TF-1细胞凋亡相关基因的研究

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TFF1在肺癌中的表达及作用研究

TFF1在肺癌中的表达及作用研究刘开泰;陆妙珍;褚宇东【摘要】目的研究三叶因子1(TFF1)在肺癌组织及肺癌细胞株中的表达、甲基化状态及其功能.方法选取98例非小细胞肺癌患者手术切除的肺癌组织,采用RT-PCR、MSP检测TFF1在肺癌组织中的表达与甲基化状态,分析其与患者临床特点的相关性,以及检测肺癌细胞株H23、H1299、L78、H446、H157及95D中TFF1的表达和甲基化状态以及5-aza-2'-deoxycy-tidine(DAC)处理后细胞株中TFF1表达的变化.TFF1转染H23细胞株,MTT法及克隆形成实验检测其增殖能力和克隆能力的变化,细胞凋亡实验及Western blot检测凋亡相关蛋白Caspase3表达水平.结果 TFF1在肺癌组织中的甲基化率为56.1％(55/98).TFF1甲基化与肺癌患者的TNM分期和淋巴结转移显著相关(P＜0.001).TFF1在H23和H157中无表达,在H1299、H446及95D中表达较低,在L78中表达较高.DAC处理后,细胞株TFF1表达的变化分别为表达恢复(H23、H157),表达增强(H1299,H446,95D)和无明显改变(L78).恢复表达TFF1后,H23细胞株的增殖及克隆形成能力明显减弱(P＜0.05),细胞凋亡率和凋亡相关蛋白Caspase3活性片段的表达明显增加.结论 TFF1在肺癌组织中的甲基化状态与肺癌的TNM分期和转移密切相关.TFF1在肺癌细胞中的表达受DNA甲基化的调控.肺癌细胞株H23中恢复表达TFF1可以抑制其增殖和克隆能力,促进其凋亡.【期刊名称】《浙江医学》【年(卷),期】2015(037)004【总页数】5页(P283-286,294)【关键词】TFF1;肺癌;DNA甲基化;表观遗传学【作者】刘开泰;陆妙珍;褚宇东【作者单位】315040 宁波市医疗中心李惠利医院放疗科;315040 宁波市医疗中心李惠利医院放疗科;315040 宁波市医疗中心李惠利医院肾内科【正文语种】中文1.1 人组织标本及细胞系 98例肺癌及癌旁组织来自宁波李惠利医院胸外科手术切除的肺癌患者，其中Ⅰ～Ⅱ期38例，Ⅲ～Ⅳ期60例。

北医免疫课程PDCD5-研究思路

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Construction of pcDITFAR19
fi ori Bgl II
CMV Intro
Nhe I EcoRI
LacZ
pGEM-TTFAR19
EcoR I
TFAR19
pcDI-IL-6
Sal I
IL-6 cDNA
EcoRI
EcoR I
ori
NeoR
XhoI BGH poly A
EcoR I
MS2
PDCD5 ORF PCR Product (410bp)
Xho I
pMTY-4
EcoR I Thrombin Linker Stu I
Xho I Pst I Klenow Xho I T4 Ligase pL Stu I Xho I
MS2
pMTY-TFAR19
EcoR I Thrombin Linker
TAA AAA AAT AAA CTT GTT ATG CAA AAA AAA AAA AAA AAA AAA AAA 540
PDCD5编码蛋白结构比较简单,全长125个氨基酸，不含二硫键， pI 5.65 ; 无信号肽，无跨膜序列，与已知人类蛋白无同源性;S118可以被CK2磷酸化
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PDCD5基因结构图
EcoRI
NeoR
NeoR 13
PDCD5过表达促进 HeLa和 MGC-803 细胞撤除血清诱导的凋亡
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PDCD5对各种细胞具有促凋亡效应,但单独不能诱导凋亡
细胞种类
TF-1 HL60，MGC-803，HEK293，HeLa Jurkat
诱导凋亡因素
GM-CSF withdrawal Serum withdrawal Serum withdrawal, VP16, FAS Ab

程序性细胞死亡分子5(PDCD5)研究进展

·综述·程序性细胞死亡分子５（ＰＤＣＤ５）研究进展杨丰赫１　叶菁菁２　郑　铭１，△（１北京大学医学部基础医学院生理学与病理生理学系，北京１００１９１；２北京大学人民医院中心实验室和临床分子生物学研究所，北京１０００４４）摘要　程序性细胞死亡分子５（ｐｒｏｇｒａｍｍｅｄｃｅｌｌｄｅａｔｈ５，ＰＤＣＤ５）在人体各种组织中广泛存在，可以通过多种凋亡通路促进细胞凋亡，其在大部分肿瘤中低表达，对肿瘤化疗具有增敏效应。

近年发现，除了在细胞凋亡过程中发挥作用外，ＰＤＣＤ５在多种疾病的病理进程中发挥重要作用，如：肿瘤、自身免疫性疾病、炎症性疾病、脑缺血再灌注损伤及动脉粥样硬化等。

ＰＤＣＤ５蛋白结构简单，无二硫键，其重组蛋白药物开发较便利，具较高的药物基因组学应用价值。

本文主要介绍近２０年来ＰＤＣＤ５的研究进展及其在肿瘤、免疫系统疾病、心血管系统疾病中可能的应用、存在的不足、以及在其他方面的应用前景。

关键词　ＰＤＣＤ５；凋亡；肿瘤；心血管系统疾病中图分类号　Ｒ３６３ＰｒｏｇｒｅｓｓｉｎＰＤＣＤ５ＲｅｓｅａｒｃｈａｎｄＩｔｓＰａｔｈｏｐｈｙｓｉｏｌｏｇｉｃａｌＲｅｌｅｖａｎｃｅ　ＹＡＮＧＦｅｎｇＨｅ１，ＹＥＪｉｎｇＪｉｎｇ２，ＺＨＥＮＧＭｉｎｇ１，△（１ＤｅｐａｒｔｍｅｎｔｏｆＰｈｙｓｉｏｌｏｇｙａｎｄＰａｔｈｏｐｈｙｓｉｏｌｏｇｙ，ＳｃｈｏｏｌｏｆＢａｓｉｃＭｅｄｉｃａｌＳｃｉｅｎｃｅｓ，ＨｅａｌｔｈＳｃｉｅｎｃｅＣｅｎｔｅｒ，ＰｅｋｉｎｇＵｎｉｖｅｒｓｉｔｙ，Ｂｅｉｊｉｎｇ１００１９１Ｃｈｉｎａ；２ＤｅｐａｒｔｍｅｎｔｏｆＣｅｎｔｒａｌＬａｂｏｒａｔｏｒｙ＆ＩｎｓｔｉｔｕｔｅｏｆＣｌｉｎｉｃａｌＭｏｌｅｃｕｌａｒＢｉｏｌｏｇｙ，ＰｅｋｉｎｇＵｎｉｖｅｒｓｉｔｙＰｅｏｐｌｅ＇ｓＨｏｓｐｉｔａｌ，Ｂｅｉｊｉｎｇ１０００４４Ｃｈｉｎａ）Ａｂｓｔｒａｃｔ　ＰｒｏｇｒａｍｍｅｄＣｅｌｌＤｅａｔｈ５（ＰＤＣＤ５）ｉｓｕｂｉｑｕｉｔｏｕｓｌｙｅｘｐｒｅｓｓｅｄｉｎｖａｒｉｏｕｓｈｕｍａｎｔｉｓｓｕｅｓ，ｐｒｏｍｏｔｉｎｇｃｅｌｌａｐｏｐｔｏｓｉｓｔｈｒｏｕｇｈｍｕｌｔｉｐｌｅａｐｏｐｔｏｔｉｃｐａｔｈｗａｙｓ；ＰＤＣＤ５ｈａｓｌｏｗｅｘｐｒｅｓｓｉｏｎｌｅｖｅｌｉｎｍｏｓｔｋｉｎｄｓｏｆｔｕｍｏｒｓ，ａｎｄｉｔｃａｎｉｎｃｒｅａｓｅｔｈｅｓｅｎｓｉｔｉｚａｔｉｏｎｏｆｔｕｍｏｒｓｉｎｒｅｓｐｏｎｓｅｔｏｃｈｅｍｏｔｈｅｒａｐｙ．Ｉｎｃｒｅａｓｉｎｇｅｖｉｄｅｎｃｅｓｈｏｗｅｄｔｈａｔ，ｉｎａｄｄｉｔｉｏｎｔｏｐａｒｔｉｃｉｐａｔｉｎｇｉｎｃｅｌｌａｐｏｐｔｏｓｉｓ，ＰＤＣＤ５ｐｌａｙｓｉｍｐｏｒｔａｎｔｒｏｌｅｓｉｎａｖａｒｉｅｔｙｏｆｄｉｓｅａｓｅｓ，ｓｕｃｈａｓｃａｎｃｅｒｓ，ｉｍｍｕｎｅｓｙｓｔｅｍｄｉｓｅａｓｅｓ，ｉｎｆｌａｍｍａｔｏｒｙｄｉｓｅａｓｅｓ，ｉｓｃｈｅｍｉａｒｅｐｅｒｆｕｓｉｏｎｉｎｊｕｒｉｅｓ，ａｎｄａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ．ＴｈｅｓｔｒｕｃｔｕｒｅｏｆＰＤＣＤ５ｐｒｏｔｅｉｎｉｓｑｕｉｔｅｓｉｍｐｌｅｗｉｔｈｏｕｔｄｉｓｕｌｆｉｄｅｂｏｎｄ，ｔｈｅｒｅｆｏｒｅ，ｉｔｉｓｖｅｒｙｃｏｎｖｅｎｉｅｎｔｔｏｄｅｖｅｌｏｐａｎｄｐｒｏｄｕｃｅｔｈｅｒｅｃｏｍｂｉｎａｎｔｐｒｏｔｅｉｎｆｏｒｔｈｅｒａｐｅｕｔｉｃｐｕｒｐｏｓｅｉｎｔｈｅｆｕｔｕｒｅ．ＴｈｉｓｒｅｖｉｅｗｍａｉｎｌｙｆｏｃｕｓｅｓｏｎｔｈｅｒｅｓｅａｒｃｈａｄｖａｎｃｅｏｆＰＤＣＤ５ｉｎｔｈｅｒｅｃｅｎｔｔｗｏｄｅｃａｄｅｓ，ｔｈｅｐｏｔｅｎｔｉａｌａｐｐｌｉｃａｔｉｏｎａｎｄｄｅｆｅｃｔｓｏｆＰＤＣＤ５ａｓｔｈｅｒａｐｅｕｔｉｃｓｔｒａｔｅｇｉｅｓｆｏｒｃａｎｃｅｒｓ，ｉｍｍｕｎｅｓｙｓｔｅｍｄｉｓｅａｓｅｓａｎｄｃａｒｄｉｏｖａｓｃｕｌａｒｄｉｓｅａｓｅｓ，ａｓｗｅｌｌａｓｐｅｒｓｐｅｃｔｉｖｅｓｏｆｔｈｅｖａｌｕｅｏｆＰＤＣＤ５ｉｎｏｔｈｅｒｄｉｒｅｃｔｉｏｎｓ．Ｋｅｙｗｏｒｄｓ　ＰＤＣＤ５；ａｐｏｐｔｏｓｉｓ；ｔｕｍｏｒ；ｃａｒｄｉｏｖａｓｃｕｌａｒｄｉｓｅａｓｅｓ１９９９年，北京大学人类疾病基因研究中心利用ｃＤＮＡ差示分析法（ｒｅｐｒｅｓｅｎｔａｔｉｖｅｄｉｆｆｅｒｅｎｃｅｓａｎａｌｙｓｉｓ），从诱导凋亡的ＴＦ１细胞用中成功克隆了在凋亡时表达上调的人类新基因，命名为ＴＦＡＲ１９（ＴＦ１ｃｅｌｌａｐｏｐｔｏｓｉｓｒｅｌａｔｅｄｇｅｎｅ１９）（Ｌｉｕ等．１９９９）。

