Name: a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals
Uploaded: 2018-06-25T12:30:00
Description: Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

这项工作得到了由 NIEHS 和中国国家自然科学基金 (31572626 号) 赞助的 CREH 中心的试点项目赠款的支持。

所有动物实验都是按照所有相关的指导方针、规章和管理机构执行的。正在示范的《议定书》是在德克萨斯一个 & M 基因组医学研究所的机构动物护理和使用委员会 (IACUC) 的指导和批准下进行的。
1. 解决方案准备
<ol><li>将10x 汉克的平衡盐溶液稀释为 1x, 双蒸馏 H2O, 并贮存在4摄氏度。</li>
	<li>通过添加 HEPES (10 毫米)、氯化镁2 (1 毫米) 和葡萄糖 (5 毫米) 来制备?...

<ol>
	<li>Maruthur, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+prevalence+of+type+2+diabetes:+increased+incidence+or+improved+survival?.">The growing prevalence of type 2 diabetes: increased incidence or improved survival?.</a> Current diabetes reports. 13 (6), 786-794 (2013).</li><li>Houstis, N., Rosen, E. D., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+have+a+causal+role+in+multiple+forms+of+insulin+resistance.">Reactive oxygen species have a causal role in multiple forms of insulin resistance.</a> Nature. 440 (7086), 944-948 (2006).</li><li>Ma, Z. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+alters+mitochondrial+activation+and+insulin+secretion+in+pancreatic+beta+cells.">Hydrogen peroxide alters mitochondrial activation and insulin secretion in pancreatic beta cells.</a> Journal of Biological Chemistry. 274 (39), 27905-27913 (1999).</li><li>Gao, 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=The+effects+of+palmitate+on+hepatic+insulin+resistance+are+mediated+by+NADPH+Oxidase+3-derived+reactive+oxygen+species+through+JNK+and+p38MAPK+pathways.">The effects of palmitate on hepatic insulin resistance are mediated by NADPH Oxidase 3-derived reactive oxygen species through JNK and p38MAPK pathways.</a> Journal of Biological Chemistry. 285 (39), 29965-29973 (2010).</li><li>Efendi&#263;, S., 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=Pancreastatin+and+islet+hormone+release.">Pancreastatin and islet hormone release.</a> Proceedings of the National Academy of Sciences. 84 (20), 7257-7260 (1987).</li><li>Tian, Y., Ke, S., Denison, M. S., Rabson, A. B., Gallo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ah+receptor+and+NF-&#954;B+interactions,+a+potential+mechanism+for+dioxin+toxicity.">Ah receptor and NF-&#954;B interactions, a potential mechanism for dioxin toxicity.</a> Journal of Biological Chemistry. 274 (1), 510-515 (1999).</li><li>Cui, H., 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=Pregnane+X+receptor+regulates+the+AhR/Cyp1A1+pathway+and+protects+liver+cells+from+benzo-[&#945;]-pyrene-induced+DNA+damage.">Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[&#945;]-pyrene-induced DNA damage.</a> Toxicology Letters. 275, 67-76 (2017).</li><li>Li, L. A., Wang, P. 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(7), e255 (2007).</li><li>Kirstetter, P., Lagneau, F., Lucas, O., Krupa, Y., Marty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelium+in+the+modulation+of+isoflurane-induced+vasodilatation+in+rat+thoracic+aorta.">Role of endothelium in the modulation of isoflurane-induced vasodilatation in rat thoracic aorta.</a> British journal of anaesthesia. 79 (1), 84-87 (1997).</li><li>Brown, E., Umino, Y., Loi, T., Solessio, E., Barlow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+can+cause+sustained+hyperglycemia+in+C57/BL6J+mice.">Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.</a> Visual neuroscience. 22 (5), 615-618 (2005).</li><li>Vaupel, D., McCoun, D., Cone, E. 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积累的证据表明, 接触环境化学物质对 T2DM 的发展起着重要的作用。外来化合物诱导的 ROS 已被认为是一个潜在的致病因素, 有助于 T2DM 的发展。人类接触到广泛的 xenobiotic 化学物质, 有很大的需要, 新的研究技术, 以有效地识别胰腺毒物和研究的毒性机制, 特异的胰腺细胞。
在这项研究中, 根据已发布的程序14, 我们已经制定了一个协议, 以隔离鼠胰岛和屏幕的?...

