Name: an efficient and simple method to establish nk and t cell lines from patients with chronic active epstein-barr virus infection
Uploaded: 2018-03-30T13:00:00
Description: Watch this Scientific Journal Video about An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection at JoVE.com

这项工作得到了中国科学院重点项目 (KFZD-SW-205)、中国科学院战略生物资源技术支持系统 (CZBZX-1 和 ZSSB-004) 的支持, 并获得中国国家科学基金会资助 (81401640) 和上海自然科学基金 (14ZR1434800)。

这项研究获得中国科学院遗传学与发展生物学研究所伦理委员会的批准, 该议定书遵循人类福利的机构指南。
注意: 有关工作流的示意图, 请参见图 1 。
1. CAEBV 患者 PBMC 的分离
<ol><li>根据制造商的指示, 通过梯度 (例如, Ficoll 菌斑) 离心法从 CAEBV 患者的2毫升全血中纯化原 PBMC。或者, 用 RPMI 1640 培养基从液氮中解冻冷冻 PBMC。</l...

<ol>
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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=Preferential+expansion+of+Vgamma9-JgammaP/Vdelta2-Jdelta3+gammadelta+T+cells+in+nasal+T-cell+lymphoma+and+chronic+active+Epstein-Barr+virus+infection.">Preferential expansion of Vgamma9-JgammaP/Vdelta2-Jdelta3 gammadelta T cells in nasal T-cell lymphoma and chronic active Epstein-Barr virus infection.</a> Am J Pathol. 162 (5), 1629-1638 (2003).</li><li>Imadome, 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=Novel+mouse+xenograft+models+reveal+a+critical+role+of+CD4++T+cells+in+the+proliferation+of+EBV-infected+T+and+NK+cells.">Novel mouse xenograft models reveal a critical role of CD4+ T cells in the proliferation of EBV-infected T and NK cells.</a> PLoS Pathog. 7 (10), e1002326 (2011).</li><li>Shibuya, A., Nagayoshi, K., Nakamura, K., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine+requirement+for+the+generation+of+natural+killer+cells+from+CD34++hematopoietic+progenitor+cells.">Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells.</a> Blood. 85 (12), 3538-3546 (1995).</li><li>Spits, H., Yssel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+Human+T+and+Natural+Killer+Cells.">Cloning of Human T and Natural Killer Cells.</a> Methods. 9 (3), 416-421 (1996).</li><li>Liu, X., Xu, C., Duan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Red+Blood+Cell+Lysis+Method+for+the+Establishment+of+B+Lymphoblastoid+Cell+Lines.">A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines.</a> J Vis Exp. 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在该协议中, 建立了 CAEBV 患者全血 NK/T 细胞系的新方法。与现有方法相比, 该方法的主要优点是其简单性和对少量血液的要求, 同时具有较高的成功率和良好的细胞生存条件。此外, 可以通过培养 PBMC 来建立 NK/T 细胞系, 而不确定 eb 病毒潜伏预先感染的细胞类型, 因为这种测定在培养前会消耗更多的血液和时间。另外, 细胞系可以通过细胞系建立后的流式细胞术进行分析, 从而纯化细胞的不同表型。?...

eb 病毒是无处不在的, 不仅感染了 B 细胞, 而且传染了 T 和自然杀手 (nk) 细胞, 这导致了一些 eb 病相关的 NK/T 淋巴疾病 (LPD) 和淋巴瘤/白血病, 如 eb 联合噬血细胞lymphohistiocytosis, hydroa vacciniforme 淋巴瘤, 淋巴结外 nk/T 细胞淋巴瘤, 鼻型和侵袭性 NK 细胞白血病 1, 2, 3.其中包括严重的慢性主动 eb 病毒 (SCAEBV) 疾病, 主要发生在东亚, 现在被认为是由 eb 病毒感染的 T 或 NK 细胞的克隆扩张引起的 LPD4,5,6,7, 但没有明显的免疫缺陷存在于传染性传染性 (IM) 类似的症状, 包括发烧, 肿大, 淋巴结肿大, 肝功能障碍持续或反复, 以及高 eb 病毒-DNA 负荷在外围血液8,9。CAEBV 患者预后不佳10,11, 其发病机制和 eb 病毒的作用尚不清楚。因此, 从 eb 病毒相关的 nk/t 淋巴疾病和淋巴瘤的细胞系是非常有用的细胞模型, 以澄清 eb 病毒诱发 NK 或 t 细胞增殖机制及其与高发病率的白血病或淋巴.
到目前为止, 已经用不同的技术建立了几个单元格线12,13,14,15,16。人类 nk 细胞系由 nk 细胞淋巴瘤/白血病建立, 通过与小鼠基质细胞系进行共培养, 并在 rhIL-2 的浓度为每毫升15。KAI3 是由患有严重蚊子过敏或 SCAEBV 的患者与自体永生细胞系 (拼箱)、乙肝病毒转化的 B 细胞、作为饲养细胞以及在 100 u/毫升16中添加 rhIL-2 的病人建立的另一种 NK 细胞系。SNK6 和 SNT8 通过添加大剂量的 rhIL-2 (700 u/毫升)12, 从鼻 NK/T 细胞淋巴瘤患者的肿瘤组织中获得。与类似的技术, SNK-1 细胞是从 CAEBV 患者, 培养从 PBMC 通过去除 T 细胞和添加 700 u/毫升 rhIL-213,17。SNT13 和 SNT15 是通过删除 CD4+ 和 CD8+ 单元格18建立的。到目前为止, 其他 t 细胞和 nk 细胞系从 eb 病毒-NK/T LPD 患者都开发了此方法19。
上述现有方法的弊端包括: 饲养细胞的使用、高剂量 IL-2 的要求、多达10毫升全血的利用率, 或对 NK/T 细胞的净化, 这在临床上是非常有挑战性的, 因为在开始培养之前, 必须验证 eb 病毒潜伏感染的细胞类型。由于 CAEBV 主要发生在亚洲儿童身上, 所以在所有地区都不容易获得10毫升血液。在本研究中, 我们开发了一种新的简单的方法, 成功率高, 建立 NK 和/或 T 细胞系, 通过培养 PBMC 从 CAEBV 患者使用低剂量的 rhIL2 和2毫升的全血容量, 没有喂食细胞。结果表明, 该方法效率高, 节省时间。