足细胞凋亡与膜性肾病的研究进展

塞旦匿堂銎查垫！Ｑ生签堑鲞箜１２塑
２２４３
中存在足细胞凋亡，而导致足细胞凋亡的主要原因则为糖尿病肾病氧化应激的产物ＲＯＳ。目前认为ＲＯＳ的来源主要有两条途径．一为ＮＡＤＰＨ氧化酶途径，二为线粒体途径。Ｓｕｓｚｔａｋ等［１０］研究表明．在体外培养的条件性永生足细胞中加入高糖（３０ｍｍｏｌ／Ｌ）可刺激细胞膜ＮＡＤＰＨ氧化酶和线粒体两条途径产生大量ＲＯＳ．ＲＯＳ又通过ｐ３８ＭＡＰＫ信号
２．Ｉ．２
ＴＧＦ．Ｂ１介导的足细胞凋亡ＴＧＦ．
Ｂｌ（转化生长因子Ｂ１）是一种多功能的细胞因子．在损伤的肾小球内。呈典型上调。越来越多的研究表明ＴＧＦ．１３１介导了肾小球足细胞的凋亡。在肾小球硬化过程中发挥着重要作用。目前认为ＴＧＦ．Ｂｌ主要通过以下两条信号转导通路介导足细胞的凋亡。
２．１．１
ＴＧＦ．Ｂ１／ｐ３８ＭＡＰＫ信号通路
ＴＧＦ．１３１除激活Ｓｍａｄ通路外．还激活丝裂原活化的蛋白激酶（ＭＡＰＫＳ），如ｐ３８ＭＡＰＫ通路。ｐ３８ＭＡＰＫ为ＴＧＦ．１３ｌ系统下游的一个通路。ＴＧＦ．１３１通过与其Ｉ型和Ⅱ型丝／苏氨酸激酶受体的不同成分相互作用，将信号转入细胞内，刺激激活ＭＫＫＫ（ＭＡＰＫ激酶激酶），转而激活ＭＫＫ（ＭＡＰＫ激酶）主要为ＭＫＫ３和ＭＫＫ６．然后通过双位点磷酸化激活ｐ３８ＭＡＰＫ．活化的ｐ３８ＭＡＰＫ信号诱导促凋亡因子Ｂａｘ（Ｂｃｌ．２家族成员）蛋白合成，Ｂａｘ与含ＢＨ３结构域的另外的Ｂｃｌ．２家族成员作用。导致前者的寡聚并插入线粒体膜，引起线粒体膜通透性的改变．跨膜电位丢失，释放细胞色素Ｃ和其他蛋白［８］。释放的细胞色素Ｃ与Ａｐａｆ－Ｉ（凋亡促进因子）结合引起Ａｐａｆ－１寡聚。从而募集并激活Ｃａｓｐａｓｅ．９．由Ｃａｓｐａｓｅ．９激活Ｃａｓｐａｓｅ－３，使细胞进入凋亡［引．此即凋亡的线粒体通路。这一过程在Ｓｃｈｉｆｆｅｒ等㈧的研．究中被证实：在足细胞内，ＴＧＦ一１３１刺激

细胞凋亡的检测(来自北京大学人类疾病基因研究中心

摘自北京大学人类疾病基因研究中心/news/apoptosis.htm细胞凋亡的检测北京大学人类疾病基因研究中心陈英玉细胞凋亡与坏死是两种完全不同的细胞凋亡形式，根据死亡细胞在形态学、生物化学和分子生物学上的差别，可以将二者区别开来。

细胞凋亡的检测方法有很多，下面介绍几种常用的测定方法。

一、细胞凋亡的形态学检测根据凋亡细胞固有的形态特征，人们已经设计了许多不同的细胞凋亡形态学检测方法。

1 光学显微镜和倒置显微镜（1）未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体。

贴壁细胞出现皱缩、变圆、脱落。

（2）染色细胞：常用姬姆萨染色、瑞氏染色等。

凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割成块状和凋亡小体等典型的凋亡形态。

2 荧光显微镜和共聚焦激光扫描显微镜一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。

常用的DNA特异性染料有：HO 33342 (Hoechst 33342)，HO 33258 (Hoechst 33258), DAPI。

三种染料与 DNA的结合是非嵌入式的，主要结合在DN A的A-T碱基区。

紫外光激发时发射明亮的蓝色荧光。

Hoechst是与DNA特异结合的活性染料，储存液用蒸馏水配成1mg/ml的浓度，使用时用PBS稀释成终浓度为2~5mg/ml。

DAPI为半通透性，用于常规固定细胞的染色。

储存液用蒸馏水配成1mg/ml的浓度，使用终浓度一般为0.5 ~1mg/ml。

结果评判：细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样（creased），部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块，产生凋亡小体(图1)。

3 透射电子显微镜观察结果评判：凋亡细胞体积变小，细胞质浓缩。

凋亡Ⅰ期（pro-apoptosis nuclei）的细胞核内染色质高度盘绕，出现许多称为气穴现象（cavitations）的空泡结构(图2)；Ⅱa期细胞核的染色质高度凝聚、边缘化；细胞凋亡的晚期，细胞核裂解为碎块，产生凋亡小体。

atf2转录因子 -回复

atf2转录因子-回复转录因子（Transcription Factors，简称TFs）是一类能够调控基因转录的蛋白质分子，它们通过结合到基因的启动子区域上，与RNA聚合酶和其他转录激活或抑制因子相互作用，从而影响基因的表达。

在细胞发育和生理过程中，TFs起到了至关重要的作用，能够调控基因的启动和停止、确定基因表达的细胞、组织和器官特异性，以及参与激素信号传导等。

本篇文章将深入探讨ATF2转录因子及其在细胞信号转导中的功能。

ATF2 （Activating Transcription Factor 2）是一个重要的转录因子家族成员，其编码基因位于人类染色体2q32-q34区域。

ATF2是一个受丝裂原活化蛋白激酶（MAPK）信号通路调节的双重转录因子，能够通过调控基因的转录起到生物学功能。

该转录因子由两个功能域组成：N末端的ATF 家族保守结构域（bZIP结构域）和C末端的加上杆。

ATF2参与了多种信号通路的调控，例如T细胞受体（TCR）信号通路、线粒体途径和糖代谢途径等。

在TCR活化过程中，ATF2被丝裂原活化激酶家族蛋白激活，进而促使其定位到TCR信号通路的启动子区域，通过结合到响应元件上，与其他转录因子相互作用，从而调控下游基因的转录。

