活化Jurkat细胞cDNA文库的构建及其初步筛选

本文报道以ＴＰＡ活化Ｊｕｒｋａｔ细胞ｍＲＮＡ为模板，逆转录合成ｃＤＮＡ第１链，在ＤＮＡ聚合酶Ⅰ作用下合成第２链，加上ＥｃｏＲＩ磷酸化接头，用离心式柱层析去除小于５００ｂｐ的ｃＤＮＡ片段和多余接头，再与去磷酸化λｇｔ（１１）ＤＮＡ连接，体外包装后感染大肠杆菌Ｙ１０９０。该文库效价５×１０５，重组率＞９０％。ＰＣＲ法鉴定阳性重组子均可见插入片段。用ＰＴＡ１ＭｃＡｂ为探针，对文库进行了免疫学筛选，初步获得４个阳性克隆。该文库的构建为ＰＴＡ１基因克隆奠定了基础。     

交联抗人DR5单抗诱导的Jurkat细胞凋亡的信号转导

目的探讨抗肿瘤坏死因子相关凋亡诱导配体（TRAIL）的死亡受体5（DR5）单抗——YM366EC对Jurkat细胞增殖的影响，阐明抗DR5抗体的抗肿瘤效应机制。方法MTF方法检测抗体对Jurkat细胞存活率，FrlE—AnnexinV／PI标记流式细胞仪检测交联YM366EC对Jurkat细胞凋亡率影响。交联YM366EC作用于Jurkat细胞2、4、6、8、10、12h收集细胞，提取蛋白Westernblot检测Bcl-2、Cyt—C、Caspase-8、Caspase-3、Bax、Caspase-9的变化。结果抗DR5抗体单独应用不能抑制高表达DR5分子的Jurkat细胞增殖，但应用抗小鼠IgG交联DR5抗体后可直接诱导TRAIL敏感的Jurkat细胞凋亡改变。并且Westernblot检测到随着抗体诱导时间的延长Jurkat细胞Bel-2蛋白表达减少；Cyt—C、Bax，有活性的Caspase-8、Caspase-3和Caspase-9蛋白的表达增加。结论交联抗DR5抗体YM366EC对Jur—kat细胞增殖有明显的抑制作用，诱导的Jurkat细胞凋亡的分子机制涉及到Cyt-C、Bax、Caspase．8、Caspase-3、Caspase-9。     

己烯雌酚诱导T细胞肿瘤Jurkat细胞系凋亡中转录因子Oct-1的表达(英文)

目的研究己烯雌酚对 T细胞肿瘤的细胞凋亡诱导作用 ,以及调亡过程中转录因子 Oct- 1的表达。方法采用DNA梯形片段化 ,荧光染色流式细胞技术分析细胞凋亡 ,以电泳泳动度迁移率法 (EMSA)检测 Oct- 1的表达。结果己烯雌酚 (5 .0 mg/L )可诱导 T细胞出现细胞凋亡 ,并有典型的 DNA梯形片段化 ,10μg己烯雌酚诱导 12 h后 ,凋亡过程中细胞数达 30 %以上。己烯雌酚诱导 T细胞凋亡过程与 Oct- 1转录因子的表达有关。结论己烯雌酚可诱导 T细胞肿瘤凋亡 ,并与转录因子 Oct- 1的表达有关。     

T细胞激活剂对Jurkat细胞重组激活基因表达的影响

目的:研究T细胞激活剂PHA,抗-CD3 mAb, PMA A23187对人T细胞白血病细胞株Jurkat重组激活基因(RAGs )mRNA表达水平的影响. 方法:采用RT-PCR法检测不同T细胞激活剂作用不同时间后Jurkat 细胞中RAG-1, RAG-2 mRNA,以β肌动蛋白(β-actin)的表达作为内参照进行灰度分析,比较不同组Jurkat细胞RAGs mRNA的表达水平 . 结果:三种T细胞激活剂刺激诱导的Jurkat细胞RAG-1 mRNA表达量明显低于对照组(P＜0.05),且RAG-1 mRNA表达量的下降与刺激诱导作用时间有关;PHA刺激诱导后RAG-2表达量显著低于对照组(P＜0.05);PMA A23187刺激诱导6, 12 h均未检测出RAG-2 mRNA的表达; 而抗-CD3 mAb作用12 h组RAG-2 mRNA表达量显著高于对照组(P＜0.05). 结论:Jurkat细胞同时表达RAG1, RAG2 mRNA,并在一定诱导下RAGs表达量发生改变,因此有可能成为研究外周T细胞RAGs表达调节的一个潜在的细胞模型.     

