Name: a proximal culture method to study paracrine signaling between cells
Uploaded: 2018-08-28T14:30:00
Description: Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

我们感谢患者参与这些实验的组织收集。国防部 OCRP 卵巢癌学院奖 (W81XWH-15-0253) 和一个试点奖从科琳的梦想基金会 Anirban k. 米特拉支持这项研究。

该议定书遵循印第安纳大学机构管理委员会的指导方针。
1. 细胞制剂
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人原发性间皮细胞的分离与培养
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<li>从人网膜中分离人原发性间皮细胞 (HPMCs), 如前所述17、18和生长在完全生长培养基 [Dulbecco 的改良鹰的培养基 (DMEM) 含有10% 胎牛血清, 1%青霉素-链霉素, 1% 非必需氨基酸, 1% 维生素] 在37摄氏度和 5% CO2</s...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+interactions+in+lymph+node+development.">Cellular interactions in lymph node development.</a> The Journal of Immunology. 174 (1), 21-25 (2005).</li><li>Suvas, S. <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+Substance+P+Neuropeptide+in+Inflammation,+Wound+Healing,+and+Tissue+Homeostasis.">Role of Substance P Neuropeptide in Inflammation, Wound Healing, and Tissue Homeostasis.</a> The Journal of Immunology. 199 (5), 1543-1552 (2017).</li><li>Gnecchi, M., Danieli, P., Malpasso, G., Ciuffreda, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+Mechanisms+of+Mesenchymal+Stem+Cells+in+Tissue+Repair.">Paracrine Mechanisms of Mesenchymal Stem Cells in Tissue Repair.</a> Methods in Molecular Biology. , 123-146 (2016).</li><li>Lionetti, V., Bianchi, G., Recchia, F. A., Ventura, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+autocrine+and+paracrine+myocardial+signals:+an+emerging+therapeutic+strategy+in+heart+failure.">Control of autocrine and paracrine myocardial signals: an emerging therapeutic strategy in heart failure.</a> Heart Failure Reviews. 15 (6), 531-542 (2010).</li><li>Nicosia, R. F., Zorzi, P., Ligresti, G., Morishita, A., Aplin, A. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juxtacrine+activation+of+epidermal+growth+factor+(EGF)+receptor+by+membrane-anchored+heparin-binding+EGF-like+growth+factor+protects+epithelial+cells+from+anoikis+while+maintaining+an+epithelial+phenotype.">Juxtacrine activation of epidermal growth factor (EGF) receptor by membrane-anchored heparin-binding EGF-like growth factor protects epithelial cells from anoikis while maintaining an epithelial phenotype.</a> Journal of Biological Chemistry. 282 (45), 32890-32901 (2007).</li><li>Swartz, M. 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=Tumor+microenvironment+complexity:+emerging+roles+in+cancer+therapy.">Tumor microenvironment complexity: emerging roles in cancer therapy.</a> Cancer Research. 72 (10), 2473-2480 (2012).</li><li>Mitra, A. 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=MicroRNAs+reprogram+normal+fibroblasts+into+cancer-associated+fibroblasts+in+ovarian+cancer.">MicroRNAs reprogram normal fibroblasts into cancer-associated fibroblasts in ovarian cancer.</a> Cancer Discovery. 2 (12), 1100-1108 (2012).</li><li>Nieman, K. 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=Adipocytes+promote+ovarian+cancer+metastasis+and+provide+energy+for+rapid+tumor+growth.">Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.</a> Nature Medicine. 17 (11), 1498-1503 (2011).</li><li>Salimian Rizi, B., 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=Nitric+oxide+mediates+metabolic+coupling+of+omentum-derived+adipose+stroma+to+ovarian+and+endometrial+cancer+cells.">Nitric oxide mediates metabolic coupling of omentum-derived adipose stroma to ovarian and endometrial cancer cells.</a> Cancer Research. 75 (2), 456-471 (2015).</li><li>Mitra, A. 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=Microenvironment-induced+downregulation+of+miR-193b+drives+ovarian+cancer+metastasis.">Microenvironment-induced downregulation of miR-193b drives ovarian cancer metastasis.</a> Oncogene. 34 (48), 5923-5932 (2015).</li><li>Tomar, 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=ETS1+induction+by+the+microenvironment+promotes+ovarian+cancer+metastasis+through+focal+adhesion+kinase.">ETS1 induction by the microenvironment promotes ovarian cancer metastasis through focal adhesion kinase.</a> Cancer Letters. 414, 190-204 (2018).</li><li>Ovarian Cancer Metastasis: A Unique Mechanism of Dissemination. Tumor Metastasis Available from: <a target="_blank" href="https://www.intechopen.com/books/tumor-metastasis/ovarian-cancer-metastasis-a-unique-mechanism-of-dissemination">https://www.intechopen.com/books/tumor-metastasis/ovarian-cancer-metastasis-a-unique-mechanism-of-dissemination</a> (2016)</li><li>Iwanicki, M. P., 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=Ovarian+cancer+spheroids+use+myosin-generated+force+to+clear+the+mesothelium.">Ovarian cancer spheroids use myosin-generated force to clear the mesothelium.</a> Cancer Discovery. 1 (2), 144-157 (2011).</li><li>Kenny, H. 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=Mesothelial+cells+promote+early+ovarian+cancer+metastasis+through+fibronectin+secretion.">Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.</a> Journal of Clinical Investigation. 124 (10), 4614-4628 (2014).</li><li>Boelens, M. 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了解细胞间分泌和 juxtacrine 信号的机制对于培养对正常组织稳态和疾病状况的更好的认识是至关重要的,7,8。大多数分泌信号的研究是通过收集条件培养基从一个细胞类型和使用它来治疗其他细胞类型。这种方法在其固有的简单性方面具有优势。然而, 它并没有准确地重述细胞微环境中分泌因子的局部浓度, 或所涉及因素的产生和消耗的动力学。虽然前者...