这一过程在免疫细胞的激活和应激反应中起到了重要的作用。

此外，ATF2还与肿瘤的发生和发展密切相关。

研究发现，ATF2能够调控细胞周期和凋亡相关基因的表达，进而影响细胞的增殖和生存。

ATF2的过度表达可能导致细胞增殖水平的提高，从而推动癌症的发展。

另一方面，ATF2的功能丧失可能导致凋亡信号通路的紊乱，从而促进肿瘤细胞的增殖和转移。

因此，ATF2有望成为治疗癌症的重要靶点。

除了在细胞信号转导中的功能，ATF2还参与了细胞的应激反应。

在环境胁迫、氧化应激等情况下，ATF2能够被激活，进而调控一系列应激反应基因的转录。

这些基因的表达可以使细胞适应环境的变化，并促进细胞的存活。

TFF与消化系统肿瘤关系的研究进展

TFF与消化系统肿瘤关系的研究进展冯丽英;马丽;秦玉彩【摘要】Trefoil factor (TFF) peptides are a family of small molecular regulatory protein which is mainly expressed in gastrointestinal mucus membrane,including TFF1 (PS2), TFF2 (SP)and TFF3(ITF). Its main function is to keep the integrity of mucous membranes and promote the repair of epithelial damage. Recent studies have found that trefoil factor family has connection with the occurrence and development of digestive tumors. At the same time it has a certain impact on cell proliferation and apoptosis. Therefore further study on mechanism of trefoil factor family in gastrointestal tumor will provide new approaches for the diagnosis and treatment of gastrointestal cancer.%TFF是一族主要表达于胃肠道黏膜的小分子多肽,包括乳腺癌相关肽、解痉多肽和肠三叶因子,其主要功能是维持黏膜的完整性和促进损伤黏膜的修复.近年研究发现TFF与消化系统肿瘤的发生、发展有关,同时对细胞的增殖和凋亡有一定的影响,因此,进一步研究TFF在消化系肿瘤中的作用机制,将为消化系肿瘤的诊断与治疗开辟新的途径.【期刊名称】《临床误诊误治》【年(卷),期】2011(024)009【总页数】4页(P82-85)【关键词】三叶因子;肽类;消化系统肿瘤;细胞增殖;细胞凋亡【作者】冯丽英;马丽;秦玉彩【作者单位】0000石家庄,河北医科大学第二医院消化科;0000石家庄,河北医科大学第二医院消化科;0000石家庄,河北医科大学第二医院消化科【正文语种】中文【中图分类】R735TFF(trefoil factor family，TFF)是新近发现的一类小分子多肽，又称三叶肽。

细胞凋亡及检测方法

细胞凋亡细胞凋亡是指由基因所决定的细胞自动结束生命的过程，由于细胞凋亡受到严格的由遗传机制决定的程序性调控，所以也常常称为细胞编程性死亡(programmed cell death,PCD)。

具体指细胞遇到内、外环境因子刺激时，受基因调控启动的自杀保护措施，包括一些分子机制的诱导激活和基因编程，通过这种方式去除体内非必需的细胞或即将发生特化的细胞。

例如，蛙的个体发育过程中，四肢的形成过程中尾巴消失等现象，都涉及细胞的自动死亡。

在形态学上把细胞凋亡分为三个阶段：第一个阶段是凋亡的开始，此阶段只是进行数分钟，细胞中所表现的特征是：微绒毛消失，细胞间接触消失，但是质膜保持完整性，线粒体大体完整，核糖体逐渐与内质网脱离，内质网囊腔膨胀，并与质膜发生融合，染色质固缩等等;第二阶段是形成凋亡小体，核染色质发生断裂，形成许多的片段，与一些细胞器聚集在一起，然后被细胞质膜包围，形成凋亡小体；第三阶段是凋亡小体被吞噬细胞所吞噬，而其残留物质被消化后重新使用。

细胞凋亡是一个主动性自杀过程，所以它是一个耗能的过程，需要ATP提供能量。

【细胞凋亡是指为维持内环境稳定，由基因控制的细胞自主的有序的死亡。

细胞凋亡与细胞坏死不同，细胞凋亡不是一件被动的过程，而是主动过程，它涉及一系列基因的激活、表达以及调控等的作用；它并不是病理条件下，自体损伤的一种现象，而是为更好地适应生存环境而主动争取的一种死亡过程。

细胞发生凋亡时，就像树叶或花的自然凋落一样，对于这种生物学观察，借用希腊“Apoptosis”来表示，意思是像树叶或花的自然凋落，可译为细胞凋亡。

人体内的细胞注定是要死亡的，有些死亡是生理性的，有些死亡则是病理性的，有关细胞死亡过程的研究，近年来已成为生物学、医学研究的一个热点，到目前为此，人们已经知道细胞的死亡起码有两种方式，即细胞坏死与细胞凋亡(apoptosis)。