The effects and mechanism of action of Prunella vulgaris L extract on Jurkat human T lymphoma cell proliferation

Objective： The aim of this study was to observe the effect of the Prunella vulgaris L extract on the Jurkat human T lymphoma cell line. Methods： Jurkat cells were cultivated with different concentrations of the extract from Prunella vulgaris L. The MTT assay and flow cytometry were employed to determine the cells＇ proliferation inhibition ratio and the apoptosis rates, respectively. Agarose gel electrophoresis was used to observe cellular DNA fragmentation, and western blotting was used to observe changes in Bcl-2 and Bax protein expression. Results： The Prunella vulgaris L extract remarkably inhibited the proliferation of Jurkat cells. This inhibition exhibited dose dependence, with an IC50 of 20.23 ± 0.31 μg/mL. Agarose gel electrophoresis showed that the apoptosis strap became wider and brighter, and flow cytometry showed that the apoptosis rate increased in a concentration-dependent manner. Western blotting showed that Bcl-2 protein was down-regulated and Bax protein was up-regulated during apoptosis. Conclusion： The extract from Prunella vulgaris L induced apoptosis of Jurkat cells by down-regulating Bcl-2 protein and up-regulating Bax protein. These actions inhibited the growth of Jurkat cells.     

甲基乙二醛对Jurkat细胞氧化应激及分泌细胞因子的影响

目的:探讨甲基乙二醛(MGO)对Jurkat细胞氧化应激及分泌细胞因子的影响。方法:不同浓度MGO作用PHA预刺激的Jurkat细胞,MTT检测细胞生长,流式细胞检测细胞内活性氧(ROS),Western blot检测p38 MAPK、JNK磷酸化水平,ELISA检测TNF-α、IFN-γ。结果:15~60μmol/L MGO对Jurkat细胞生长无影响,但能使ROS呈浓度依赖性升高;N-乙酰-L-半胱氨酸(NAC)和氨基胍(AG)能明显抑制此作用。MGO作用30分钟,pp38/p38、pJNK/JNK明显升高。MGO诱导Jurkat细胞分泌TNF-α、IFN-γ;P38、JNK抑制剂及NAC能降低TNF-α、IFN-γ的分泌。结论:MGO能诱导Jurkat分泌TNF-α、IFN-γ;其机制可能通过氧化应激、P38、JNK信号通路。     

Ahi-1基因导入Jurkat细胞中表达及功能的研究

本研究目的是探讨Ahi-1基因及其编码蛋白在人T淋巴细胞白血病Jurkat细胞中的表达、细胞内定位以及Ahi-1基因对Jurkat细胞生长的调控作用。分别应用Northernblot、Werstemblot检测Ahi-1基因的mRNA及蛋白表达水平。构建Ahi-1的真核表达质粒并导入Jurkat细胞,用G418筛选获得稳定过表达Ahi-1基因的Jurkat-A细胞,用XTT法检测细胞的增殖活性,半固体培养法检测细胞集落形成能力。结果表明：正常人外周血淋巴细胞（PBL）、Jurkat细胞以及HUT78细胞均表达6．5、4．2以及2kb的Ahi-1基因mRNA转录本。PBL及Jurkat细胞均表达140kD的Ahi-1蛋白且主要定位于细胞浆内,但在细胞核内未见表达。PBL尚表达120kD的Ahi-1蛋白,主要存在于细胞核内,Jurkat细胞中120kD的Ahi-1蛋白表达水平低下。甲异靛、阿糖胞苷（Ara-C）、高三尖杉酯碱（HHT）、甲氨喋呤（MTX）以及鬼臼乙叉甙（VP16）可诱导Jurkat细胞核内140kD的Ahi-1蛋白表达增强,甲异靛降低细胞浆内Ahi-1蛋白表达。与转染质粒对照Jurkat细胞（Jurkat-C）相比,高表达外源性Ahi一1的Jurkat—A细胞的生长及集落形成能力显著低于Jurkat—C细胞;细胞总c-myb蛋白表达水平无改变,但Jurkat-A细胞中磷酸化c-myb蛋白表达增强;细胞中AKT磷酸化水平无明显变化。结论：Jurkat细胞表达6．5、4．2以及2kb的Ahi-1转录本;140kD的Ahi-1蛋白主要定位于细胞浆中,多种化疗药物可诱导细胞核内140kD的Ahi．1蛋白表达增强;过表达外源性全长Ahi-1可抑制Jurkat细胞的生长及集落形成能力,并诱导c-myb蛋白磷酸化。     