癌细胞与肿瘤微环境的相互作用在肿瘤进展中的作用已经得到了很好的确立, 并已成为癌症生物学研究的主要重点1。在伤口愈合、免疫应答、血管生成、干细胞龛位和发育过程中, 双向信号的相似情况是至关重要的,2、3、4、5、6,7,8. 在所有这些生物过程中, 一个共同的主题是细胞以各种方式对其微环境中的细胞外信号作出反应, 这决定了体细胞的命运、组织生理学和疾病进展。因此, 重点日益转向发展对这种细胞通讯所涉及的机制的更好的了解。大多数这种相互作用涉及细胞之间的分泌或 juxtacrine 信号。分泌信号涉及特定信号因子的分泌由一个细胞, 这是由相应的受体在附近的另一个细胞感知, 触发反应, 在它9,10, 而 juxtacrine信号要求两个细胞的细胞成分之间的直接接触涉及11,12。
这种信号是组织稳态以及肿瘤微环境中的重要组成部分。癌细胞从肿瘤基质细胞中的分泌和 juxtacrine 因子中受益, 包括肿瘤相关的成纤维细胞 (咖啡馆)、免疫细胞和脂肪瘤13、14、15、16。分泌信号可以由生长因子、细胞因子、趋化因子等介导, 而 juxtacrine 信号则包括在凹槽信号中并列配体和受体, 或整合与其各自的相互作用细胞外基质蛋白。我们已经证明了卵巢癌细胞与咖啡馆在肿瘤进展和转移中相互作用的重要性14。同样, 转移卵巢癌细胞与覆盖转移部位的间皮细胞的相互作用, 调节癌细胞的关键 microRNAs 和转录因子, 促进转移性殖民化17,18。
大多数关于分泌信号的研究涉及使用从一个细胞类型收集的条件培养基来治疗第二细胞类型。虽然这种方法已被广泛使用, 但它并没有有效地复制在接收细胞微环境中分泌因子的局部高浓度水平。它也无法重现由一个细胞产生的分泌因子连续流动的动力学, 相邻细胞接收。分泌信号是有效的短距离, 因为分泌因子是在所需浓度仅在源细胞附近, 并倾向于扩散和稀释, 随着距离的增加。这种局部高浓度的分泌因子对于触发受体细胞的反应至关重要。此外, 受体细胞的反应也依赖于新分泌因子的平衡, 以及它们在受体细胞中的退化、束缚和内化以及从源细胞扩散的持续损耗。条件培养基可以集中在微环境中存在的较高的局部浓度, 但不能准确地复制准确的浓度。此外, 它不能模仿生产的自然动力学和所涉及的因素的耗尽。为了更准确地复制分泌信号并将其与 juxtacrine 信号机制分开, 我们设计了一种新的近距离培养方法, 它涉及在多孔膜的任一表面上生长两种细胞类型。毛孔小到足以防止 juxtacrine 相互作用, 但允许在局部高浓度下交换分泌因子。这样, 系统就保留了生产的动力学和分泌因素的损耗。

