Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs.     

Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines

Solanum nigrum is used in various traditional medical systems for antiproliferative, antiinflammatory, antiseizure and hepatoprotective activities. We have evaluated organic solvent and aqueous extracts obtained from berries of Solanum nigrum for antiproliferative activity on leukemic cell lines, Jurkat and HL-60 (Human promyelocytic leukemia cells). The cell viability after the treatment with Solanum nigrum extract was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Results indicated increased cytotoxicity with increasing extract concentrations. Comparative analysis indicated that 50% inhibitory concentration value of methanol extract is the lowest on both cell lines.     

CD59-CD2对T细胞信号转导的作用

目的将构建的CD59-linker-CD2融合基因真核表达载体在Jurkat细胞中稳定表达,研究CD59与CD2分子在T细胞信号转导中的相互作用,探讨CD59向T细胞内传递信号的相关机制。方法酶切鉴定构建的pIRES-CD59-linker-CD2融合基因真核表达载体,脂质体法转染Jurkat细胞,G418筛选建立稳定转染细胞系,免疫酶法检测转染细胞表面CD59的表达,Western blot检测转染细胞中CD59蛋白的表达;用CD59单克隆抗体作用于各组Jurkat细胞,使用激光共聚焦显微镜检测细胞浆内的Ca2+,ELISA检测细胞上清中白介素2(IL-2)的变化,比较各组差异。结果经酶切鉴定证实携带CD59-linker-CD2融合基因的重组质粒扩增成功;免疫酶法和Western blot证实CD59在转染细胞中稳定表达,且转染组L-Jurkat细胞比正常组Ju-rkat细胞CD59表达量增加,二者比较其差别有统计学意义(P<0.05);与正常细胞相比,胞浆内Ca2+和IL-2在转染细胞中均高表达,两组细胞比较其差别有统计学意义(P<0.05)。结论 pIRES-CD59-linker-CD2融合基因真核表达载体可在Jurkat细胞中稳定表达,CD59mAb交联刺激后,CD59可通过CD2向细胞内传递信号,引起细胞内信号分子的一系列变化,为研究CD59与CD2在T细胞信号转导中的协同作用及进一步探讨CD59向T细胞内传递信号的机制奠定基础。     

夏枯草提取物诱导B、T淋巴瘤细胞凋亡的实验研究

目的:研究夏枯草提取物对人B淋巴瘤Raji细胞及T淋巴瘤Jurkat细胞增殖的影响及其差异,探讨夏枯草提取物体外抗淋巴瘤的作用机制。方法:不同浓度的夏枯草提取物分别作用于Raji细胞及Jurkat细胞;48 h后分别收集各组细胞,应用MTT法、琼脂糖凝胶电泳法及流式细胞技术分别检测每组细胞的增殖抑制率、细胞凋亡率及细胞凋亡情况;Western blot检测BCL-2及BAX蛋白表达变化。结果:不同浓度的夏枯草提取物对Raji及Jurkat细胞均有明显的增殖抑制作用(P0.01),且对Raji细胞的抑制作用大于Jurkat,其对Raji细胞IC50(18.01±0.92)μg/mL显著低于Jurkat细胞(25.47±0.96)μg/mL(P0.05);凝胶电泳检测结果表明夏枯草提取物处理Raji及Jurkat细胞后均出现凋亡相关DNA Ladder;随夏枯草提取物浓度的增加,Raji及Jurkat细胞的早期凋亡率均显著增加,与空白对照组比较差异显著(P0.01),浓度为15、20、25μg/mL的夏枯草提取物作用于Raji及Jurkat细胞后的早期凋亡率分别为(9.4±0.25)%、(21.68±0.46)%、(35.03±0.35)%和(4.06±0.14)%、(13.59±0.23)%、(22.92±0.20)%,同浓度条件下,Raji细胞的早期凋亡率显著高于Jurkat细胞(P0.01);随夏枯草提取物浓度增加,实验组细胞中BCL-2蛋白的表达逐渐减弱,BAX蛋白的表达逐渐增强,与空白对照组比较均有显著性差异(P0.05),同浓度条件下,Raji细胞内BCL-2蛋白表达的下降程度、BAX蛋白表达的增加程度显著高于Jur-kat细胞(P0.05)。结论:夏枯草提取物具有抑制淋巴瘤细胞增殖的作用,其机制可能与调节BCL-2和Bax蛋白的表达以诱导细胞凋亡有关,且夏枯草提取物对Raji细胞的抑制作用大于Jurkat细胞。     

