Name: evaluation of the interplay between the complement protein c1q and hyaluronic acid in promoting cell adhesion
Uploaded: 2019-06-15T13:00:00
Description: Watch this Scientific Journal Video about Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion at JoVE.com

我们感谢伊万·多纳蒂提供HA、莱昂纳多·阿马迪奥、加布里埃拉·齐托(IRCCS"Burlo Garofolo"妇科,意大利里雅斯特)和安德里亚·罗曼诺(意大利里雅斯特卡蒂纳医院解剖和病理组织学临床科) 用于组织样本收集。我们也感谢尼科洛·莫罗西尼在视频准备和亚历克斯·科波拉的声音帮助。这项工作得到了妇幼保健研究所、IRCCS"Burlo Garofolo"、意大利里雅斯特(RC20/16)和R.Bulla的丰达齐奥内·卡萨迪·里萨米奥·里雅斯特的资助。

意大利里雅斯特大学医院机构委员会经道德考虑后,经知情同意后采集了患者的组织样本。
1. 肿瘤细胞分离与培养(第1天)
<ol><li>从MPM固体标本中分离出人类中皮瘤细胞。用切割机精细切碎组织,获得约2-3 mm2大小的片段,并在由汉克斯平衡盐溶液(HBSS)组成的5 mL消化溶液中孵育,辅以0.5 mM Ca 2+/Mg2+,0.5%胰蛋白酶和50μg/mLDNase I,在4°C过夜。</li>
	<li>肿瘤细胞分离?...

<ol>
	<li>Zamboni, F., Vieira, S., Reis, R. L., Oliveira, J. M., Collins, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+hyaluronic+acid+in+immunoprotection+and+immunomodulation:+Chemistry,+processing+and+function.">The potential of hyaluronic acid in immunoprotection and immunomodulation: Chemistry, processing and function.</a> Progress in Materials Science. 97, 97-122 (2018).</li><li>Lorusso, G., Ruegg, 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+tumor+microenvironment+and+its+contribution+to+tumor+evolution+toward+metastasis.">The tumor microenvironment and its contribution to tumor evolution toward metastasis.</a> Histochemistry and Cell Biology. 130 (6), 1091-1103 (2008).</li><li>Ricklin, D., Hajishengallis, G., Yang, K., Lambris, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement:+a+key+system+for+immune+surveillance+and+homeostasis.">Complement: a key system for immune surveillance and homeostasis.</a> Nature Immunology. 11 (9), 785-797 (2010).</li><li>Bulla, 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=C1q+acts+in+the+tumour+microenvironment+as+a+cancer-promoting+factor+independently+of+complement+activation.">C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation.</a> Nature Communications. 7, 10346 (2016).</li><li>Winslow, S., Leandersson, K., Edsjo, A., Larsson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+stromal+gene+signatures+in+breast+cancer.">Prognostic stromal gene signatures in breast cancer.</a> Breast Cancer Research. 17, 23 (2015).</li><li>Collins, M. N., Birkinshaw, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+solutions:+A+processing+method+for+efficient+chemical+modification.">Hyaluronic acid solutions: A processing method for efficient chemical modification.</a> Journal of Applied Polymer Science. 130 (1), 145-152 (2013).</li><li>Itano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+primary+structure,+complex+turnover+regulation+and+multiple+roles+of+hyaluronan.">Simple primary structure, complex turnover regulation and multiple roles of hyaluronan.</a> Journal of Biochemistry. 144 (2), 131-137 (2008).</li><li>Agostinis, C., 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+alternative+role+of+C1q+in+cell+migration+and+tissue+remodeling:+contribution+to+trophoblast+invasion+and+placental+development.">An alternative role of C1q in cell migration and tissue remodeling: contribution to trophoblast invasion and placental development.</a> Journal of Immunology. 185 (7), 4420-4429 (2010).</li><li>Agostinis, C., 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=Complement+Protein+C1q+Binds+to+Hyaluronic+Acid+in+the+Malignant+Pleural+Mesothelioma+Microenvironment+and+Promotes+Tumor+Growth.">Complement Protein C1q Binds to Hyaluronic Acid in the Malignant Pleural Mesothelioma Microenvironment and Promotes Tumor Growth.</a> Frontiers in Immunology. 8, 1559 (2017).</li><li>Bossi, 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=C1q+as+a+unique+player+in+angiogenesis+with+therapeutic+implication+in+wound+healing.">C1q as a unique player in angiogenesis with therapeutic implication in wound healing.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (11), 4209-4214 (2014).</li><li>Hosszu, K. 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=Cell+surface+expression+and+function+of+the+macromolecular+c1+complex+on+the+surface+of+human+monocytes.">Cell surface expression and function of the macromolecular c1 complex on the surface of human monocytes.</a> Frontiers in Immunology. 3, 38 (2012).</li><li>Laurent, T. C., Fraser, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan.">Hyaluronan.</a> Journal of the Federation of American Societies for Experimental Biology. 6 (7), 2397-2404 (1992).</li><li>Gaboriaud, C., 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+crystal+structure+of+the+globular+head+of+complement+protein+C1q+provides+a+basis+for+its+versatile+recognition+properties.">The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties.</a> Journal of Biological Chemistry. 278 (47), 46974-46982 (2003).</li><li>Lam, J., Truong, N. F., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+cell-matrix+interactions+in+hyaluronic+acid+hydrogel+scaffolds.">Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds.</a> Acta Biomaterialia. 10 (4), 1571-1580 (2014).</li></ol>

