 &#956;g/mL rh Insulin, 5 &#956;g/mL Apo- Transferrin, and 100 ng/mL Hydrocortisone Hemisuccinate in Collagen-1 coated culture flasks (BioCoat, Corning, Tewksbury, MA, USA).</td>
		</tr>
		<tr>
			<td>Live Dead calcien and ethidium homodimer</td>
			<td>Invitrogen, Burlington, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MesoScale Discovery (MSD) QuickPlex SQ 120 instrument</td>
			<td>Rockville, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSD Human Proinflammatory Panel II (4-Plex) V-Plex assay</td>
			<td>Rockville, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100x concentration so add 1 mL to each 99 mL of media</td>
		</tr>
		<tr>
			<td>Primary Corneal Epithelial Cells</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>SCCE016</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax fluorescence multi-well plate reader</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express (cell disassociation solution)</td>
			<td>Fisher Scientific</td>
			<td>12605036</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining the toxicity of uv radiation and chemicals on primary and immortalized human corneal epithelial cells

本文介绍了测量紫外线 （UV） 辐射和眼毒素对原发性 （pHCEC） 和永生化 （iHCEC） 人角膜上皮细胞培养物的毒性的方法。细胞暴露于紫外线辐射和有毒剂量的苯扎氯铵（BAK），过氧化氢（H 2 O2）和十二烷基硫酸钠（SDS）。使用代谢测定法测量代谢活性。使用多重白细胞介素（IL）-1β，IL-6，IL-8和肿瘤坏死因子-α（TNF-α）测定法测量炎症细胞因子的释放，并使用荧光染料评估细胞的活力。
紫外线对细胞代谢活性和细胞因子释放的破坏性影响发生在iHCEC的紫外线暴露5分钟和pHCEC的20分钟。在暴露于BAK，H2O2 或SDS后，iHCEC和pHCEC的代谢活性下降百分比相似，IL-6和IL-8的细胞因子释放变化最显着。荧光染色的iHCEC和pHCEC BAK暴露细胞的显微镜检查显示，在0.005%BAK暴露时细胞死亡，尽管iHCEC的乙锭染色程度高于pHCEC。利用多种方法评估使用显微镜的毒性作用，评估代谢活性和细胞因子产生，可以确定原代和永生化细胞系的紫外线辐射和化学毒素的毒性。

细胞大小 用三种荧光染料可视化原代和永生化的HCEC，它们反映了细胞活力的三个不同阶段。活细胞为绿色（钙黄绿素-AM），死细胞为红色（乙锭同源二聚体-1），凋亡细胞为黄色（膜联蛋白V-计算机调整的颜色，以便更好地可视化荧光信号）。活细胞在细胞质中含有酯酶，并将钙黄绿素-AM转化为钙黄绿素。死细胞具有可渗透乙锭同源二聚体-1的细胞膜。凋亡细胞在细胞膜处有染色，...

Watch this Scientific Journal Video about Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells at JoVE.com