MSP扩增中3种DNA提取法的比较

目的比较标准酚-氯仿、TripureDNA提取法、强碱法3种DNA提取法,研究哪种方法更简单方便,更适合做DNA甲基化特异性PCR（MSP）。方法分别用标准酚-氯仿、TripureDNA提取法、强碱法提取Jurkat细胞株中的DNA,并对3种方法提取的DNA含量、纯度及所需时间进行比较。结果3种方法提取DNA的纯度和完整性都较好,标准酚-氯仿法提取的DNA含量最低,且最费时;强碱法提取的DNA含量最高,并最省时。结论这3种DNA提取法提取的DNA都能够达到做MSP的要求,比较而言,强碱法更具优越性。     

丙戊酸钠对白血病细胞Jurkat的增殖抑制作用

 目的　探讨丙戊酸钠（VPA）对Jurkat细胞增殖抑制作用及对组蛋白乙酰化修饰调控的影响。方法　采用CCK-8法检测VPA对Jurkat细胞增殖的抑制作用;流式细胞术检测VPA作用前后Jurkat细胞周期的变化情况;半定量RT-PCR检测VPA作用前后Jurkat细胞中组蛋白去乙酰化酶1（HDAC1）mRNA的表达变化;Western blotting检测VPA作用前后HDAC1及组蛋白H3、H4乙酰化蛋白水平的变化。结果　VPA对Jurkat细胞的增殖抑制作用呈时间-浓度依赖性;不同浓度的VPA处理细胞48 h后,细胞周期检测显示随浓度增加,G0／G1期比例增高,S期比例下降,细胞被阻滞在G0／G1期（P＜0.05）;RT-PCR证实VPA能够抑制HDAC1 mRNA水平的表达;Western blotting方法分析证实,不同浓度VPA作用细胞48 h后降低HDAC1蛋白水平,提高组蛋白H3、H4乙酰化表达水平。结论　VPA能抑制Jurkat细胞增殖,阻滞细胞周期于G0／G1期,这可能与VPA抑制HDAC1表达,上调组蛋白H3、H4乙酰化水平有关。     

雷公藤红素对白血病HL-60和Jurkat细胞增殖及凋亡的影响

探讨雷公藤红素对人急性髓性白血病HL-60细胞、急性T淋巴细胞白血病Jurkat细胞增殖及凋亡的影响。采用MTT法、AnnexinV-FITC/PI双染法、PI染色法、透射电镜观察不同浓度雷公藤红素作用于两种细胞后对其生长增殖、凋亡、周期及形态等方面的影响。结果表明雷公藤红素能显著抑制HL-60细胞、Jurkat细胞的增殖,降低其存活率。给药24 h后,半抑制浓度(IC₅₀)分别为(0.46±1.05)μmol/L和(0.88±1.13)μmol/L。以剂量依赖方式诱导两种细胞凋亡,细胞周期分布G₁期比例增加,S期比例降低(P<0.05),且伴有典型的细胞凋亡形态学改变。雷公藤红素能显著抑制HL-60细胞、Jurkat细胞的生长增殖,并诱导细胞凋亡。     

MIP-1α enhances Jurkat cell transendothelial migration by up-regulating endothelial adhesion molecules VCAM-1 and ICAM-1