我们描述了一种简单的方法来研究补充组分C1q如何与透明质酸相互作用,如何调节从人类肿瘤组织分离的初级细胞的行为。HA 和 C1q 在生理和病理条件下在组织微环境中均大量存在,参与多个细胞生物过程。例如,C1q已被证明存在于胎盘的微环境中,它有利于在胎盘8期间对母体脱胎酶的外型营养器入侵。C1q也沉积在伤口愈合的皮肤,它促进组织再生和修复10所需的血管...

肿瘤微环境(TME)影响癌症的发展和进展,因为它可以为细胞生存、生长和入侵提供一个宽松的利基。TME 中新关键参与者的识别可能有助于发现用于靶向治疗的新分子工具。TME 包括一个复杂且动态的非恶性细胞网络,如内皮细胞、成纤维细胞和免疫系统的细胞,嵌入周围的细胞外基质 (ECM) 组件中,包括胶原蛋白、层压、纤维蛋白、孕激素和透明质。肿瘤和非肿瘤细胞合成和分泌ECM成分,以及细胞因子、化学因子、生长因子和炎症和基质重塑酶,这些酶整体上改变了TME的物理、化学和信号特性。在这些成分中,透明质酸(HA)在肿瘤生物学中起着至关重要的作用。尽管HA的化学成分简单,但连同其HA结合分子(透明环酸),可以调节血管生成、免疫系统反应和ECM重塑,其大小和浓度依赖性1。
补充(C)系统也是当地TME的一部分,最近受到越来越多的关注。C 系统包括一组可溶性和膜结合蛋白,这些蛋白质涉及对非自细胞、不需要的宿主元素和病原体的第一道防线。在功能上,C连接与生俱来和自适应系统的双效应臂,以促进直接细胞杀死或安装炎症反应2。C活化可以通过破坏癌细胞或抑制其外生长来抑制肿瘤生长,但越来越明显的是,它可以通过维持慢性炎症,促进建立一个促进肿瘤的活动免疫抑制环境,诱导血管生成,并激活癌症相关信号通路3。在此背景下,C1q是C系统经典通路的第一个识别分子,它独立于C活化4在肿瘤微环境中发挥着重要的作用。C1q已被证明由一系列人类恶性肿瘤在当地表达,除了血管生成和转移5外,它还能有利于癌细胞的粘附、迁移和增殖。有趣的是,C1q 与 ECM 的主要组成部分(如 HA)交互。
我们开发了一种从肿瘤质量中分离原发癌细胞的技术。此外,我们创建了能刺激肿瘤微环境的基质,特别是C1q与高分子量透明质酸之间的相互作用。与HA结合的C1q能够诱导肿瘤细胞的粘附。

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evaluation of the interplay between the complement protein c1q and hyaluronic acid in promoting cell adhesion

肿瘤微环境在肿瘤生长和转移中起着积极的作用。通过不同的途径,肿瘤细胞可以通过分泌刺激因子、化学因子和细胞因子来有效地招募基质细胞、免疫细胞和内皮细胞。反过来,这些细胞可以通过释放促进生长的信号、代谢物和细胞外基质成分来改变微环境的信号特性,以维持高增殖和转移能力。在此背景下,我们确定,补充组分 C1q,由一系列人类恶性肿瘤在局部高度表达,在与细胞外基质透明质酸相互作用时,强烈影响从人类肿瘤中分离的初级细胞的行为标本。在这里,我们描述了一种测试C1q与透明质酸(HA)结合如何影响肿瘤细胞粘附的方法,其基础是细胞外基质的关键成分(在本例中为HA)的生物特性可以通过对肿瘤的生物活性信号来塑造进展。

HA是一种带负电荷的高分子量多糖,由重复(β,1-4)-D-葡萄糖酸-(β,1-3)-N-乙酰-D-葡萄糖胺脱糖单位(图1B)7组成。利用生物仿薄HA(生物-HA)对96孔板的HA结合的发生及其固定效率进行了测试。生物HA的不同浓度,从10微克/mL到1mg/mL,在100mM碳酸盐/碳酸氢盐缓冲液pH9.6中重新悬浮,并在4°C下孵育过夜。
经?...