细胞坏死是早已被认识到的一种细胞死亡方式，而细胞凋亡则是近年逐渐被认识的一种细胞死亡方式。

细胞凋亡相关功能基因列表

Entrez Gene ID Current Symbol 25ABL1134ADORA1135ADORA2A142PARP1143PARP4148ADRA1A186AGTR2196AHR199AIF1207AKT1208AKT2239ALOX12317APAF1318NUDT2328APEX1329BIRC2330BIRC3331BIRC4332BIRC5333APLP1351APP355FAS356FASLG387RHOA388RHOB472ATM572BAD573BAG1578BAK1580BARD1581BAX596BCL2597BCL2A1598BCL2L1599BCL2L2602BCL3604BCL6608TNFRSF17627BDNF637BID638BIK652BMP4662BNIP1663BNIP2664BNIP3666BOK672BRCA1673BRAF695BTK727C5729C6730C7731C8A732C8B733C8G735C9823CAPN1 827CAPN6 834CASP1835CASP2 836CASP3 837CASP4 838CASP5 839CASP6 840CASP7 841CASP8 842CASP9 843CASP10 857CAV1 858CAV2 867CBL914CD2922CD5L 929CD14 939CD27 940CD28 943TNFRSF8 944TNFSF8 949SCARB1 952CD38 958CD40 959CD40LG 970CD70 984CDC2L1 993CDC25A 1026CDKN1A 1029CDKN2A 1032CDKN2D 1082CGB 1147CHUK 1149CIDEA 1191CLU 1201CLN3 1268CNR1 1269CNR2 1270CNTF 1432MAPK14 1434CSE1L 1499CTNNB1 1509CTSD 1537CYC11603DAD1 1611DAP1612DAPK1 1613DAPK3 1616DAXX 1630DCC1647GADD45A 1649DDIT3 1676DFFA 1677DFFB 1773DNASE1 1776DNASE1L3 1777DNASE2 1793DOCK1 1848DUSP6 1849DUSP7 1869E2F1 1871E2F3 1875E2F5 1982EIF4G2 2012EMP1 2013EMP2 2014EMP3 2033EP3002068ERCC2 2071ERCC32081ERN1 2147F22149F2R2175FANCA 2185PTK2B 2277FIGF2308FOXO1 2309FOXO3 2355FOSL2 2549GAB1 2620GAS2 2668GDNF 2765GML2876GPX1 2879GPX4 2885GRB2 2932GSK3B 2934GSN2936GSR2944GSTM1 2950GSTP1 2953GSTT2 2999GZMH 3001GZMA3002GZMB 3004GZMM 3064HD3065HDAC1 3309HSPA5 3336HSPE1 3375IAPP 3440IFNA2 3456IFNB1 3480IGF1R 3481IGF2 3482IGF2R 3483IGFALS 3485IGFBP2 3487IGFBP4 3488IGFBP5 3489IGFBP6 3551IKBKB 3552IL1A 3553IL1B 3558IL23559IL2RA 3562IL33563IL3RA 3586IL10 3588IL10RB 3604TNFRSF9 3605IL17A 3607FOXK2 3659IRF1 3725JUN 3726JUNB 3727JUND 3906LALBA 3956LGALS1 3987LIMS1 4049LTA 4050LTB 4055LTBR 4064CD180 4069LYZ 4137MAPT 4170MCL1 4193MDM2 4194MDM4 4214MAP3K1 4215MAP3K3 4217MAP3K5 4294MAP3K10 4318MMP9 4353MPO4599MX14602MYB4605MYBL2 4609MYC4616GADD45B 4671NAIP4790NFKB1 4791NFKB2 4792NFKBIA 4793NFKBIB4794NFKBIE4803NGFB4804NGFR4832NME34842NOS14843NOS2A 4869NPM14908NTF34914NTRK1 4982TNFRSF11B 5023P2RX1 5055SERPINB2 5058PAK15062PAK25074PAWR 5077PAX35081PAX75133PDCD1 5134PDCD2 5153PDE1B 5288PIK3C2G 5366PMAIP1 5371PML5447POR5519PPP2R1B 5551PRF15562PRKAA1 5578PRKCA 5581PRKCE 5590PRKCZ 5594MAPK1 5596MAPK4 5598MAPK7 5599MAPK8 5602MAPK10 5604MAP2K1 5607MAP2K5 5608MAP2K6 5610EIF2AK2 5663PSEN1 5664PSEN25728PTEN 5733PTGER3 5741PTH5743PTGS25747PTK2 5777PTPN6 5783PTPN13 5885RAD21 5887RAD23B 5894RAF1 5925RB1 5926ARID4A 5927JARID1A 5928RBBP4 5929RBBP5 5930RBBP6 5933RBL1 5934RBL25970RELA 5971RELB 5977DPF2 6285S100B 6300MAPK12 6347CCL2 6416MAP2K4 6422SFRP1 6423SFRP2 6425SFRP5 6446SGK 6477SIAH1 6494SIPA1 6622SNCA 6647SOD1 6651SON6696SPP16753SSTR3 6772STAT1 6788STK3 6868ADAM176885MAP3K7 6997TDGF1 7009TEGT 7027TFDP1 7029TFDP2 7040TGFB1 7072TIA17073TIAL17078TIMP37097TLR2 7122CLDN5 7124TNF7126TNFAIP1 7127TNFAIP2 7128TNFAIP3 7130TNFAIP6 7132TNFRSF1A 7133TNFRSF1B 7157TP537159TP53BP2 7161TP737184HSP90B1 7185TRAF1 7186TRAF2 7187TRAF3 7188TRAF5 7189TRAF6 7262PHLDA27292TNFSF47293TNFRSF4 7416VDAC1 7423VEGFB 7424VEGFC7529YWHAB 7531YWHAE 7532YWHAG 7533YWHAH 7534YWHAZ 7818DAP3 8065CUL5 8087FXR1 8193DPF1 8382NME5 8451CUL4A 8452CUL3 8453CUL2 8454CUL1 8477GPR65 8507ENC1 8517IKBKG 8535CBX4 8539API58565YARS8567MADD8600TNFSF11 8626TP73L8678BECN1 8717TRADD 8718TNFRSF25 8727CTNNAL1 8737RIPK18738CRADD 8739HRK8740TNFSF14 8741TNFSF13 8742TNFSF12 8743TNFSF10 8744TNFSF98764TNFRSF148767RIPK28771TNFRSF6B 8772FADD8784TNFRSF18 8792TNFRSF11A 8793TNFRSF10D8794TNFRSF10C 8795TNFRSF10B 8797TNFRSF10A 8835SOCS2 8837CFLAR 8841HDAC3 8870IER38887TAX1BP1 8915BCL10 8993PGLYRP1 8995TNFSF18 8996NOL38997KALRN 9020MAP3K14 9021SOCS3 9093DNAJA3 9131AIFM19140ATG12 9141PDCD5 9166EBAG9 9191DEDD9214FAIM39220TIAF19262STK17B 9263STK17A 9275BCL7B9352TXNL1 9446GSTO1 9450LY869459ARHGEF6 9474ATG5 9516LITAF 9529BAG5 9530BAG4 9531BAG3 9532BAG2 9538EI249540TP53I3 9542NRG2 9577BRE9616RNF7 9618TRAF4 9633MTL5 9647PPM1F 9700ESPL1 9774BCLAF1 9966TNFSF15 9994CASP8AP2 10000AKT3 10010TANK 10016PDCD6 10017BCL2L10 10018BCL2L11 10116FEM1B 10134BCAP31 10201NME6 10224ZNF443 10285SMNDC1 10293TRAIP 10296MAEA 10392NOD1 10420TESK2 10497UNC13B 10507SEMA4D 10549PRDX4 10553HTATIP2 10572SIVA1 10668CGRRF1 10673TNFSF13B 10753CAPN9 10787NCKAP1 10884MRPS30 10885WDR3 10892MALT1 10912GADD45G 10913EDAR 10922FASTK10971YWHAQ 11009IL2411035RIPK3 11124FAF1 11131CAPN11 11235PDCD10 22861NLRP1 22900CARD8 22984PDCD11 23017FAIM2 23064SETX 23087TRIM35 23531MMD 23542MAPK8IP2 23567ZNF346 23581CASP14 2359139174 23604DAPK2 23636NUP62 23645PPP1R15A 23647ARFIP2 23786BCL2L13 23787MTCH1 25816TNFAIP8 25824PRDX5 26471NUPR1 26574AATF 27018NGFRAP1 27141CIDEB 27242TNFRSF21 27250PDCD4 27429HTRA2 28986MAGEH1 29108PYCARD 29775CARD10 51079NDUFA13 51129ANGPTL4 51275C12orf47 51283BFAR 51330TNFRSF12A 51374C2orf28 51428DDX41 51454GULP1 51567TTRAP 51651PTRH2 51747CROP 51750RTEL1 54205CYCS 54466SPIN2A 54476TRIAD3 54764ZRANB154840APTX 55504TNFRSF19 55655NLRP2 55973BCAP29 56616DIABLO 56848SPHK2 56940DUSP22 57099AVEN 57142RTN4 57448BIRC6 57761TRIB3 58484NLRC4 59082ICEBERG 59347FKSG2 63924CIDEC 63970P53AIP1 64065PERP 64127NOD2 64170CARD9 64651AXUD1 64919BCL11B 79092CARD14 79370BCL2L14 79444BIRC7 79960PHF17 83596BCL2L12 84306PDCD2L 84433CARD11 84674CARD6 91662NLRP12 112401BIRC8 114548NLRP3 114770PGLYRP2 283849EXOC3L 399687MYO18A 440081DDX12 728642CDC2L2Refseq TranscriptNM_007313 NM_005157NM_000674 NM_001048230NM_000675NM_001618NM_006437NM_033304 NM_033303 NM_033302 NM_000680NM_000686NM_001621NM_001623 NM_032955NM_001014431 NM_005163 NM_001014432NM_001626NM_000697NM_013229 NM_181868 NM_181861 NM_181869 NM_001160NM_147172 NM_001161 NM_147173NM_080649 NM_080648 NM_001641NM_001166NM_182962 NM_001165NM_001167NM_001012271 NM_001168 NM_001012270NM_001024807 NM_005166NM_201413 NM_201414 NM_000484NM_152871 NM_152877 NM_000043 NM_152876 NM_152874 NM_152873 NM_152875 NM_152872NM_000639NM_001664NM_004040NM_000051 NM_138292NM_004322 NM_032989NM_004323NM_001188NM_000465NM_004324 NM_138764 NM_138763 NM_138761 NM_138765NM_000657 NM_000633NM_004049NM_138578 NM_001191NM_004050NM_005178NM_138931 NM_001706NM_001192NM_170733 NM_170734 NM_170731 NM_001709 NM_170735 NM_170732NM_001196 NM_197966 NM_197967NM_001197NM_130850 NM_130851 NM_001202NM_013979 NM_013978 NM_001205 NM_013980NM_004330NM_004052NM_032515NM_007300 NM_007297 NM_007304 NM_007294 NM_007296 NM_007299 NM_007303 NM_007302 NM_007295 N NM_004333NM_000061NM_001735NM_000065NM_000587NM_000562NM_000066NM_000606NM_001737NM_005186NM_014289NM_033293 NM_033292 NM_001223 NM_033295 NM_033294NM_032983 NM_032982NM_032991 NM_004346NM_001225 NM_033306NM_004347NM_001226 NM_032992NM_001227 NM_033339 NM_033338 NM_033340NM_001080125 NM_033355 NM_033358 NM_033356 NM_001080124 NM_001228 NM_001229 NM_032996NM_001230 NM_032977 NM_032974NM_001753NM_001233 NM_198212NM_005188NM_001767NM_005894NM_000591 NM_001040021NM_001242NM_006139NM_001243 NM_152942NM_001244NM_001082959 NM_005505NM_001775NM_001250 NM_152854NM_000074NM_001252NM_033488 NM_033492 NM_033486 NM_033489 NM_033487 NM_033493NM_201567 NM_001789NM_078467 NM_000389NM_000077 NM_058195 NM_058197NM_079421 NM_001800NM_000737NM_001278NM_001279 NM_198289NM_001831 NM_203339NM_001042432 NM_000086NM_016083 NM_033181NM_001841NM_000614NM_139012 NM_139014 NM_139013 NM_001315NM_001316NM_001098210 NM_001904 NM_001098209NM_001909NM_001916NM_001344NM_004394NM_004938NM_001348NM_001350NM_005215NM_001924NM_004083NM_004401 NM_213566NM_004402NM_005223NM_004944NM_001375NM_001380NM_022652 NM_001946NM_001947NM_005225NM_001949NM_001083589 NM_001951 NM_001083588NM_001418 NM_001042559NM_001423NM_001424NM_001425NM_001429NM_000400NM_000122NM_152461 NM_001433NM_000506NM_001992NM_001018112 NM_000135NM_004103 NM_173176 NM_173175 NM_173174 NM_004469NM_002015NM_001455 NM_201559NM_005253NM_002039 NM_207123NM_005256 NM_177553NM_199231 NM_000514 NM_199234NM_002066NM_201397 NM_000581NM_002085 NM_001039847 NM_001039848NM_002086 NM_203506NM_002093NM_000177 NM_198252NM_000637NM_000561 NM_146421NM_000852NM_000854NM_033423NM_006144NM_004131NM_005317NM_002111NM_004964NM_005347NM_002157NM_000415NM_000605NM_002176NM_000875NM_001007139 NM_000612NM_000876NM_004970NM_000597NM_001552NM_000599NM_002178NM_001556NM_000575NM_000576NM_000586NM_000417NM_000588NM_002183NM_000572NM_000628NM_001561NM_002190NM_004514 XM_001134363 XM_001134364NM_002198NM_002228NM_002229NM_005354NM_002289NM_002305XM_001129158 NM_004987NM_000595NM_009588 NM_002341NM_002342NM_005582NM_000239NM_016834 NM_016835 NM_016841 NM_005910NM_021960 NM_182763NM_006881 NM_002392 NM_006879 NM_006878 NM_006882 NM_002393XM_042066 XM_001128827NM_203351 NM_002401NM_005923NM_002446NM_004994NM_000250NM_002462NM_005375NM_002466XM_001129520 NM_002467NM_015675NM_022892 NM_004536NM_003998NM_001077494 NM_001077493 NM_002502NM_020529NM_001001716 