Name: determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and balb/c mouse macrophages
Uploaded: 2013-05-30T13:00:00
Description: Watch this Scientific Journal Video about Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages at JoVE.com

我们感谢保科沃斯和埃夫罗斯瓦西利乌博士实验室的协助，并提供小鼠巨噬细胞，分别。我们也感谢我们的实验室经理Richard Criasia与材料的指导和帮助。最后，作者感谢Ramanpreet，考尔的帮助视频制作。

1。组织文化<ol><li>使用小家鼠巨噬细胞（BALB / c小鼠）对霍乱毒素的测定。最初文化M.肌细胞按照厂商的说明。 </li><li>传播的BALB / c小鼠细胞的Dulbecco改性鹰的中等与L-谷氨酰胺10％胎牛血清完成，并用10％胎牛血清，5％大号-谷氨酰胺和1完成1％抗生素/抗真菌剂或RPMI 1640基地媒体％抗生素/抗真菌试剂。 </li><li>培养细胞在75 厘米康宁烧瓶，在37℃，95％空气和5％CO 2通?...

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建议浓度脂肪酸和霍乱毒素虽然脂肪酸如何增强粘膜免疫的确切机制不明，一些研究试图探讨其有益的影响。本研究的目的是提供方法来确定细胞的脂肪酸，可以容忍，以及霍乱毒素的最大浓度没有显着影响细胞的存活细胞可以容忍的最大浓度。 为了测定浓度的三脂肪酸（油酸，亚油酸和亚麻酸），以及霍乱毒素用于治疗的细胞没有显着的不利影响，对细?...

脂肪酸，如油酸，亚油酸和亚麻酸对健康的好处已经并继续记录。例如，油酸有助于渗透的亲脂性药物在体内1,2冠状动脉心脏病，降低了24％时，饱和脂肪酸3取代的，并且是用于治疗代谢性疾病如肾上腺脑白质营养不良4是X-连锁的脂肪酸代谢的遗传性疾病。一个必要的前体在哺乳动物中花生四烯酸，亚油酸（不像油酸）时由体内合成，必须通过外部信息源，例如由亚麻籽消耗。5研究表明一些有益的健康效应如亚油酸：对皮肤的抗老化性能;抗发炎的特性;降低大肠癌和前列腺癌细胞增殖;和能力对抗肥胖和推广Øf心血管健康。9 ​​亚麻酸发挥了作用，减少牙周发炎，10和调节血栓素和前列环素的生物合成。 12阿尔皮塔（Arpita）研究胆汁脂肪酸和胆固醇的影响的五霍乱弧菌的毒力因子的表达和活力。山崎13日表示，红辣椒，以及其他自然提取的化合物，甲醇提取物有可能降低生产霍乱毒素。可以想象的是考虑使用在上面的脂肪酸（如亚麻种子）通过粘膜免疫的传染病，如霍乱的预防和减轻丰富的食品。我们进行了调查，以溶解脂肪酸和确定，使用细胞增殖实验，最高浓度与人肠上皮细胞中的脂肪酸，可以承受的，没有不利影响策LL可行性。我们假设，油酸，亚油酸和亚麻酸，在较低浓度对细胞活力提供了有益的效果，但是，在较高浓度时，它们将是对细胞有毒。而且，我们溶解的霍乱毒素和霍乱毒素B亚单位的确定的最大浓度，BALB / C小鼠巨噬细胞的细胞存活率没有显着下降，可以容忍。我们推测对细胞活性的霍乱毒素的毒性作用，即使在非常低的水平。增溶霍乱毒素的方法，用它来确定的毒素，细胞没有显着下降的生存能力可以容忍的最大金额为今后的研究提供了一个优点。例如，可以使用上述的方法的组合，以确定是否脂肪酸提供抗霍乱感染的粘膜免疫的细胞。据我们所知，这种理性和方法尚未探索。 我们二scuss我们的初步数据如何可以用在以后的调查，以确定是否油酸，亚油酸和亚麻酸提供细胞粘膜抗感染霍乱。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells/Reagent</td>
		 </tr>
		 <tr>
			<td>Mus musculus macrophages</td>
			<td>ATCC</td>
			<td>ATCC RAW 264.7</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dulbecco's Modified Eagle's Medium</td>
			<td>ATCC</td>
			<td>30-2002</td>
			<td></td>
		 </tr>
		 <tr>
			<td>L-glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal bovine serum </td>
			<td>Bio-west</td>
			<td>S0250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotic/antimycotic </td>
			<td>Hyclone</td>
			<td>SV3007901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human intestinal epithelial cells </td>
			<td>ATCC</td>
			<td>ATTC CCL-241</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HybriCare media </td>
			<td>ATCC</td>
			<td>46-X</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Oleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>O1008</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linoleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-1376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linolenic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-2376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cholera toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>BD Falcon 96-Well Cell Culture Plates</td>
			<td>BD Biosciences </td>
			<td>351172</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spectrophotometer with Dynex Revelations 4.22 software</td>
			<td>Dynex</td>
			<td>91000101</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and balb/c mouse macrophages

脂肪酸在预防和减轻人类和非人类疾病的积极作用已经并将继续被广泛记载。这些角色包括感染性和非感染性疾病，包括预防炎症和传染病的粘膜免疫的影响。霍乱是由霍乱弧菌引起的急性肠道疾病。它发生在发展中国家，如果不及时治疗，可导致死亡。虽然霍乱疫苗存在时，它们并不总是有效的，需要其他的预防方法。我们容许浓度的三脂肪酸（油酸，亚油酸和亚麻酸）和霍乱毒素，分别使用小鼠BALB / C小鼠巨噬细胞和人类肠道上皮细胞，以确定。溶解上述脂肪酸和使用细胞增殖实验，以确定的浓度范围和特定浓度的脂肪酸，没有吨有损于人类肠道上皮细胞的存活率。增溶的霍乱毒素B亚单位，在一个试验中，并用它来确定的浓度范围和霍乱毒素，在统计学上不降低细胞存活率在BALB / C小鼠的巨噬细胞中的特定浓度的。 我们发现最佳的脂肪酸浓度介于1-5纳克/微升，和霍乱毒素&lt;30纳克每处理的。这些数据可能有助于未来的研究，旨在找到脂肪酸在预防或减轻感染霍乱的保护黏膜的作用。

脂肪酸的最佳浓度测定脂肪酸的最适浓度被定义为细胞生长的最大浓度可媲美或超过对照组细胞，在结果中的相对低的变异。 ，以确定最佳浓度的油酸，亚油酸和亚麻酸的细胞最初以较大的增量，并与不同浓度的各脂肪酸购买较小的增量处理， 图1示出的生存的细胞作为阳性对照的治疗使用的函数描绘脂肪酸浓度的增加。显而易见的是，细胞不耐受超过100毫微克/微...

Watch this Scientific Journal Video about Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages at JoVE.com

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

我们容许浓度的三脂肪酸（油酸，亚油酸和亚麻酸）和霍乱毒素没有显着不利影响细胞生存脂肪酸和毒素溶解，并利用他们在细胞存活实验来确定。

使用人类肠道上皮细胞和BALB / C小鼠巨噬细胞的容忍脂肪酸和霍乱毒素浓度的测定

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

Research

JoVE Journal

Immunology and Infection

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

肠道细菌侵入肠上皮细胞<em&gt;体外</em&gt;使用垂直扩散室模型，显着增强

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

哺乳动物细胞细菌挑战后应激颗粒形成的免疫荧光分析

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

饱和脂肪酸诱导神经酰胺相关巨噬细胞死亡

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

小鼠和人源性胃上皮单层培养对再生的研究

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...