转染人livinα基因对Jurkat细胞增殖及凋亡的影响

目的：研究人livinα基因表达对Jurkat细胞增殖、凋亡的影响。方法基因转染获得稳定表达livinα的Jurkat细胞克隆（Jurkat/livinα）;MTT法测定细胞增殖,流式细胞仪分析细胞凋亡率,通过观察细胞生长和凋亡情况,探讨livinα对Jurkat细胞增殖及凋亡的影响;RT-PCR检测细胞中livinα和caspase-3 mRNA的表达。结果Jurkat/livinα组livinαmRNA表达高于对照组（Jurkat细胞组）（P＜0.05）,转染后caspase-3 mRNA受到抑制,表达减弱,细胞生长曲线显示Jurkat/livinα组细胞生长速度增快,倍增时间缩短9-10 h（P＜0.05）,细胞凋亡率计算显示Jurkat/livinα组细胞凋亡率较Jurkat细胞组明显减少（P＜0.01）。结论 livin α基因能促进Jurkat细胞生长,可能通过抑制细胞凋亡而在急性T淋巴细胞白血病发生发展中起重要作用,有望成为白血病治疗的新分子靶点。     

AGEs通过RAGE促进Jurkat细胞分泌肿瘤坏死因子-α

目的:探讨晚期糖化终产物(AGEs)与晚期糖化终产物受体(RAGE)结合对Jurkat细胞分泌肿瘤坏死因子-α(TNF-α)的影响及相关信号分子的变化。方法:不同浓度AGEs作用于植物血球凝集素(PHA)预刺激的Jurkat细胞24小时,MTT检测AGEs对细胞生存率的影响,ELISA检测AGEs诱导Jurkat细胞分泌TNF-α;Western blot检测AGEs诱导Jurkat细胞表达RAGE和p38MAPK、JNK磷酸化水平。结果:AGEs作用于PHA预刺激Jurkat细胞24小时对细胞生存率无明显影响;AGEs促进Jurkat细胞表达RAGE,增加炎症因子TNF-α分泌,抗RAGE抗体明显抑制AGEs诱导的TNF-α分泌。AGEs可引起p38MAPK、JNK磷酸化,p38MAPK、JNK的抑制剂明显抑制AGEs诱导的TNF-α表达。结论:AGEs通过RAGE促进PHA预刺激的Jurkat细胞分泌炎症因子TNF-α,其机制与激活p38MAPK、JNK途径有关。     

Synthesis and characterization of a new labeled gastrin ligand, 125I-BH-[Leu15]-gastrin-(5-17), on binding to canine fundic mucosal cells and Jurkat cells

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we have synthesized and characterized a new labeled gastrin ligand, ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) [(3-[¹²⁵I]iodo-4-hydroxyphenyl)-propionyl-[Leu¹⁵]-gastrin-(5-17)]. Binding of ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) to isolated canine fundic mucosal cells was specific, saturable and of high affinity. ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) and ¹²⁵I-BH-CCK-8 [(3-[¹²⁵I]iodo-4-hydroxyphenyl)-propionyl-CCK-8] interact with isolated canine fundic mucosal cells with small differences in maximal binding capacities and affinities, 3800 ± 900 binding sites/cell (Kd = 0.52 ± 0.23 nM) and 6200 ± 1100 binding sites/cell (K_d= 0.31 ± 0.18 nM), respectively. The relative order of potencies for gastrin and CCK analogs in displacing ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) binding correlated well with those obtained using ¹²⁵I-BH-CCK-8. Selective CCK/gastrin antagonists L-364,718 (MK-329) and L-365,260 also inhibited ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) binding. These results indicate that ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) binds to gastrin receptors in isolated canine fundic mucosal cells. We have also characterized ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) binding to the human Jurkat lymphoblastic cell line (Jurkat cells) known to express the CCK-B/gastrin receptor. Saturation experiments have shown that both ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) and ¹²⁵I-BH-CCK-8 interact with a single class of high-affinity binding sites in the Jurkat cell line. Binding characteristics of ¹²⁵I-BH-[Leu¹⁵]-Gastrin-(5-17) and ¹²⁵I-BH-CCK-8 were estimated to be about 2500 ± 400 binding sites/cell (Kd= 0.19 ± 0.09 nM) and 2400 ± 350 binding sites/cell (Kd= 0.06 ± 0.02 nM), respectively. Furthermore, the apparent affinities of CCK analogs and selective antagonists MK-329 and L-365,260 for the sites labeled by both probes were identical. The biological activity of cold ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) and [Leu¹⁵]-gastrin-(5-17) was demonstrated by their ability to increase intracellular calcium concentration in Jurkat cells. All these experiments showed that ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) provides a convenient ligand for gastrin receptor binding studies. © Munksgaard 1994.