Name: a novel feeder-free system for mass production of murine natural killer cells in vitro
Uploaded: 2018-01-09T14:00:00
Description: Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

这项研究得到香港研究资助局 (GRF 468513、CUHK3/CRF/12R) 及香港创新科技基金 (ITS/227/15、其 InP/164/16、ITS-InP/242/16)、中大 (2016.035) 及香港学者的直接资助。程序.
/设计和监督所有的实验, 并为手稿的编写做出贡献。康普茶进行了实验, 分析了数据并为稿件的编写做出了贡献。t。彼莱, 俱乐部, M.W., 和 G.-/收集动物标本, 并参与动物实验。右侧和 K-f.t 为手稿的编写做出了贡献。

获得和区分骨髓源性 nk 细胞的协议是基于以前发布的方法20,21,22。所有与老鼠有关的程序已获香港中文大学动物伦理实验委员会 (AEEC) 批准。
1. OP9 条件培养基的制备
<ol><li>培养小鼠基质细胞系 OP9 在α-记忆中含有 20% FBS, 100 U/毫升青霉素 G 和100µg/毫升链霉素, 在37° c 空气湿润大气/CO2 (95%: 5%)。<ol>
<...

<ol>
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在目前的工作中, 我们描述了一种新的方法生产骨髓源性小鼠 NK 细胞体外。原始系统21、22中的单元格进纸器被 OP9 单元的条件介质成功替换, 这极大地提高了微分系统的稳定性。此外, 该系统还能为体外的成熟 nk 细胞产生高纯度的增殖和纯化, 并能促进人体疾病中 nk 细胞的机械研究和转化研究. 
所描述的方法已?...

癌症进展主要依赖于肿瘤微环境1,2, 包括主机派生的免疫,, NK 细胞。几项研究表明, 肿瘤的 NK 细胞呈负相关的癌症进展3,4。此外, 临床研究表明 NK 细胞过继疗法是一种可能的治疗癌症的策略5,6,7,8,9。NK 细胞癌免疫治疗是最近被认为是一种治疗选择的固体肿瘤, 但存在的挑战, 由于免疫抑制剂的分泌因子和下调激活配体在微环境中的实体肿瘤10,11。转化生长因子β (TGF β) 已被认为起抑制作用的致癌作用, 但矛盾的癌细胞也产生 TGF-β1支持肿瘤的发展12,13,14,15. TGF β信号通过下调干扰素反应性和 CD16-mediated 干扰素-γ-γ (干扰素) 产生抑制 NK 细胞的细胞活性体外16,17, 18。
虽然 TGF β信号在肿瘤微环境中的破坏可能是消除癌症的一种可能的方法, 完全阻断 TGF β信号将导致自身免疫性疾病, 由于其抗炎作用, 证明了发展不良副作用包括全身炎症、心血管缺陷和自身免疫在小鼠模型中的19。因此, 了解 TGF β介导的免疫抑制的工作机制将导致确定一个治疗癌症的辅助性治疗靶。
为了阐明 nk 细胞发育所需的分子事件, 威廉斯et al.建立了一个用于将小鼠骨髓造血干细胞分化为 nk 细胞的体外系统20。该系统在很大程度上促进了 nk 细胞发育的机制研究, 包括对 nk 细胞的新祖的鉴定21。然而, 骨髓祖细胞应在系统中培养, 以支持 OP9 基质干细胞为饲养层20,21, 而这种异构的细胞群在很大程度上限制了基因中断工具的进一步应用(例如, siRNA 介导的基因沉默) 专门用于区分 NK 细胞。
在这里, 我们描述一个无馈线系统, 已经开发的进一步修改的在体外系统的威廉姆斯et al20。在我们的系统中, 不需要 OP9 基质饲养细胞, 而使用 OP9 条件培养基, 而不影响 NK 细胞的分化在体外, 这最近导致我们发现, TGF β能够促进癌症进展通过抑制 E4bp4-dependent NK 细胞在肿瘤微环境中的发育22。该系统成功地提供了一种无背景的方法来阐明在特定条件下 NK 细胞发育的分子机制 (例如, 高 TGF β, siRNA 介导的基因沉默, etc。体外。

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">OP9 cell line</td>
			<td style="width:129px;">ATCC</td>
			<td style="width:344px;">ATCC&#174; CRL-2749&#8482;</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MEM &#945;, no nucleosides</td>
			<td>Gibco</td>
			<td>22561021</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-7</td>
			<td>PEPROTECH</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine SCF</td>
			<td>PEPROTECH</td>
			<td>250-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine Flt3-Ligand</td>
			<td>PEPROTECH</td>
			<td>250-31L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-2</td>
			<td>PEPROTECH</td>
			<td>212-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamin RNAiMAX Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>1377815</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IC Fixation Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-8222-49</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow Cytometry Staining Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-4222-26</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-conjugated anti-mouse CD244</td>
			<td>eBioscience</td>
			<td>12-2441-83</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cy3-conjugated anti-mouse NKp46</td>
			<td>Bioss</td>
			<td>bs-2417R-cy3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nonsense control (NC)</td>
			<td>Ribobio</td>
			<td>siN05815122147</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">siRNA against mouse E4BP4 mRNA</td>
			<td>Ribobio</td>
			<td>N/A</td>
			<td>5&#8242;-GAUGAGGGUGUA 
			GUGGGCAAGUCUU-3&#8242;</td>
		</tr>
	</tbody>
</table>

a novel feeder-free system for mass production of murine natural killer cells in vitro

自然杀手 (NK) 细胞属于先天免疫系统, 是一线抗癌免疫防御;然而, 它们在肿瘤微环境中被压制, 其基本机制仍然是未知的。nk 细胞缺乏一致、可靠的来源, 限制了 nk 细胞免疫的研究进展。在这里, 我们报告一个体外系统, 可以提供高质量和数量的骨髓来源的小鼠 NK 细胞在无饲养条件下。更重要的是, 我们还证明, siRNA 介导的基因沉默成功地抑制了 E4bp4-dependent NK 细胞成熟, 利用该系统。因此, 这本新的体外NK 细胞分化系统是一种生物材料免疫研究的解决方案。

根据所述协议得到了代表性的结果。在无饲养诱导分化系统下培养的总骨髓悬浮细胞11天;7天, 与0天 (图 1A) 的总细胞数相比, 增殖率显著增加。在系统 (图 1B) 中, 6 天发现了成熟的 NK 细胞, 细胞核胞质比和富含颗粒的细胞质形态。
为了阐明 TGF-β1/Smad3 信号在 ...

Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

在这里, 我们提出了一个 mass-produce 基因沉默小鼠 NK 细胞的协议, 使用无馈线分化系统的机械研究体外和在体内。

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Research

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Immunology and Infection

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

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制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

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人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

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In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...