NM_002503NM_004556NM_002506NM_002507NM_002513NM_000620NM_000625NM_001037738 NM_199185 NM_002520NM_002527NM_001012331 NM_002529 NM_001007792NM_002546NM_002558NM_002575NM_002576NM_002577 XM_001126110NM_002583NM_181457 NM_181460 NM_181459 NM_013942 NM_181461 NM_181458 NM_000438NM_013945 NM_002584NM_005018NM_144781 NM_002598NM_000924NM_004570NM_021127NM_033238 NM_033250 NM_033246 NM_033249 NM_033240 NM_033244 NM_033247 NM_002675 NM_033239 NM_000941NM_002716 NM_181699NM_001083116 NM_005041NM_006251 NM_206907NM_002737NM_005400NM_002744 NM_001033581 NM_001033582NM_138957 NM_002745NM_002747NM_139033 NM_139032 NM_002749 NM_139034NM_139046 NM_139049 NM_139047 NM_002750NM_138980 NM_002753 NM_138982 NM_138981NM_002755NM_002757 NM_145160NM_002758NM_002759NM_000021NM_012486 NM_000447NM_000314NM_198716 NM_198713 NM_198715 NM_198712 NM_198719 NM_198717 NM_198714 NM_000957 NM_198718 NM_000315NM_000963NM_153831 NM_005607NM_002831 NM_080548NM_080685 NM_006264 NM_080684 NM_080683NM_006265NM_002874NM_002880NM_000321NM_023001 NM_002892 NM_023000NM_001042603 NM_005056NM_005610NM_005057NM_032626 NM_018703 NM_006910NM_002895 NM_183404NM_005611NM_021975NM_006509NM_006268NM_006272NM_002969NM_002982NM_003010NM_003012NM_003013NM_003015NM_005627NM_003031 NM_001006610NM_006747 NM_153253NM_000345 NM_007308NM_000454NM_032195 NM_138927NM_000582 NM_001040060 NM_001040058NM_001051NM_007315 NM_139266NM_006281NM_003183NM_145332 NM_145333 NM_003188 NM_145331NM_003212NM_003217 NM_001098576NM_007111NM_006286NM_000660NM_022037 NM_022173NM_003252 NM_001033925NM_000362NM_003264NM_003277NM_000594NM_021137NM_006291NM_006290NM_007115NM_001065NM_001066NM_000546NM_005426 NM_001031685NM_005427NM_003299NM_005658NM_021138NM_003300 NM_145726 NM_145725NM_145759 NM_001033910 NM_004619 NM_004620 NM_145803NM_003311NM_003326NM_003327NM_003374XM_001128909 NM_003377NM_005429NM_139323 NM_003404NM_006761 XM_001126863NM_012479NM_003405NM_003406 NM_145690NM_033657 NM_004632NM_003478NM_005087 NM_001013439 NM_001013438 NM_004647NM_003551NM_003589 NM_001008895NM_003590NM_003591NM_003592NM_003608NM_003633NM_003639NM_003655NM_006595NM_003680NM_130476 NM_130475 NM_130470 NM_003682 NM_130473 NM_130474 NM_130471 NM_130472 NM_003701 NM_033012NM_003722NM_003766NM_003789NM_148967 NM_003790 NM_001039664 NM_148965 NM_148970 NM_148966NM_003798NM_003804NM_003805NM_003806NM_003807 NM_172014NM_172088 NM_003808 NM_172087NM_003809NM_003810NM_003811NM_003820NM_003821NM_032945 NM_003823NM_003824NM_004195 NM_148902 NM_148901NM_003839NM_003840NM_003841NM_147187 NM_003842NM_003844NM_003877NM_003879NM_003883NM_003897NM_006024 NM_001079864NM_003921NM_005091NM_005092NM_003946NM_007064 NM_003947 NM_001024660NM_003954NM_003955NM_005147NM_145813 NM_004208 NM_145812NM_004707NM_004708NM_198120 NM_004215NM_001039712 NM_001039711 NM_032998NM_005449NM_004740NM_004226NM_004760NM_001707NM_004786NM_004832NM_004271NM_004840NM_004849NM_004862NM_001015048 NM_004873 NM_001015049NM_004874NM_004281NM_004282NM_004879 NM_001007277NM_004881 NM_147184NM_013981 NM_013982 NM_013983 NM_004883NM_199191 NM_004899 NM_199194 NM_199192 NM_199193NM_183237 NM_014245NM_004295NM_004923 NM_001039656NM_014634NM_012291NM_001077441 NM_001077440 NM_014739NM_005118NM_012115NM_181690 NM_005465NM_133484 NM_004180NM_013232NM_020396NM_207002 NM_138621 NM_006538NM_015322NM_005745NM_005793NM_005815NM_005871NM_005879NM_005882 NM_001017405NM_006092NM_007170NM_006377NM_006378NM_006406NM_001098520 NM_001098523 NM_001098522 NM_001098521 NM_006410 NM_021709 NM_006427NM_006568NM_006573NM_006615 NM_016452NM_013436 NM_205842NM_016640NM_006784NM_006785 NM_173844NM_006705NM_022336NM_033015 NM_006712NM_006826NM_006850 NM_181339NM_006871NM_007051NM_007058NM_007217 NM_145860 NM_145859NM_014922 NM_033006 NM_001033053 NM_033007 NM_033004 NM_014959NM_014976NM_012306NM_015046NM_171982NM_012329NM_016431 NM_012324NM_012279NM_012114XR_017911 XR_017759NM_014326NM_016553 NM_153719 NM_012346 NM_153718NM_014330NM_012402NM_015367NM_014341NM_001077654 NM_014350NM_181651 NM_012094 NM_181652NM_012385 NM_001042483NM_012138NM_014380 NM_206917 NM_206915NM_014430NM_014452NM_145341 NM_014456NM_013247 NM_145074NM_014061NM_145182 NM_013258NM_014550NM_015965NM_139314 NM_001039667XR_017874 XR_017973NM_016561NM_016639NM_080592 NM_016085NM_016222NM_016315NM_016614NM_016077NM_006107 NM_016424NM_016434 NM_032957NM_018947NM_019003NM_207111 NM_207116NM_017580NM_175073 NM_175069 NM_175071 NM_175072 NM_017692 NM_018647 NM_148957NM_017852NM_001008406 NM_018844 NM_001008407 NM_001008405 NM_138930 NM_138929 NM_019887NM_020126NM_020185 XM_001132925NM_020371NM_007008 NM_153828 NM_020532 NM_207521 NM_207520 NM_016252NM_021158NM_021209NM_021571NM_021631NM_022094NM_022112NM_022121NM_022162NM_052813NM_033027NM_022898 NM_138576NM_024110 NM_052819NM_138722 NM_138724 NM_030766 NM_138723NM_022161 NM_139317NM_199320 NM_024900NM_138639 NM_001040668NM_032346NM_032415NM_032587NM_033297 NM_144687NM_033341NM_183395 NM_001079821 NM_004895NM_052890NM_178516NM_203318 NM_078471XM_495908 XM_931833NM_033529 NM_024011Description1v-abl Abelson murine leukemia viral oncogene homolog 12adenosine A1 receptor3adenosine A2a receptor4poly (ADP-ribose) polymerase family, member 15poly (ADP-ribose) polymerase family, member 46adrenergic, alpha-1A-, receptor7angiotensin II receptor, type 28aryl hydrocarbon receptor9allograft inflammatory factor 110v-akt murine thymoma viral oncogene homolog 111v-akt murine thymoma viral oncogene homolog 212arachidonate 12-lipoxygenase13apoptotic peptidase activating factor 114nudix (nucleoside diphosphate linked moiety X)-type motif 215APEX nuclease (multifunctional DNA repair enzyme) 116baculoviral IAP repeat-containing 217baculoviral IAP repeat-containing 318baculoviral IAP repeat-containing 419baculoviral IAP repeat-containing 5 (survivin)20amyloid beta (A4) precursor-like protein 121amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) 22Fas (TNF receptor superfamily, member 6)23Fas ligand (TNF superfamily, member 6)24ras homolog gene family, member A25ras homolog gene family, member B26ataxia telangiectasia mutated (includes complementation groups A, C and D) 27BCL2-antagonist of cell death28BCL2-associated athanogene29BCL2-antagonist/killer 130BRCA1 associated RING domain 131BCL2-associated X protein32B-cell CLL/lymphoma 233BCL2-related protein A134BCL2-like 135BCL2-like 236B-cell CLL/lymphoma 337B-cell CLL/lymphoma 6 (zinc finger protein 51)38tumor necrosis factor receptor superfamily, member 1739brain-derived neurotrophic factor40BH3 interacting domain death agonist41BCL2-interacting killer (apoptosis-inducing)42bone morphogenetic protein 443BCL2/adenovirus E1B 19kDa interacting protein 144BCL2/adenovirus E1B 19kDa interacting protein 245BCL2/adenovirus E1B 19kDa interacting protein 346BCL2-related ovarian killer47breast cancer 1, early onset48v-raf murine sarcoma viral oncogene homolog B149Bruton agammaglobulinemia tyrosine kinase50complement component 551complement component 652complement component 753complement component 8, alpha polypeptide54complement component 8, beta polypeptide55complement component 8, gamma polypeptide56complement component 957calpain 1, (mu/I) large subunit58calpain 659caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase)60caspase 2, apoptosis-related cysteine peptidase (neural precursor cell expressed, developmentally down-regulated 2)61caspase 3, apoptosis-related cysteine peptidase62caspase 4, apoptosis-related cysteine peptidase63caspase 5, apoptosis-related cysteine peptidase64caspase 6, apoptosis-related cysteine peptidase65caspase 7, apoptosis-related cysteine peptidase66caspase 8, apoptosis-related cysteine peptidase67caspase 9, apoptosis-related cysteine peptidase68caspase 10, apoptosis-related cysteine peptidase69caveolin 1, caveolae protein, 22kDa70caveolin 271Cas-Br-M (murine) ecotropic retroviral transforming sequence72CD2 molecule73CD5 molecule-like74CD14 molecule75CD27 molecule76CD28 molecule77tumor necrosis factor receptor superfamily, member 878tumor necrosis factor (ligand) superfamily, member 879scavenger receptor class B, member 180CD38 molecule81CD40 molecule, TNF receptor superfamily member 582CD40 ligand (TNF superfamily, member 5, hyper-IgM syndrome)83CD70 molecule84cell division cycle 2-like 1 (PITSLRE proteins)85cell division cycle 25 homolog A (S. pombe)86cyclin-dependent kinase inhibitor 1A (p21, Cip1)87cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)88cyclin-dependent kinase inhibitor 2D (p19, inhibits CDK4)89chorionic gonadotropin, beta polypeptide90conserved helix-loop-helix ubiquitous kinase91cell death-inducing DFFA-like effector a92clusterin93ceroid-lipofuscinosis, neuronal 3, juvenile (Batten, Spielmeyer-Vogt disease) 94cannabinoid receptor 1 (brain)95cannabinoid receptor 2 (macrophage)96ciliary neurotrophic