The aim of this study is to evaluate the expression of macrophage inflammatory protein-1α (MIP-1α) in Jurkat cells and its effect on transendothelial migration. In the present study, human acute lymphoblastic leukemia Jurkat cells (Jurkat cells) were used as a model of T cells in human T-cell acute lymphoblastic leukemia (T-ALL), which demonstrated significantly higher MIP-1α expression compared with that in normal T-cell controls. The ability of Jurkat cells to cross a human brain microvascular endothelial cell (HBMEC) monolayer was almost completely abrogated by MIP-1α siRNA. In addition, the overexpression of MIP-1α resulted in the up-regulated expression of endothelial adhesion molecules, which enhanced the migration of Jurkat cells through a monolayer of HBMEC. MIP-1α levels in Jurkat cells appeared to be an important factor for its transendothelial migration, which may provide the theoretical basis to understand the mechanisms of brain metastases of T-ALL at cellular and molecular levels.     

miR-20a海绵载体构建及在白血病细胞株Jurkat中的表达

本研究构建miR-20a基因的miRNA海绵载体,建立Jurkat-S稳定细胞株,为进一步研究的miR-20a功能及应用干扰技术治疗奠定基础。设计、合成1对含有2个重复针对miR-20a成熟序列第9-12碱基错配的序列,并带有酶切位点,退火后连接到pCDNA3.0-L表达载体上;载体双酶切后再与退火产物连接,重复4次后进行酶切及荧光素酶活性鉴定;亚克隆到慢病毒表达载体,与psPAX2、PMD2G共转染HEK 293T细胞,包装产生慢病毒颗粒并测定病毒滴度,感染Jurkat细胞,建立稳定细胞株;应用实时PCR和Western blot技术分别检测Jurkat-S稳定细胞中P21及E2F1 mRNA和蛋白的表达,并与对照组进行比较。结果表明,成功构建了针对miR-20a基因的海绵载体,病毒滴度为5×107TU/ml;建立了稳定转染的Jurkat-S细胞株。有效干扰验证显示,miR-20a海绵能明显上调P21及E2F1的mRNA及蛋白表达水平,差异有统计学意义(P<0.05)。结论:成功构建miR-20a基因的miRNA海绵载体,建立稳定干扰miR-20a表达的Jurkat-S细胞株。     

丝裂霉素诱发Jurkat细胞DNA损伤修复机制中blm基因的作用研究

本研究通过丝裂霉素(mitomycin C,MMC)诱发Jurkat细胞凋亡,了解Jurkat细胞DNA损伤后blm mRNA水平变化与细胞周期和凋亡之间关系及意义。用流式细胞术检测凋亡率和细胞周期变化;用半定量RT-PCR检测blm mRNA的表达水平。结果表明:0.4 g/L MMC分别诱导Jurkat细胞和正常成纤维细胞后,Jurkat细胞于24小时凋亡率为(11.42±0.013)%,此时(66.08±1.60)%的Jurkat细胞受阻于G2/M期;48小时时Jurkat细胞凋亡率反而下降(8.08±0.27)%,停滞于G2/M期Jurkat细胞下降为(33.96±1.05)%;正常成纤维细胞诱导后24小时与48小时凋亡率变化不明显,G2/M期细胞、G0-G1、S期细胞在24小时和48小时时变化不显著。2组细胞凋亡率和各期细胞所占比例的变化幅度比较显示,其差异显著有统计学意义(p0.01)。Jurkat细胞blm mRNA表达水平不仅明显高于正常成纤维细胞(p0.01),诱导48小时后blm mRNA表达水平明显较诱导24小时后增高,2组细胞间blm mRNA表达水平变化幅度比较差异显著(p0.01)。结论:blm基因在MMC诱发Jurkat细胞DNA损伤修复过程中发挥重要作用,其mRNA表达异常可能是导致白血病耐药的原因之一。