Name: exploring the potential of mesenchymal stem cell sheet on the development of hepatocellular carcinoma in vivo
Uploaded: 2018-09-11T12:30:00
Description: Watch this Scientific Journal Video about Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo at JoVE.com

作者感谢沙特国王大学医学院实验性手术和动物实验室的工作人员的合作与支持, 特别是侯赛因 Almukhayzim 和 Hisham Aloudah。作者还要感谢沙特国王-阿齐兹大学的媒体小组准备了视觉材料, 特别是 Muath Ghannam 和 Abdulwahab Alsulami。

该议定书遵循沙特国王大学伦理委员会的动物保育准则。在动物身上使用的外科手术、麻醉剂和其他药物都是由沙特国王大学的伦理委员会批准的。所有实验工作都由受过适当训练的人员完成。
1. 细胞板结构
<ol><li>用未稀释的胎牛血清 (血清) 涂上培养皿 (3.5 厘米温度敏感的培养皿), 确保覆盖所有的盘子表面。 注意: 这一步对细胞表的分离很重要, 因为它支持单层细胞的生...

<ol>
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S., Tomchuck, S. L., Henkle, S. L., Betancourt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mesenchymal+stem+cell+(MSC)+paradigm:+polarization+into+a+pro-inflammatory+MSC1+or+an+Immunosuppressive+MSC2+phenotype.">A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype.</a> PLoS One. 5 (4), e10088 (2010).</li><li>Waterman, R. S., Henkle, S. L., Betancourt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+1+(MSC1)-based+therapy+attenuates+tumor+growth+whereas+MSC2-treatment+promotes+tumor+growth+and+metastasis.">Mesenchymal stem cell 1 (MSC1)-based therapy attenuates tumor growth whereas MSC2-treatment promotes tumor growth and metastasis.</a> PLoS One. 7 (9), e45590 (2012).</li><li>Curtin, L. I., 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=Evaluation+of+buprenorphine+in+a+postoperative+pain+model+in+rats.">Evaluation of buprenorphine in a postoperative pain model in rats.</a> Comparative Medicine. 59 (1), 60-71 (2009).</li><li>. Guidelines on Anesthesia and Analgesia in Rats - ULAM Guidelines and SOPs - Michigan Medicine Confluence Available from: <a target="_blank" href="https://wiki.med.umich.edu/display/ULAMGSOP/Guidelines+on+Anesthesia+and+Analgesia+in+Rats">https://wiki.med.umich.edu/display/ULAMGSOP/Guidelines+on+Anesthesia+and+Analgesia+in+Rats</a> (2017)</li><li>Alshareeda, A. T., Sakaguchi, K., Abumaree, M., Mohd Zin, N. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preclinical+new+drug+research+program+of+the+National+Cancer+Institute.">The preclinical new drug research program of the National Cancer Institute.</a> Cancer Treatment Reports. 68 (1), 63-76 (1984).</li><li>Suzuki, R., Aruga, A., Kobayashi, H., Yamato, M., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+in+vivo+cancer+model+using+cell+sheet+engineering.">Development of a novel in vivo cancer model using cell sheet engineering.</a> Anticancer Research. 34 (9), 4747-4754 (2014).</li><li>Chen, G., 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=Application+of+the+cell+sheet+technique+in+tissue+engineering.">Application of the cell sheet technique in tissue engineering.</a> Biomedical Reports. 3 (6), 749-757 (2015).</li><li>Matsuura, K., Haraguchi, Y., Shimizu, T., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sheet+transplantation+for+heart+tissue+repair.">Cell sheet transplantation for heart tissue repair.</a> Journal of Controlled Release. 169 (3), 336-340 (2013).</li><li>Matsuura, K., Shimizu, T., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+development+of+bioengineered+human+three-dimensional+vascularized+cardiac+tissue+using+cell+sheet+technology.">Toward the development of bioengineered human three-dimensional vascularized cardiac tissue using cell sheet technology.</a> International Heart Journal. 55 (1), 1-7 (2014).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+evidence+that+tumors+are+angiogenesis+dependent?.">What is the evidence that tumors are angiogenesis dependent?.</a> Journal of the National Cancer Institute. 82 (1), 4-6 (1990).</li></ol>

大量的研究致力于开发一种与人类癌症相似的活体前临床动物模型。目前, 用于创建癌症动物模型的主要方法涉及基因工程和细胞移植11。转基因动物模型是识别和验证目标基因的好工具, 也是了解药物诱导毒性的分子机制的良好手段。但是, 该模型与某些局限性相关;例如, 对某一特定基因的修饰并不总是导致预期的表型12。细胞移植法更常用于诱发动物肿...