<table>
	<tbody>
		<tr>
			<td>Hu HLA-DR FITC</td>
			<td>BD</td>
			<td>560944</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu CD4 FITC</td>
			<td>BD</td>
			<td>555346</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu/NHP CD8 PE</td>
			<td>BD</td>
			<td>557086</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu CD3 PE</td>
			<td>BD</td>
			<td>555340</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu CD16 FITC 3G8</td>
			<td>BD</td>
			<td>555406</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu/NHP CD56 PE MY31</td>
			<td>BD</td>
			<td>556647</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>hu/CD19 PE-Cy7</td>
			<td>BD</td>
			<td>560728</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>human IL-2</td>
			<td>Roche</td>
			<td>11147528001</td>
			<td></td>
		</tr>
		<tr>
			<td>human serum</td>
			<td>MRC</td>
			<td>CCC118-125</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>SANYO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Techcomp</td>
			<td>CT6T</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>OLYMPUS CKX53</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Countstar</td>
			<td>IC 1000</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>24 well cell culture cluster</td>
			<td>Corning Incorporated</td>
			<td>3524</td>
			<td>Polystyrene plates</td>
		</tr>
		<tr>
			<td>25cm2 cell culture flask</td>
			<td>Nest</td>
			<td>707001</td>
			<td>Polystyrene</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270</td>
			<td>Component of cell medium</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>life</td>
			<td>22400089</td>
			<td>For cell medium</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Amresco</td>
			<td>374</td>
			<td>Component of cell medium</td>
		</tr>
		<tr>
			<td>100 x streptomycin penicillin solution BioRoYeeBRY-2309</td>
			<td>BioRoYee</td>
			<td>BRY-2309</td>
			<td>Component of cell medium</td>
		</tr>
		<tr>
			<td>Ficoll paque plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td>For in vitro isolation of lymphocyte</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>For freezing cells</td>
		</tr>
		<tr>
			<td>flow cytometer</td>
			<td>BD</td>
			<td>BD FACSAria II</td>
			<td></td>
		</tr>
		<tr>
			<td>soft ware</td>
			<td>BD</td>
			<td>BD FACSDiva soft ware</td>
			<td>FACS analysis</td>
		</tr>
	</tbody>
</table>

an efficient and simple method to establish nk and t cell lines from patients with chronic active epstein-barr virus infection

从淋巴瘤或淋巴综合征患者中, 我们已经描述了一些方法来建立 NK/T 细胞系。这些方法使用饲养细胞, 纯化 NK 或 T 细胞多达10毫升的血液, 或高剂量的 IL-2。本研究提出了一种通过添加重组人 IL-2 (rhIL-2), 通过培养外周血单个核细胞 (PBMC) 来建立 NK 和 T 细胞系的新方法, 并使用2毫升的全血。细胞可以在两周内迅速增殖并维持3月以上。用该方法建立了 7 NK 或 T 细胞系, 成功率高。该方法简便、可靠, 适用于多例 CAEBV 或 NK/T 细胞淋巴瘤细胞株的建立。

在建立细胞系3天后, 多态细胞开始出现 (图 2)。7天后, 细胞生长迅速, 因为细胞的数量和生存能力都以较高的速率增加 (图 3)。当细胞浓度可超过 3-6 x 106时, 10-14 天后, 小簇细胞明显可见。在这一时期, 细胞应该被分成两到三口的培养板。大约一个月后, 一旦单元格号达到 3-5×107, 计数就足够高, 可以保存。
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Watch this Scientific Journal Video about An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection at JoVE.com

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

采用高效、少量外周血、低剂量 IL-2 的方法, 建立了 CAEBV 患者的 NK 和 T 细胞克隆的简便算法。

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A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Epstein - Barr病毒生长转化的淋巴细胞系的建立

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...