Name: generation of induced regulatory t cells from primary human na&iuml;ve and memory t cells
Uploaded: 2012-04-16T12:00:00
Description: Watch this Scientific Journal Video about Generation of Induced Regulatory T Cells from Primary Human NaÃƒÂ¯ve and Memory T Cells at JoVE.com

<p class="jove_content"作者希望感谢他们协助流式细胞仪分析和排序詹妮弗怪和格雷格·鲍曼。这项工作得到了NIH的资助从NCRR 2P20 RR020171和大学由肯塔基启动资金调频; GIE位承认总统的研究生奖学金和肯塔基机遇奖学金的支持。</p

1。分离人外周血单个核细胞（单核细胞从白膜） <ol><li>从医院或附近的血液中心位置，获得一个单位白膜。我们的血液中心提供约40-60毫升单位正常献血者中获得的棕黄色大衣。 </li><li>倒入蒸压500毫升玻璃瓶含无菌PBS稀释白膜血。最终体积的PBS +白膜应该是250毫升。 </li><li>装满十50​​ ml锥形管各20毫升Lymphoprep的解决方案。 </li><li>轻轻覆盖25毫升稀释白膜Lymphoprep解决方案，小心不扰乱Lymphoprep?...

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虽然拥有巨大的调节性T细胞的转移在打击自身免疫排斥反应和其他免疫或炎症介导的疾病，其高效发电和维护稳定的方法治疗的承诺尚未开发。由于只有1-5％的循环人类T细胞是调节性T细胞，他们控制的扩张和分化克服这种缺乏作为实施调节性T细胞进入临床的主要威慑。另一方面，我们从审判13 TGN1412了解到，这是科学和伦理的必要深入了解分子事件，编排实施任何治疗方案之前，人类T?...

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> lymphoprep </td><td>轴盾</td><td> 1114547 </td><td>保持室温</td></tr><tr><td>冷冻台式离心机</td><td> Eppendorf公司</td><td> 5810R </td><td></td></tr><tr><td>亮线血球</td><td>豪塞尔科学</td><td> 3110 </td><td></td></tr><tr><td> EasySep人类CD4 + T细胞富集试剂盒</td><td>干细胞技术</td><td> 19052 </td><td></td></tr><tr><td> EasySep人类记忆CD4 + T细胞富集试剂盒</td><td>干细胞技术</td><td> 19157 </td><td></td></tr><tr><td> EasySep人类朴素的CD4 + T细胞富集试剂盒</td><td>干细胞技术</td><td> 19155 </td><td></td></tr><tr><td>大易EasySep MAG净</td><td>干细胞技术</td><td> 18001 </td><td>银</td></tr><tr><td> 5 mL圆底聚苯乙烯管</td><td>屋宇署猎鹰</td><td> 352008 </td><td></td></tr><tr><td> 15 mL聚丙烯离心管</td><td>厂商VWR </td><td> 89004-368 </td><td></td></tr><tr><td> 14 mL聚丙烯圆底管</td><td>屋宇署猎鹰</td><td> 352059 </td><td></td></tr><tr><td> 50 mL聚丙烯离心管</td><td>厂商VWR </td><td> 89004-364 </td><td></td></tr><tr><td>人类抗CD3抗体</td><td>生物X手机</td><td> BE0001-2 </td><td>克隆：OKT3治疗</td></tr><tr><td> 24孔细胞聚苯乙烯培养板</td><td>屋宇署猎鹰</td><td> 353047 </td><td></td></tr><tr><td> 96井圆底培养板</td><td>格雷尼尔生物一</td><td> 650180 </td><td></td></tr><tr><td> Glutamax-I和HEPES缓冲的RPMI 1640 </td><td> Gibco公司</td><td> 72400 </td><td></td></tr> &lt;TR&gt; <td>胎牛血清（FBS）， </td><td> Gibco公司</td><td> 16000 </td><td></td></tr><tr><td> β-巯基乙醇</td><td>西格玛</td><td> M7522 </td><td></td></tr><tr><td>重组人转化生长因子-β1 </td><td> eBioscience </td><td> 14-8348-62 </td><td></td></tr><tr><td>重组人IL-2 </td><td> eBioscience </td><td> 14-8029-63 </td><td></td></tr><tr><td>牛血清白蛋白（BSA） </td><td> MP Biomedicals公司</td><td> 810531 </td><td></td></tr><tr><td> 0.5 M的EDTA </td><td> AMRESCO </td><td> E177 </td><td></td></tr><tr><td>鼠抗人CD25 - PE </td><td>美天旎生物技术</td><td> 130-091-024 </td><td>克隆：4E3 </td></tr><tr><td>鼠抗人CD45RA的PE-Cy5的</td><td> eBioscience </td><td> 15-0458-42 </td><td>克隆：HI100 </td></tr><tr><td>鼠抗人CD127-APC </td><td>美天旎生物技术</td><td> 130-094-890 </td><td>克隆：MB15-18C9 </td></tr><tr><td> DNA酶二</td><td>中号带够Biomedicals公司</td><td> 190370 </td><td></td></tr><tr><td> MoFlo流式细胞仪</td><td>贝克曼</td><td></td><td></td></tr><tr><td> FlowJo软件</td><td>树星公司</td><td></td><td></td></tr><tr><td>细胞任务Pro软件</td><td> BD公司</td><td></td><td></td></tr><tr><td>新生牛血清</td><td> Gibco公司</td><td> 16010 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司</td><td> 25030 </td><td></td></tr><tr><td> AIM-V的</td><td> Gibco公司</td><td> 0870112 </td><td></td></tr><tr><td> CellTrace CFSE标记细胞增殖试剂盒</td><td> Invitrogen公司</td><td> C34554 </td><td></td></tr><tr><td> Treg细胞抑制督察珠</td><td>美天旎生物技术</td><td> 130-092-909 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Gibco公司</td><td> 15140 </td><td></td></tr><tr><td> 40微米尼龙细胞株呃</td><td>屋宇署猎鹰</td><td> 352340 </td><td></td></tr></table>