肝癌是原发性肝癌, 与预后差有显著相关性。每年, 几乎有一半的新病人被诊断为肝癌, 占全世界1的肝癌患者的85%。肝癌不是一种单一形式的疾病;相反, 它是一个疾病的集合, 有不同的病理学特征和基因和基因组变异, 除了不同的预后结果1。因此, 对肝癌的有效治疗策略的发展面临的主要挑战是对肝癌生物学的认识有限, 缺乏合适的实验动物模型来帮助了解这种复杂的疾病。模拟人类癌症的体内动物模型对于选择候选基因和确定与癌症诱导相关的预后/预测指标以及调查可能影响癌症反应的不同因素都是必要的。治疗药物。
体外肿瘤的研究仍与主要的局限性有关。这是由于癌症细胞在培养时失去了许多体内的特性。细胞体外发生的变化是由于体内的组织生理没有形成。癌细胞对细胞的相互作用 (基质、免疫、血管、上皮等) 在肿瘤微环境中对癌细胞的特征有很大的反映2。除了 angiogenetic 和转移电位外, 肿瘤微环境可以改变癌细胞的基因/蛋白表达和表型特征。二维 (2 维)体外培养系统也缺乏合适的组织基质, 对肿瘤进展有一定的调节作用。因此, 由于这些限制,体内模型应始终用于支持的初步结果的体外模型。在本研究中, 我们利用细胞板技术开发了一种活体动物模型, 阐明了肝癌的完整生物学过程。
十年前, 冈野实验室建立了一种基于细胞片技术的组织工程新方法3。这种技术利用热响应的培养塑料, 以允许可逆的细胞粘附/脱离通过控制表面疏水性。这种方法允许在完整的三维 (3 维) 格式 (即细胞片) 中温和地收获培养细胞, 并保持良好的细胞外基质 (ECM) 和细胞对细胞的相互作用。细胞板技术需要预涂的文化菜肴与温度反应聚合物聚 (N-丙烯酰胺) (琵琶 Am), 这是商业上可用和准备使用。在摄氏20摄氏度以下的温度下, 琵琶的聚合物变得水合并溶解于水溶液中, 而在较高温度 (37 摄氏度) 时, 聚合物变得脱水, 转化成混浊沉淀。该聚合物含有亲水性酰胺链和疏水性侧链 (异丙基团)。在高温下, 水分子的布朗运动被强化, 而在低温下, 水合结构击穿的异丙基群周围的水分子和疏水性异丙基集群因为疏水性的相互作用。因此, 整个链的聚合物聚合和沉淀4。
在本文的研究中, 采用该技术开发了三种不同细胞片的肝癌动物模型。第一张工作表仅由肝癌细胞系细胞组成, 而另两片则由两种不同来源的肝癌细胞系细胞和 mscs 组成: 骨髓间充质干细胞 (BMMSCs) 和脐带 mscs (UCMSCs)。MSCs 是非造血基质细胞, 能够区分 intocell 衍生物的间充质谱系, 包括脂肪细胞, 骨, 软骨, 和肌细胞5。我们在创建癌细胞表时使用这些细胞的原因是关于 MSCs 对癌症的影响的报告不一致。有人建议, MSCs 可以有两个不同的表型: "MSC1", 促炎型, 和 "MSC2", 免疫抑制表6。MSCs 表达的类似的收费受体 (TLRs)。TLR4 启动的 MSCs 增加其分泌的促炎因子, 而 TLR3 启动增加其分泌免疫抑制因子6。对这两种表型的体外研究报告说, MSC1 与癌细胞株的共培养会削弱癌细胞的生长, 而 MSC2 共培养则有相反的作用7。这表明, MSCs 可以是亲癌或抗癌, 取决于他们的表型。因此, 除了利用细胞板技术开发肝癌动物模型外, 我们还要探讨 MSC 移植对肿瘤发展的影响, 以及使用这些细胞是否会增强或减少该模型的发展。

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exploring the potential of mesenchymal stem cell sheet on the development of hepatocellular carcinoma in vivo

一种模拟人类癌症的体内动物模型可以有各种应用来提供重要的临床信息。目前用于体内肿瘤模型发展的技术有很大的局限性。因此, 本研究旨在实现细胞板技术在体内肿瘤模型的建立。肝细胞癌 (hcc) 是在裸鼠中成功开发的, 使用细胞片创建的癌细胞系细胞。癌细胞片是通过细胞内黏附和形成的分层结构, 由细胞外基质控制。这使得肝癌片移植到肝脏, 并在一个月内创建一个肿瘤动物模型。此外, 研究了骨髓间充质干细胞 (MSC) 在肿瘤模型发展中的作用。除了肝癌细胞线外, 还创建了另外两个细胞片: 一张肝癌细胞和骨髓间充质干细胞 (BMMSCs) 和一张肝癌细胞和脐带 MSCs (UCMSCs)。有肝癌细胞和骨髓间充质干细胞组合的床单也能产生一种含肿瘤的动物。然而, 骨髓间充质干细胞的增加减少了肿瘤的大小, 这对肿瘤发育的不利影响取决于所使用的 mscs 的来源。这表明用某些 MSC 亚型制成的细胞片可以用于肿瘤的管理和控制。

大鼠移植细胞致瘤性的研究:
移植后一个月, 在大鼠肝脏移植的所有细胞片都已发育成肿瘤 (图 3)。被开发的肿瘤的平均大小从 HepG2, HepG2/BMMSC 和 HepG2/UCMSC 细胞片是 4.5 cm, 4 cm 和 2.5 cm, 分别10。
肝组织组织学分析:
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Watch this Scientific Journal Video about Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo at JoVE.com

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

在这里, 我们提出了一个使用细胞板技术开发体内肿瘤模型的协议。这种模型对肿瘤治疗的评价有很大的参考价值。

骨髓间充质干细胞片对肝癌体外发育的潜能探讨

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

Research

JoVE Journal

Bioengineering

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

科研

生物工程

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

创伤显微注射和分析<em&gt;在体内</em&gt;表皮的伤口本地化响应记者在<em&gt;果蝇</em&gt;胚胎。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

发展多深的圆形横截面微内皮化的一个芯片的程序

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Optimizing Attachment of Human Mesenchymal Stem Cells on PolyÃƒÅ½Ã‚Âµ-caprolactone Electrospun Yarns

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

山羊窒息关节髌下脂肪垫间充质干细胞的分离、扩增和分化

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...