T2DM 患病率的上升已成为近年来全球卫生危机, 对公共卫生构成严重威胁1。许多因素被发现与 T2DM 的发展有因果关系, 其中, 反复发现表明, 这些因素的一个共同的收敛点是诱导氧化应激导致过量 ROS 的产生2,3。
广泛的环境化学物质, 包括多氯联苯, 二恶英和 BaP 被发现诱导氧化应激, 这可能会损害胰β细胞的功能, 导致胰岛素抵抗和 T2DM4。虽然 ros 的生理水平在细胞功能中起着重要作用, 但暴露于活性氧超过抗氧化剂系统的能力会导致细胞/组织的损伤, 导致疾病5。胰β细胞表达的抗氧化酶水平较低, 因此是一个敏感的目标为氧化应激介导的损害6,7。慢性暴露于高水平的 ROS 已经表明, 导致应力诱发胰腺细胞功能障碍5以及胰岛素抵抗的肝脏和脂肪组织8。
这个项目的总目标是建立一个细胞为基础的检测, 以筛选化学物质的致糖尿病潜能的基础上, 他们诱导的活性氧在胰腺细胞。胰腺缺乏代谢解毒, 是 xenobiotic 致损伤6、7的敏感靶。因此, 通过直接测定胰腺细胞中产生的 ROS, 可以直接对胰腺内化学诱发的损伤进行近似分析。为了开发这种方法, 我们分离了小鼠胰岛, 用化学物质在细胞培养条件下培养出孤立的胰岛, 并利用化学诱导的 ROS 生成作为读数。该方法简便、有效地鉴别孤立胰岛中的活性氧诱导化学物质;它可以进一步发展, 以调查的毒性机制, 具体的胰腺的体外。

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">10&#215;Hank&#8217;s balanced salt solution&#160;</td>
			<td style="width:283px;">GIBCO</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type 4</td>
			<td>Worthington Biochemical Corporation</td>
			<td>CLS-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polysucrose/sodium diatrizoate solution&#160;</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#8217;,7&#8217;-dichlorofluorescein (DCFH-DA)</td>
			<td>Sigma</td>
			<td>D6883-50MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fluorescence microplate reader&#160;</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Sigma</td>
			<td>G8540-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">streptomycin</td>
			<td>GIBCO</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>GIBCO</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>GIBCO</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">avertin</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat/Mouse Insulin ELISA Kit</td>
			<td>Millipore</td>
			<td>EZRMI-13K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Sorval</td>
			<td>Sorval RT7</td>
			<td>for 96-well plate centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td>Biotek</td>
			<td>Epoch 2</td>
			<td>for fluorescence reading</td>
		</tr>
	</tbody>
</table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

暴露在人类和动物中的某些环境化学物质已经被发现会导致胰腺β细胞的细胞损伤, 这将导致2型糖尿病 (T2DM) 的发展。虽然化学诱导β细胞损伤的机制不清楚, 而且很可能是复杂的, 但一个反复发现是这些化学物质诱导氧化应激导致产生过量的活性氧 (ROS), 从而导致损害β细胞。为了识别潜在的致糖尿病环境化学物质, 我们从 C57BL/6 小鼠和培养的胰岛细胞中分离出了96井细胞培养皿中的胰腺细胞;然后, 用化学试剂对胰岛细胞进行了剂量测定, 用 2 ', 7 '-dichlorofluorescein (DCFH) 荧光染料检测了 ROS 的产生。使用这种方法, 我们发现双酚 a (BPA), 苯并 [a] 芘 (BaP) 和多氯联苯, 可能会诱发高水平的 ROS, 表明它们可能会导致胰岛细胞的损害。该方法应用于筛选致糖尿病外来化合物。此外, 培养的胰岛细胞也可用于胰腺细胞化学诱导毒性的体外分析。

图 2显示了健康的孤立胰岛的显微图像, 其中胰岛具有圆形或椭圆形, 尺寸相对均匀 (虽然大小均匀性可以不同于应变)。我们接下来研究胰岛在试管中的作用, 通过分离胰岛和刺激胰岛素分泌的培养胰岛。图 3显示了我们对由3.3 毫米和16.7 毫米葡萄糖9诱导的 C57BL/6 小鼠孤立胰岛 GSIS 检测的典型分析。
<p ...

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

在这里, 我们提出了一个协议, 以隔离小鼠胰岛细胞筛选的 ROS 归纳的外来化合物, 以确定潜在的致糖尿病 xenobiotic 化学品。

小鼠胰岛细胞筛选致糖尿病环境化学物质的研究

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Research

JoVE Journal

Immunology and Infection

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Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

科研

免疫与感染

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

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The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

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Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

人胰岛自身抗体的电化学发光测定

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

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RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...