factor97mitogen-activated protein kinase 1498CSE1 chromosome segregation 1-like (yeast)99catenin (cadherin-associated protein), beta 1, 88kDa100cathepsin D101cytochrome c-1102defender against cell death 1103death-associated protein104death-associated protein kinase 1105death-associated protein kinase 3106death-associated protein 6107deleted in colorectal carcinoma108growth arrest and DNA-damage-inducible, alpha109DNA-damage-inducible transcript 3110DNA fragmentation factor, 45kDa, alpha polypeptide111DNA fragmentation factor, 40kDa, beta polypeptide (caspase-activated DNase) 112deoxyribonuclease I113deoxyribonuclease I-like 3114deoxyribonuclease II, lysosomal115dedicator of cytokinesis 1116dual specificity phosphatase 6117dual specificity phosphatase 7118E2F transcription factor 1119E2F transcription factor 3120E2F transcription factor 5, p130-binding121eukaryotic translation initiation factor 4 gamma, 2122epithelial membrane protein 1123epithelial membrane protein 2124epithelial membrane protein 3125E1A binding protein p300126excision repair cross-complementing rodent repair deficiency, complementation group 2 (xeroderma pigmentosum D)127excision repair cross-complementing rodent repair deficiency, complementation group 3 (xeroderma pigmentosum group B complementing)128endoplasmic reticulum to nucleus signalling 1129coagulation factor II (thrombin)130coagulation factor II (thrombin) receptor131Fanconi anemia, complementation group A132PTK2B protein tyrosine kinase 2 beta133c-fos induced growth factor (vascular endothelial growth factor D)134forkhead box O1135forkhead box O3136FOS-like antigen 2137GRB2-associated binding protein 1138growth arrest-specific 2139glial cell derived neurotrophic factor140GPI anchored molecule like protein141glutathione peroxidase 1142glutathione peroxidase 4 (phospholipid hydroperoxidase)143growth factor receptor-bound protein 2144glycogen synthase kinase 3 beta145gelsolin (amyloidosis, Finnish type)146glutathione reductase147glutathione S-transferase M1148glutathione S-transferase pi149glutathione S-transferase theta 2150granzyme H (cathepsin G-like 2, protein h-CCPX)151granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3)152granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) 153granzyme M (lymphocyte met-ase 1)154huntingtin (Huntington disease)155histone deacetylase 1156heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)157heat shock 10kDa protein 1 (chaperonin 10)158islet amyloid polypeptide159interferon, alpha 2160interferon, beta 1, fibroblast161insulin-like growth factor 1 receptor162insulin-like growth factor 2 (somatomedin A)163insulin-like growth factor 2 receptor164insulin-like growth factor binding protein, acid labile subunit165insulin-like growth factor binding protein 2, 36kDa166insulin-like growth factor binding protein 4167insulin-like growth factor binding protein 5168insulin-like growth factor binding protein 6169inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta170interleukin 1, alpha171interleukin 1, beta172interleukin 2173interleukin 2 receptor, alpha174interleukin 3 (colony-stimulating factor, multiple)175interleukin 3 receptor, alpha (low affinity)176interleukin 10177interleukin 10 receptor, beta178tumor necrosis factor receptor superfamily, member 9179interleukin 17A180forkhead box K2181interferon regulatory factor 1182jun oncogene183jun B proto-oncogene184jun D proto-oncogene185lactalbumin, alpha-186lectin, galactoside-binding, soluble, 1 (galectin 1)187LIM and senescent cell antigen-like domains 1188lymphotoxin alpha (TNF superfamily, member 1)189lymphotoxin beta (TNF superfamily, member 3)190lymphotoxin beta receptor (TNFR superfamily, member 3)191CD180 molecule192lysozyme (renal amyloidosis)193microtubule-associated protein tau194myeloid cell leukemia sequence 1 (BCL2-related)195Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse)196Mdm4, transformed 3T3 cell double minute 4, p53 binding protein (mouse)197mitogen-activated protein kinase kinase kinase 1198mitogen-activated protein kinase kinase kinase 3199mitogen-activated protein kinase kinase kinase 5200mitogen-activated protein kinase kinase kinase 10201matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)202myeloperoxidase203myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) 204v-myb myeloblastosis viral oncogene homolog (avian)205v-myb myeloblastosis viral oncogene homolog (avian)-like 2206v-myc myelocytomatosis viral oncogene homolog (avian)207growth arrest and DNA-damage-inducible, beta208NLR family, apoptosis inhibitory protein209nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105)210nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100) 211nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha 212nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, beta213nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon214nerve growth factor, beta polypeptide215nerve growth factor receptor (TNFR superfamily, member 16)216non-metastatic cells 3, protein expressed in217nitric oxide synthase 1 (neuronal)218nitric oxide synthase 2A (inducible, hepatocytes)219nucleophosmin (nucleolar phosphoprotein B23, numatrin)220neurotrophin 3221neurotrophic tyrosine kinase, receptor, type 1222tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin)223purinergic receptor P2X, ligand-gated ion channel, 1224serpin peptidase inhibitor, clade B (ovalbumin), member 2225p21/Cdc42/Rac1-activated kinase 1 (STE20 homolog, yeast)226p21 (CDKN1A)-activated kinase 2227PRKC, apoptosis, WT1, regulator228paired box gene 3 (Waardenburg syndrome 1)229paired box gene 7230programmed cell death 1231programmed cell death 2232phosphodiesterase 1B, calmodulin-dependent233phosphoinositide-3-kinase, class 2, gamma polypeptide234phorbol-12-myristate-13-acetate-induced protein 1235promyelocytic leukemia236P450 (cytochrome) oxidoreductase237protein phosphatase 2 (formerly 2A), regulatory subunit A, beta isoform238perforin 1 (pore forming protein)239protein kinase, AMP-activated, alpha 1 catalytic subunit240protein kinase C, alpha241protein kinase C, epsilon242protein kinase C, zeta243mitogen-activated protein kinase 1244mitogen-activated protein kinase 4245mitogen-activated protein kinase 7246mitogen-activated protein kinase 8247mitogen-activated protein kinase 10248mitogen-activated protein kinase kinase 1249mitogen-activated protein kinase kinase 5250mitogen-activated protein kinase kinase 6251eukaryotic translation initiation factor 2-alpha kinase 2252presenilin 1 (Alzheimer disease 3)253presenilin 2 (Alzheimer disease 4)254phosphatase and tensin homolog (mutated in multiple advanced cancers 1) 255prostaglandin E receptor 3 (subtype EP3)256parathyroid hormone257prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)258PTK2 protein tyrosine kinase 2259protein tyrosine phosphatase, non-receptor type 6260protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)-associated phosphatase)261RAD21 homolog (S. pombe)262RAD23 homolog B (S. cerevisiae)263v-raf-1 murine leukemia viral oncogene homolog 1 264retinoblastoma 1 (including osteosarcoma)265AT rich interactive domain 4A (RBP1-like)266jumonji, AT rich interactive domain 1A267retinoblastoma binding protein 4268retinoblastoma binding protein 5269retinoblastoma binding protein 6270retinoblastoma-like 1 (p107)271retinoblastoma-like 2 (p130)272v-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene enhancer in B-cells 3, p65 (avian)273v-rel reticuloendotheliosis viral oncogene homolog B, nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 (avian)274D4, zinc and double PHD fingers family 2275S100 calcium binding protein B276mitogen-activated protein kinase 12277chemokine (C-C motif) ligand 2278mitogen-activated protein kinase kinase 4279secreted frizzled-related protein 1280secreted frizzled-related protein 2281secreted frizzled-related protein 5282serum/glucocorticoid regulated kinase283seven in absentia homolog 1 (Drosophila)284signal-induced proliferation-associated gene 1285synuclein, alpha (non A4 component of amyloid precursor)286superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult)) 287SON DNA binding protein288secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1)289somatostatin receptor 3290signal transducer and activator of transcription 1, 91kDa 291serine/threonine kinase 3 (STE20 homolog, yeast)292ADAM metallopeptidase domain 17 (tumor necrosis factor, alpha, converting enzyme)293mitogen-activated protein kinase kinase kinase 7294teratocarcinoma-derived growth factor 1295testis enhanced gene transcript (BAX inhibitor 1)296transcription factor Dp-1297transcription factor Dp-2 (E2F dimerization partner 2) 298transforming growth factor, beta 1299TIA1 cytotoxic granule-associated RNA binding protein。