Watch this Scientific Journal Video about Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion at JoVE.com

Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion

补体组分C1q是一种在组织微环境中高度表达的亲炎分子,可与细胞外基质相互作用。在这里,我们描述了一种测试C1q与透明质酸结合如何影响细胞粘附的方法。

补充蛋白C1q和透明质酸在促进细胞粘附中的相互作用评价

It has been increasingly demonstrated that the tumor microenvironment plays an active role in neoplasia growth and metastasis. Through different pathways, ...

评价补充蛋白C1q和透明质酸在促进细胞粘附方面相互作用的综合程序。我们最近定义，补充成分C1q的先天免疫系统分子在肿瘤和胎盘微环境中表达强烈，能够与细胞外基质的成分相互作用。在这种情况下，透明质酸。提出了一种研究蛋白质在整个基因组环境中的生物功能的方法。为了研究这些分子形成细胞外基质因子细胞分裂特性的能力。为了研究这种相互作用如何能被环境中的细胞感知，我们建立了这种简单的方法，从人体组织中提取的二元细胞可以粘附在与细胞外基质（本例中为透明质酸）绑定的C1q中。对于涂装工艺，第一步是将高分子量透明质酸与96井组织涂层微孔板结合。对于涂层，透明质酸以每毫升15微克的浓度在0.1摩尔碳缓冲液PH 9.6中稀释。轻轻的管道得到回报，以实现均匀的解决方案。溶液的100微升用于涂覆油井。然后，板在四摄氏度下一夜之间被转移。第二天真空吸气处理井。用 dPBS 洗一次。在相应的油井中添加 C1q 或 BSA。C1q现在以每毫升1毫克的浓度市售，每毫升25微克的浓度被稀释。和dPBS含有0.5%BSA和0.7毫摩尔钙和镁和二分质。BSA LPS 无浓度为 0.5%和日期 PBF。然后一夜之间在四摄氏度下转移板。C1q剂量依赖性相互作用与固定透明质酸是由酶链接免疫SA决定的。使用快速 DII 标记的单元格。亲脂追踪器快速DiI用于标记感兴趣的细胞。在 dPBS 中重新使用的细胞以每毫升一百万个细胞的浓度使用。Fast DiI 被稀释一到 100 直接到细胞悬浮液中。细胞悬浮液手动混合，在37摄氏度下传输15分钟。孵育五分钟后，混合细胞悬浮液，再重复两次。要去除快速 DiI 的过量，请添加 10 毫升 dPBS。轻轻上下旋转，在250克下离心7分钟。在0.1%BSA存在的情况下，将细胞颗粒重新在一毫升的人类嗜血血清自由培养基中。透明质酸C1q基质上的细胞粘附。真空从涂层井中吸入 dPBS。将 100 微升标记的电池悬浮液分发到涂层孔中。200微升标记的电池被拆开，用在一个带状管中，用于标准曲线生成。在37摄氏度下孵育板35分钟。从培养箱中取出盘子。轻轻吸气不平衡的细胞。用含有0.5%BSA和0.7毫摩尔钙和镁的dPBS洗一次。通过添加200微升的解解缓冲液来加入粘附细胞。同时在一到两次连续稀释解液缓冲液中标记的细胞后准备标准曲线。使用 544 纳米作为激发波长，使用 590 纳米作为发射波长，在无限 F 200 T-can 仪器上读取荧光信号。代表性结果。如代表性结果所示，透明质酸上的细胞粘附力明显，与HA单独或HA绑定BSA相比，C1q具有强。每个矩阵上粘附单元格的百分比根据标准曲线计算。结论。在我们的实验中，首先我们评估了补量成分C12使用虹膜金属结合细胞外基质的能力。其次，分析了沾染荧光染料的人类原细胞追求绑定透明质酸的能力。本研究中提出的模型是一种更简单、更便宜的方法，替代的是细胞外基质支架的 3D 模型，用于评估这种细胞对刺激组合的反应。

Research

JoVE Journal

Immunology and Infection

Depletion of Specific Cell Populations by Complement Depletion

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

测量50％的溶血性补充（CH 50）活性的血清

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

2'-脱氧核苷在裸体小鼠中治疗鼠胚胎胸腺的肾下囊移植

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...