generation of induced regulatory t cells from primary human na&iuml;ve and memory t cells

免疫的CD4 +调节性T细胞（Tregs）的开发和维护，促进周边的宽容，需要保持大量对人体的自我和共生抗原在免疫稳态。调节性T细胞和炎症常规T细胞之间的平衡扰动可导致免疫病理或癌症。虽然已被证明是4几个根本的分歧在人类与小鼠的调节性T细胞生物学5 1，I型糖尿病，类风湿关节炎和移植物抗宿主病，结肠炎的小鼠模型中的有效调节性T细胞治疗注射迄今排除了临床使用。由于缺乏足够数量，纯度，稳定性和特异性治疗调节性T细胞归巢的需要Treg的人类发展的动力平台上，以优化条件下体外扩增 6。 在这里，我们倒是描述一个从一个单一的人外周血献血，其中可分为四个阶段的分化诱导调节性T细胞（iTregs）分离外周血单个核细胞，CD4 + T细胞磁性选择的方法： 在体外细胞培养和荧光激活T细胞亚群细胞分选（FACS）。由于Treg的签名转录因子叉头框P3（FOXP3）是人类7激活诱导的转录因子，并没有其他存在独特的标记，标记的组合面板必须用于识别与抑制活性的T细胞。 6天培养后，细胞在我们的系统可以被划分成幼稚T细胞，记忆T细胞或基于其相对表达CD25，CD45RA的iTregs。由于内存和幼稚T细胞有不同的极化要求和可塑性8，牛逼成CD45RA +和CD45RO细胞人口的初始预分拣+亚群可以E中检查这些差异。与他人一致，CD25表达喜 CD45RA的- iTregs快递高水平的FOXP3 9，GITR和CTLA-4的第11和CD127 12的水平低。按照每个人口的流式细胞仪，由此产生的细胞可用于在评估相对延缓羧基琥珀酰亚胺酯（CFSE）标记的自体T细胞增殖能力的抑制实验。

Watch this Scientific Journal Video about Generation of Induced Regulatory T Cells from Primary Human NaÃƒÂ¯ve and Memory T Cells at JoVE.com

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

我们描述了从一个单一的人献血的监管，内存和幼稚T细胞产生的一种方法。极化调节性T细胞可以比较多种遗传同质性，这里还详细介绍了包括抑制法与遗传和功能应用中的其他子集。

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Research

JoVE Journal

Immunology and Infection

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

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 ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve CD4+ T Cells Using a TGF-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-containing Protocol

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...