细胞因子对胰岛细胞凋亡及凋亡相关基因表达的影响

ＨｉｇＬＵＭｎ，ＵＤｍｎＳｎ，Ｎａｐｎ，Ｎｎ，ＨＮＫｑｎＹＮＷｅＥＱｎ，ＩｉｇＹｅｉ，ＵＪｇＷＡＧＢｏｉｇＷＡＧＪｇＣＥｅｉ，Ｉｉｉｉ
ＤｐｒｅｔｆｎｏｒｏｏｙＧｎｒｌｏｐｔｏＴａｊｅａｔｎｏＥｄｃｉｌ，ｅｅａＨｓｉｆｉｉｄａＵｉｒｔＴｎｉ００２ＣｉｍｎｇｌａｎｎＭｅｉｌｎｖｓｙｉｊｃｅｉ，ａｎ３０５，ｈｎａＡｓｒｃｂｔｔｊｃｉＴｖｓｇｔｅｅｔｏｕｒｅｒｓｃｒ（Ｎ）ｆｎｔｒｒｎ（Ｎ）ｎｔｅｐｐｏｉａＯｂｔｅ：ｏｉｅｔａｅｆｃｆｔｍｏｃｉｆｔＴＦｏａｄｉｅｅｏＩｏｏｔｓｅｖｎｉｓｎｏｓａｏ．ｎｆＦｈａｓ
基因表达的影响。方法：应用ＴＮＬ法检测高浓度ＴＦｔＩＮ／ＵＥＮｏ和Ｆ￣培养后大鼠胰岛细胞和小鼠１Ｃ３３一细胞凋亡百分Ｔ率．用半定量Ｒ－ＣｓＲ－Ｃ检测培养后ｂｌ２ｂｘｃｍｃｐ３和ｆｓＮ应ＴＰＲ（ＱＴＰＲ）ｃ、ａ、— ｙ、５一ａＡ的表达。结果：鼠胰岛细胞经ｍＲ大
ａｄｅｐｅｓｎｏｏｔｓ —ｅａｄｇｎｓｎｉｅｅｓＭｅｈｄ：ｐｐｏｃｃｌｆｒｒｔｓｔｅｌｎＣ３ｃｌｎｘｒｓｏｆｐｐｏｉｒｌｅｅｅｌｃｉ．ｔｏｓＡｏｔｔｅｓｏｉｙｒｌｌａｄ１Ｔ一ｅｉａｓｔｉｓｔｌｉｌｐｍａａｉｅｃ３ｌ