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

本文介绍了用于评估紫外线辐射和化学毒素对原代和永生化细胞系的毒性的程序。

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

该方法可用于体外评价新眼科产品制剂的毒性。通过在体外使用眼细胞精炼化学品和产品配方的浓度，可以最大限度地减少使用动物来评估新产品的安全性。体外测定评估无法在体内评估的毒性终点，例如确定化学物质对线粒体功能、细胞膜完整性和酶活性的影响。体外评估细胞毒性是了解潜在毒性机制的关键。这对于确定眼睛中治疗性化学物质的最大可耐受剂量至关重要。展示该程序的将是琼斯实验室的研究员Nijani Nagaarudkumaran。首先将pHCEC和iHCEC都放在胶原蛋白包被的培养皿上，玻璃底盖玻片浓度为1 x 10至第5个，含有1毫升人眼上皮培养基或HOEM。在含有 5% 二氧化碳的 37 摄氏度培养箱中培养 pHCEC 和 iHCEC 1 组培养物 24 小时，另一组培养 48 小时。孵育后，用含有4微摩尔钙黄绿素，8微摩尔乙锭同源二聚体1和膜联蛋白5的500微升膜联蛋白染色缓冲液在37摄氏度下染色细胞20分钟。染色细胞后，调整共聚焦激光扫描显微镜以捕获激发和发射波长，如文本手稿中所述。按照稿件中的说明获取二维和三维图像。在 24 孔胶原蛋白 1 包被培养板的每个孔中以每毫升 HOEM 5 x 10 至 4 分之一接种细胞，并在 37 摄氏度下用 5% 二氧化碳孵育 3 小时。孵育后，将孔中培养基的体积减少至300微升。接下来，在单独的实验中，将细胞暴露在每平方米6.48瓦的UVA辐射下，在37摄氏度下以每平方米1.82瓦的UVA辐射照射5和20分钟。紫外线辐射后，向每个孔中加入200微升新鲜HOEM，并在37摄氏度下用5%二氧化碳孵育20小时。第二天，收集每个孔中的细胞上清液并将其转移到无菌的2毫升聚丙烯管中。将上清液冷冻在零下80摄氏度。要确定 pHCEC 和 iHCEC 在紫外线照射后释放的细胞因子水平，请使用多重细胞因子测定并按照试剂盒的说明量化白细胞介素 6、白细胞介素 8、白细胞介素-1 β 和 TNF-α。在DMEM和F12中制备10%代谢测定试剂。用1毫升10%代谢测定溶液替换每个孔中的培养基，并在37摄氏度下与5%二氧化碳孵育4小时。使用荧光板读数器在530纳米激发和590纳米发射波长下测量每种溶液的荧光。在24孔胶原蛋白1包被培养板的每个孔中以每毫升HOEM的5 x 10至第四种子细胞。在 37 摄氏度下与 5% 二氧化碳一起孵育 3 小时。孵育后，除去培养基并将细胞暴露于1毫升化学毒素中，其中包括PBS中的0.001%苯扎氯铵或BAK，0.01%过氧化氢和0.0025%十二烷基硫酸钠5和15分钟。暴露于化学毒素后，从井中取出毒素，用1毫升PBS冲洗，并向每个孔中加入1毫升HOEM。孵育20小时后，通过用1毫升10%代谢测定溶液替换培养基并在37摄氏度下用5%二氧化碳孵育4小时来进行代谢测定。使用荧光酶标仪在530纳米激发和590纳米发射波长下测量每种溶液的荧光。孵育20小时后将细胞上清液从孔中转移到单独的无菌2毫升聚丙烯管中，并在零下80摄氏度下冷冻。按照试剂盒的说明，使用用于评估紫外线处理细胞的细胞因子的相同多重平台，以量化白细胞介素 6、白细胞介素 8、白细胞介素-1 β 和 TNF-α。在 24 孔胶原蛋白 1 包被培养板的每个孔中接种每毫升 HOEM 1 x 10 至 5 分之一的细胞，并在 37 摄氏度下与 5% 二氧化碳孵育 24 小时。孵育后，除去培养基并将细胞暴露于 1 毫升化学毒素中，包括 PBS 中的 0.001%BAK、0.005% BAK 和 0.01% BAK 5 分钟。暴露后，去除化学毒素，用1毫升PBS冲洗孔，并向每个孔中加入1毫升HOEM。孵育20小时后，用含有钙黄绿素，乙锭同源二聚体和膜联蛋白5的500微升膜联蛋白染色缓冲溶液在37摄氏度下染色细胞20分钟。调整显微镜以测量膜联蛋白 630 和 675 纳米的激发和发射波长下的强度，钙黄绿素 AM 的 495 和 515 纳米，以及乙锭 1 染色的 528 和 617 纳米。然后使用荧光显微镜获取细胞的图像。发现iHCEC是10至20微米范围内的小细胞，pHCEC在生长24小时后在20至50微米范围内。在生长48小时后，iHCEC和pHCEC在大小范围内观察到类似的差异。与非紫外线暴露细胞相比，照射的pHCEC的代谢活性在暴露20分钟时显着降低。对于iHCEC，在5和20分钟照射的细胞的代谢活性降低。因此，与iHCEC相比，降低pHCEC的代谢活性需要更长的暴露时间。iHCEC的最大细胞因子释放是在紫外线照射5分钟时。pHCEC的最大细胞因子释放发生在紫外线照射20分钟时。就释放的炎性细胞因子总量而言，pHCEC释放的白细胞介素-1β、白细胞介素8和TNF-α明显高于iHCEC，而iHCEC释放的白细胞介素6更多。pHCEC和iHCEC在暴露于所有三种化学物质后都显示出白细胞介素6释放的变化。与原发性和永生化HCEC的对照组相比，BAK导致白细胞介素6的释放减少。过氧化氢导致iHCEC中白细胞介素6的释放增加，以及pHCEC的释放减少。pHCEC和iHCEC均受到显著损害，分别为BAK的0.005%和0.1%。在对眼细胞毒性进行体外评估后，可以进行进一步的眼毒性动物模型以确定眼部产品对角膜神经或眼部免疫系统的影响。这些技术对于确定化学和产品配方对眼睛的毒性机制至关重要，并有助于最大限度地减少动物在产品开发测试中的使用。

Research

JoVE Journal

Biology

Primary Human Bronchial Epithelial Cells Grown from Explants

从外植体生长的首要人权支气管上皮细胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

Quantification of ÃƒÅ½Ã‚Â³H2AX Foci in Response to Ionising Radiation

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

Evaluation of the Spatial Distribution of ÃƒÅ½Ã‚Â³H2AX following Ionizing Radiation

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Induction and Analysis of Epithelial to Mesenchymal Transition

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

Quantitation of Endothelial Cell Adhesiveness In Vitro

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

SCAP分析<em&gt;ñ</em&gt; -glycosylation和贩卖人体细胞

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

由上皮细胞和原发免疫细胞组成的多细胞人类Alveal模型，用于危险评估

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...