<table>
	<tbody>
		<tr>
			<td>24 mm Transwell permeable support with 0.4 &#181;m Pore Polycarbonate Membrane Insert</td>
			<td>Corning (Costar)</td>
			<td>3412</td>
			<td>&#8226; 10 &#181;m thick translucent polycarbonate membrane 
			&#8226; Treated for optimal cell attachment 
			&#8226; Packaged 6 inserts in a 6 well plate, 4 plates per case 
			&#8226; Membrane must be stained for cell visibility 
			&#8226; Sterilized by gamma radiation</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning (Falcon)</td>
			<td>353046</td>
			<td>Flat Bottom, TC-treated, sterile, with Lid</td>
		</tr>
		<tr>
			<td>15 cm culture dish</td>
			<td>Corning (Falcon)</td>
			<td>353025</td>
			<td>Sterile, TC-treated Cell Culture Dish</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning (Cellgro)</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential amino acids</td>
			<td>Corning (Cellgro)</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins</td>
			<td>Corning (Cellgro)</td>
			<td>25-020-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin, 2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipets</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>Biosafety level II cabinet</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>FN1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01549976_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFB1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs00998133_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CDH1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01023895_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs99999905_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy mini RNA isolation Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>43-688-13</td>
			<td></td>
		</tr>
		<tr>
			<td>HeyA8 ovarian cancer cells</td>
			<td>Obtained from Ernst Lengyel Lab, University of Chicago</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946; Neutralizing Antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1835-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a proximal culture method to study paracrine signaling between cells

细胞间相互作用在许多生物学过程中起着重要作用, 包括肿瘤进展、免疫反应、血管生成和发育。分泌或 juxtacrine 信号介导这种相互作用。使用条件中等和共培养研究是区分这两种相互作用的最常见的方法。然而, 在分泌相互作用过程中, 局部高浓度分泌因子在微环境中的作用不是通过条件培养基准确概括的, 因此可能导致不精确的结论。为了克服这一问题, 我们设计了一种近端培养方法来研究分泌信号。两种细胞生长在10µm 厚的聚碳酸酯膜的任何表面, 0.4 µm 毛孔。毛孔允许交换分泌因子, 同时抑制 juxtacrine 信号。可以在端点收集和裂解细胞, 以确定分泌信号的影响。除了允许分泌因子的局部浓度梯度外, 这种方法还能经受长时间的培养以及抑制剂的使用的实验。利用这种方法研究卵巢癌细胞与转移部位间皮细胞的相互作用, 可以适应任何两种黏附细胞类型, 研究分泌信号在各个领域的应用,包括肿瘤微环境、免疫学和发育。

转移卵巢癌细胞在腹膜腔内的转移部位出现间皮细胞19。生产分泌和 juxtacrine 与间皮细胞的相互作用有助于诱导卵巢癌细胞的适应性反应, 从而成功转移17,18,20,21.为了检验近端培养方法的有效性, 我们测试了 HeyA8 卵巢癌细胞与 HPMCs 的分泌相互作用。以前有报?...

Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

A Proximal Culture Method to Study Paracrine Signaling Between Cells

分泌和 juxtacrine 细胞相互作用在许多生物学过程中起着重要作用, 包括肿瘤的进展、免疫反应、血管生成和发育。在这里, 一种近端培养方法用于研究分泌信号, 其中的分泌因子的局部浓度保持, 而防止直接细胞接触。

一种研究细胞间分泌信号的近距离培养方法

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A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

血管钙的可视化 2 +信号旁分泌源性活性氧引发

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...