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生物化学与生物物理进展PROGRESS INBIOCHCMISTRYAND BIOPHYSICS1999年第1期 No.11999TF-1细胞凋亡相关基因的研究刘红涛　王玉刚　张颖妹　宋泉声　敬保迁　袁　勇　马大龙摘要　利用近年来发展起来的代表差异分析（cDNA representational differences analysis, cDNA-RDA）技术研究了在人红白血病细胞株TF-1细胞撤除细胞因子后进入凋亡时诱导表达的基因.发现了6个新基因片段.其中有三个经与GenBank nr和dbEST查询均没有发现同源性，已经向GenBank进行登记，登记号分别为U83208，U83279，U83397.此外还发现一批已知基因的表达与凋亡相关，其中包括Hou和人硫氧还原蛋白等, 提示它们在凋亡中可能起作用.这项工作为进一步研究凋亡相关基因打下了良好基础.通过RDA的研究结果，有可能发现人白血病细胞凋亡的特异标记蛋白或发挥作用的重要蛋白，以期为白血病治疗提供理论基础.关键词　TF-1细胞株，代表差异分析，凋亡学科分类号　R392.1Studies on the Apoptosis-Related Genes of TF-1 Cell Line by cDNA-RDA Technique. LIU Hong-Tao, WANG Yu-Gang, ZHANG Ying-Mei, SONG Quan-Sheng, JING Bao-Qian, YUAN Yong, MA Da-Long (Department of Immunology, Beijing Medical University, Beijing 100083, China).Abstract　The genes effecting in the process of the apoptosis of TF-1 cell line when it is deprived of the cytokine in the culture medium were studied by RDA (representational difference analysis) method. The TF-1 cell depriving of cytokines for 8 hours was selected as the Tester and normal-cultured TF-1 cell as the Driver. Seven gene fragments were found uniquely expressed or highly expressed in the process of apoptosis of TF-1 cell line which include some known genes such as Hou and thioredoxin that formerly suggested to play a role in apoptosis.There are three fragments are complete novel after searching the nr and EST catalogues of GenBank and were banked into GenBank. The accession numbers for them are U83208, U83279, U83397 respectively. From the novel gene fragments, the complete cDNA sequence of them can be fished and the bioactivity and function of them in apoptosis of TF-1 cell and other hematological tumors can be further studied. On the other hand, the function of some known genes which were not suggested formerly in the course of apoptosis can be studied.Key words　TF-1 cell line, representational difference analysis(RDA), apoptosis 细胞凋亡（apoptosis）是当前国际生物学研究的热点之一.目前已发现多种基因参与细胞凋亡，例如TNF/FAS家族，BCL2家族，ICE/CED-3家族，多种癌基因，抗癌基因，热休克蛋白等［1］.但由于细胞凋亡为多阶段，多系统，多基因参与的复杂过程，目前对其参与的全部基因尚远未了解清楚.通过进一步克隆化新的凋亡诱导基因，特别是具有启动作用的凋亡早期表达基因，将有助于阐明凋亡机理，为一些凋亡相关疾病特别是肿瘤的治疗提供新的思路. 细胞凋亡也是造血细胞自然死亡的一种主要形式，一些血液病问题也与细胞凋亡密切相关，特别在一些血液肿瘤的发生、发展中起重要作用.在CML、B-CLL、ALL、AML等肿瘤细胞中均有高水平BCL-2的表达，细胞凋亡过程的受阻是血液肿瘤形成的重要原因［2］.目前已证明抗肿瘤化疗药物，肿瘤细胞诱导分化剂（如维甲酸及其合成的衍生物HPR），某些激素（如糖皮质激素及PGE2等），某些细胞因子及某些化学药物（如砷类化合物（As2O3）［3］）也是通过诱导血液肿瘤的细胞凋亡，发挥治疗作用.因此深入研究血液肿瘤细胞凋亡的发生机制，及其基因调控并进行基因分离和克隆，可望人为地调控细胞凋亡，提高治疗效果，预防疾病发生. 本项研究以细胞因子依赖性的人红白血病细胞株TF-1细胞作为研究对象，通过撤除细胞因子诱导其凋亡，利用代表差异分析技术(cDNA-representational difference analysis,cDNA-RDA)［4］进行凋亡早期特异表达基因的克隆化，并进行功能研究.RDA 是1993年［5］发展起来的一种新方法，最初用于基因组表达差异的研究，因其简便有效性被大家推广用于cDNA的差异表达研究.1　材料和方法1.1　细胞系和细胞培养 TF-1细胞由中国药品生物制品检定所丁锡申教授惠赠，由本室长期传代培养.培养基中加入80～100 U/ml的人重组GM-CSF.细胞去细胞因子时用Hank "s液洗三遍后悬于不含GM-CSF的培养基中.1.2　DNA片段化分析为了解TF-1细胞去细胞因子后凋亡情况，分别于去细胞因子后0、8、12、24、48、80 h收集2×106 TF-1细胞，提取片段化DNA［6］.1.3　细胞总RNA及mRNA的提取细胞总RNA的提取利用GIBCO-BRL公司的TRI Z0L TM试剂.各取1×107去细胞因子8 h后的TF-1细胞及相同数目不去细胞因子的TF-1细胞，离心收集后，加入1 mlTRI ZOL TM试剂提取总RNA.以上两种细胞mRNA的提取使用Pharmacia公司的QuickPrep "Micro mRNA Purification kit.1.4　双链cDNA的合成采用GIBCO-BRL的Superscript TM Choice System for cDNA Synthesis试剂盒，利用1.3所提的不去细胞因子和去细胞因子细胞mRNA为模板，进行单链及双链cDNA的合成.1.5　RDA寡核苷酸以下是应用于RDA的寡核苷酸［4,5,7］.RBgl24, 5′-AGCACTCTCCAGCCTCTCACCGCA-3′，RBgl12, 5′-GATCTGCGGTGA-3′，JBgl24, 5′-ACCGACGTCGACTATCCATGAACA-3′，JBgl12, 5′-GATCTGTTCATG-3′，NBgl24, 5′-AGGCAAC-TGTGCTATCCGAGGGAA-3′，NBgl12, 5′-GATCTTTCCCTCG-3′ 划线的地方是每对引物配对互补的区域，以上引物由上海生工生物工程有限公司合成.1.6　RDA差示杂交操作参照文献［4,5,7］.去细胞因子和不去细胞因子组cDNA 2 μg用DpnⅡ切后连上RBgl12和RBgl24，然后以RBgl24为引物对两者进行PCR扩增，扩增产物用DpnⅡ切后，利用Glassmilk回收200～1 600 bp的片段，并定量取2 μg去细胞因子组的回收产物再连入JBgl12和JBgl24后,再取0.4 ng纯化的连接产物(被测组），与40 μg加因子的回收产物(本底组)（1∶100）进行长时间杂交，杂交后的产物以JBgl24为引物进行PCR扩增，PCR产物纯化后用绿豆核酸酶处理.终止反应后取20 μl处理后的产物为模板，以JBgl24为引物，进行18轮PCR反应.此次PCR产物纯化后用DpnⅡ切,酶切后片段为DP1（以上为第一轮杂交）.取2 μg酶切后的DP1再按以上操作连上新的Adaptor NBgl12和NBgl24（第一轮后）或JBgl12和JBgl24（第二轮后），重复RDA过程，连入新adaptor 的DNA为被测组,作PCR时分别以NBgl24或JBgl24为引物，杂交时本底组的量分别为0.1 ng(1∶400),0.01 ng(1∶4000)，分别得到DP2和DP3.取一定量的DP3与经BamHⅠ酶切并用CIP处理的载体pGEM3zf进行连接并筛选阳性克隆.1.7　测序反应测序使用Pharmacia Biotech公司的ALF TM DNA Sequencer，测序反应按Standard Annealing of Primer to Double-Stranded Template, 以荧光标记的反向引物为测序引物，使用的酶为T7 DNA聚合酶.1.8　与GenBank已有序列的比较及新序列的登记注册将测序所得到的序列以E-mail的形式或通过进行序列比较，并通过所提供的BankIt功能将在nr和dbEST中完全没有同源性的序列到GenBank进行登记［8～10］.1.9　mRNA斑点杂交和RNA印迹基本操作按文献［11］，去细胞因子和未去细胞因子组总RNA用分光光度计Beckman 640定量后，对应狭线孔中各加入20 μg总RNA，利用GIBCO-BRL公司抽滤点膜装置HXBRI-SLOT TM MANIFLD将总RNA点于DUPONT公司的Gene Screen Plus " Hybridization Transfer Membrane上.RNA印迹时［12］，总RNA经在含有甲醛的凝胶上进行的RNA电泳后，通过毛吸法转到尼龙膜上.探针的标记和杂交操作参照DUPONT公司Random Primer Fluorescein Labeling kit with Antifluorescein-HRP(DuPont NEN "NEL803)试剂盒说明书，一般X片压膜过夜后显影，定影.2　结果2.1　TF-1细胞去细胞因子后进入凋亡分别于去细胞因子0、8、12、24、48、80 h时提取TF-1细胞的DNA，并进行电泳.电泳显示（图1）去细胞因子12 h时DNA的片段化就已经非常明显，说明TF-1细胞去细胞因子后进入凋亡.一般来说，基因水平的改变TF-1细胞应该早于形态学上的改变.为此我们选取去细胞因子8 h后TF-1细胞进行RDA.图1　TF-1细胞去细胞因子后DNA片段化电泳结果1:λDNA/HindⅢ标准; 2～7: TF-1细胞去因子0、8、12、24、48、80 h.2.2　RDA 进行RDA操作时，以未去细胞因子的TF-1细胞为本底组，而以去细胞因子8 h后的TF-1细胞为被测组.进行三轮RDA，每轮中本底组:被测组分别为100∶1，400∶1，4000∶1.图2显示了杂交前不去及去细胞因子TF-1细胞cDNA，以及三轮RDA每一轮的产物，从电泳上可以看出，随着RDA进行，带型发生了改变，大部分消失而局部增强.第三轮后，可以看到明显的不同条带，此类差异带可能代表差异表达的基因.图2　利用RDA筛选在TF-1细胞去细胞因子8 h后特异表达片段的电泳结果1:λDNA/HindⅢ标准; 2: 正常培养（不去细胞因子）的TF-1细胞cDNA经DpnⅡ酶切后，PCR扩增; 3: 去细胞因子8 h时TF-1细胞cDNA经DpnⅡ酶切后，PCR扩增; 4:第一轮RDA后即DP1; 5: 第二轮RDA后即DP2; 6:第三轮RDA后即DP3.2.3　测序，检索和杂交结果三轮RDA后的产物与pGEM3zf相连后，电泳分析选择大于pGEM3zf载体的克隆进行保存并选择20个克隆用LiCl和PEG8000的方法提取质粒［12］后进行测序.测定的序列以E-mail或WWW的方式到GenBank进行检索.检索结果(表1)表明其中有7个克隆只有部分序列有同源性，可能为新的未知基因.其余13个为已知基因，将以上7个未知序列再到dbest数据库进行检索，发现其中4个有相同序列，3个没有同源序列，将这3个基因片段以EST的形式向GenBank进行登记，并获得承认.GenBank的登记号码分别是U83208，U83279，U83397.对以上克隆中的12个进行了斑点杂交以检测它们在TF-1细胞中的表达情况，杂交结果显示其中7个在凋亡的TF-1细胞中高表达（图3显示了部分结果），而在正常TF-1细胞中低表达或不表达.并用RNA印迹的方法对2个新基因转录产物(被命名为TFAR19和TFAR15(2))的长度进行了估算.结果显示（图4）TFAR15(2)长度约为1.0 kb,而TFAR19长度约为700 bp.利用Pharmacia公司的Pharmacia LKB Imagemaster DTS扫描仪扫描分析.结果显示以上两个克隆在去细胞因子组及不去细胞因子组间表达有差异(表2).表1 利用RDA技术发现在TF-1细胞去细胞因子所致凋亡中表达的基因克隆号测序长度同源性已知序列（全长）杂交结果130998%HSU32849人Hou基因(1426)H 325096%HSU54645人腺苷激酶2B(adk2b)基因(2105)H 425798%HSU21138人核糖体蛋白L9mRNA N.D51)39668%(40/68)CEF07A11 Caenorhabditis elegans cosmid F07A11(35,692)H620398%HSLDHAR人乳酸脱氢酶A(LDH-A,EC1.1.1.27)(1661)NH 724797%HUMCD24B人CD24 mRNA(2116)ND 811198%人硫氧还原蛋白mRNA(501)H 923697%人胸腺嘧啶核苷合成酶基因(18597)NH 1022098%人卫星DNA ⅢNH 11 同4ND 1328898%HSU17899人氯离子通道调节蛋白mRNA(1355)ND 141)16658%(68/117)DDP8A7 Dictyostelium discoideum P8A7 gene(2568)NH 15290　同4ND 1729096%同10NH191)29059%(71/119)HUMSFRS人剪切因子7（富含精氨酸/丝氨酸）(SFRS7)(8213)H2020099%HSU56637人帽蛋白alpha亚基同种异形体ND 231)27089%(44/49)X58779/HSHTLIP人肝三甘油酯脂酶5′非编码区ND 8(2)1)263　无同源ND 15(2)1)31274%(35/47)HUMMDR1人P糖蛋白(MDR1)mRNA(4646)H 19(2)1)25059%(71/119)同19号H 1)表示可能为新基因；ND表示没有检测；H或NH表示在去细胞因子TF-1细胞中高表达或不高表达.图3　RDA得到克隆在正常培养和去细胞因子TF-1细胞中表达的狭缝杂交结果1:β-actin;2～5: 克隆1, 5, 14, 19.图4　二个未知新基因的RNA印迹结果1: 人GAPDH; 2: TFAR15(2); 3: TFAR19.A：去细胞因子；B：加细胞因子.表2　RNA印迹扫描分析结果　去细胞因子组加细胞因子组GAPDH 1.27 1.27TFAR190.3240.132TFAR15(2)0.6960.236注：表中数值为去除背景(background)后条带的平均分光光度值(A).3　讨论 TF-1细胞株［13］是从患红白血病的病人分离建立的人前髓细胞株.TF-1细胞的生长增殖依赖于IL-3、GM-CSF、IL-13、EPO、SCF等细胞因子的单独或联合作用［13］.一旦在培养基中去除细胞因子，TF-1细胞就会进入凋亡.一般去细胞因子8 h后，几乎70％的细胞进入凋亡［14］.我们的DNA片段化电泳结果也证实了这一点.去细胞因子后培养基中加入蛋白质合成抑制剂环己酰胺后，细胞的凋亡受到抑制［15］，这说明TF-1细胞去细胞因子后的凋亡时有大分子蛋白质的合成.尽管以前的工作证明，TF-1细胞凋亡时BCL-2的mRNA和蛋白质水平均下降，而且是通过抑制PKC激酶的活性达到的［14］.但对TF-1细胞凋亡早期基因水平的变化还没有一个全面的了解. 为了更好地研究TF-1细胞去细胞因子凋亡机制并以期发现在凋亡中起作用的新基因，我们用RDA方法进行了TF-1细胞去细胞因子凋亡中基因差异表达的研究.本研究以细胞因子刺激生长的正常TF-1细胞为本底，以去除细胞因子的进入凋亡的TF-1细胞为被检查者，试图发现在凋亡早期过程中发挥作用的一系列基因.并进一步为其他血液肿瘤细胞的诱导凋亡和临床治疗提供一定的理论基础. 总的来说，凋亡涉及到从胞外至胞内一系列的信号传导过程和效应器蛋白水解酶的活化和调节两个步骤［16］.从本研究中所得到的在TF-1细胞凋亡中差异表达的基因编码的蛋白质，主要是一些核内调控因子，这与我们选择去细胞因子的时相在早期有关.一些克隆（如Thioredoxin［17］, Hou［18］等）经过检索发现以前的工作提示它们的高表达在凋亡中起作用,为我们今后进一步地研究以上已知基因在凋亡中的作用打下良好基础.更为重要的是，我们发现了数个新基因片段.而利用未知基因的片段可以从文库中筛选全长cDNA或用RACE的方法克隆出全长cDNA序列，有可能发现信号传导途径上的新蛋白或凋亡的标记蛋白.使我们对血液肿瘤细胞的凋亡的认识有进一步的提高，具有重要的理论意义和潜在的应用价值.最近，我们已克隆出TFAR15和TFAR19的全长cDNA序列在GenBank登记并被接受，登记号分别为AF022385和AF014955.作者单位：北京医科大学卫生部医学免疫学重点实验室, 北京 100083参考文献　[1]　Thompson C B. 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