沉默Sam68基因对Jurkat细胞增殖影响的研究

本研究探讨Sam68基因对人急性T淋巴细胞白血病细胞Jurkat增殖的影响.针对Sam68 mRNA 531-552靶位点设计并合成shRNA,构建pLKO-Tet-On条件性真核干扰载体,制备慢病毒并感染Jurkat细胞;应用强力霉素（Doxycycline）诱导干扰载体表达,用Real-time PCR和Western blot验证干扰效率,用改良MTT法检测沉默Sam68基因对Jurkat细胞活力的影响,用体外细胞集落形成实验观察沉默Sam68基因对细胞集落形成能力的影响,用流式细胞术分析沉默Sam68前后细胞周期的变化.结果表明：与对照组相比,实验组Sam68基因的表达被有效抑制（P＜0.05）;沉默Sam68基因降低了Jurkat细胞活力,抑制了细胞的集落形成能力并且导致S期细胞比例显著增加,G2期细胞比例显著降低（P＜0.05）.结论：利用shRNA靶向干扰Sam68基因的表达可以有效抑制人急性T淋巴细胞白血病Jurkat细胞的增殖.     

MIP-1α 诱导 Jurkat 细胞穿过人脑微血管内皮细胞单层能力分析简

本研究为探究人急性T淋巴细胞白血病（T-ALL）细胞脑转移发生中枢神经系统白血病的机制及MIP-1α在T-ALL模式细胞Jurkat穿过人脑微血管内皮细胞（HBMEC）过程中的作用.应用real-time PCR、siRNA试验、跨内皮迁移试验和细胞黏附试验,分别检测MIP-1 α的表达、Jurkat细胞穿透能力、迁移能力和黏附力.结果表明,Jur-kat细胞作为研究T-ALL细胞穿过血脑屏障（BBB）机制的模式细胞MIP-1α表达均高于正常人T淋巴细胞以及其他3种T-ALL细胞系（HSB2、CEM、SUP-T1）,Jurkat细胞分泌的MIP-1α使其穿过HBMEC单层能力增强.MIP-1 αsiRNA干扰后的Jurkat细胞对HBMEC黏附和跨内皮迁移力下降.结论：Jurkat细胞分泌的MIP-1α参与了Jurkat细胞穿过HBMEC单层的过程.     

抗原冲击致敏树突状细胞诱导特异性CTL杀伤Jurkat细胞实验研究

本研究以健康人外周血单核细胞为前体细胞，体外诱导其为树突状细胞（DC），分别负载Jurkat细胞冻融抗原及WT1多肽抗原，产生特异性细胞毒性T淋巴细胞（CTL），以探讨其对白血病Jurkat细胞的杀伤作用。联合应用rhGM—CSF、rhIL-4、rhTNF-α及rhsCD40L等细胞因子，密度梯度离心法、贴壁法从外周血获取单核细胞并进行诱导扩增，培养出DC，分别用冻融抗原及WT1多肽抗原冲击致敏DC。实验分4组：冻融抗原致敏DC组为实验组A，WT1多肽致敏DC组为实验组B，未致敏DC组为对照组A，单核细胞组为对照组B，观察CTL对Jurkat细胞的杀伤作用。结果表明：培养出了具有典型特征的DC，它高表达CD40、CD80、CD1a及CD86等表面标志，能体外诱导强烈的同种异基因混合淋巴细胞增殖反应。实验组显示高水平杀伤率，与对照组比较均具有统计学意义（P〈0.01），实验组A、B间比较无统计学意义。结论：Jurkat细胞冻融抗原及WT1多肽抗原冲击致敏DC能有效诱导T细胞抗白血病作用，为临床研制DC疫苗提供了实验依据。     

HBx蛋白对人T细胞抗炎性细胞因子分泌的影响

目的探讨HBx蛋白对人T细胞抗炎性细胞因子分泌的影响。方法构建HBx蛋白的绿色荧光蛋白真核表达载体HBx-pEGFP-C1,双酶切(HindⅢ和KpnⅠ)、DNA测序鉴定。利用脂质体转染技术将质粒HBx-pEGFP-C1瞬时转染人T细胞系Jurkat细胞株,荧光显微镜检测报告基因表达产物EGFP,RT-PCR方法检测X基因的表达情况。PHA(10μg/ml)刺激活化预转染的Jurkat细胞6h、24h和48h后,采用RT-PCR技术在mRNA水平检测Jurkat细胞分泌IL-4、IL-10、IL-13和IL-14等抗炎性细胞因子的变化。结果成功构建HBx-pEGFP-C1真核表达载体,并成功转染Jurkat细胞;与对照组相比,转染HBx蛋白组Jurkat细胞分泌IL-4、IL-10、IL-13和IL-14均明显降低(P<0.05)。结论 HBx蛋白即时高表达可降低人T细胞抗炎性细胞因子的表达。     

Molecular mechanisms in the TCR (TCR alpha beta-CD3 delta epsilon, gamma epsilon) interaction with zeta 2 homodimers: clues from a 'phenotypic revertant' clone.

The association between the TCRalphabeta-CD3gammaepsilondeltaepsilon hexamers and zeta2 homodimers in the endoplasmic reticulum (ER) constitutes a key step in TCR assembly and export to the T cell surface. Incompletely assembled TCR-CD3 complexes are degraded in the ER or the lysosomes. A previously described Jurkat variant (J79) has a mutation at position 195 on the TCR Calpha domain causing a phenylalanine to valine exchange. This results in a lack of association between TCRalphabeta-CD3gammaepsilondeltaepsilon hexamers and zeta2 homodimers. Two main hypotheses could explain this phenomenon in J79 cells: TCR-CD3 hexamers may be incapable of interacting with zeta2 due to a structural change in the TCR Calpha region; alternatively, TCR-CD3 hexamers may be incapable of interacting with zeta2 due to factors unrelated to either molecular complex. In order to assess these two possibilities, the TCR-CD3 membrane-negative J79 cells were treated with ethylmethylsulfonate and clones positive for TCR membrane expression were isolated. The characterization of the J79r58 phenotypic revertant cell line is the subject of this study. The main question was to assess the reason for the TCR re-expression. The TCR on J79r58 cells appears qualitatively and functionally equivalent to wild-type TCR complexes. Nucleotide sequence analysis confirmed the presence of the original mutation in the TCR Calpha region but failed to detect compensatory mutations in alpha, beta, gamma, delta, epsilon or zeta chains. Thus, mutated J79-TCR-CD3 complexes can interact with zeta2 homodimers. Possible mechanisms for the unsuccessful TCR-CD3 interaction with zeta2 homodimers are presented and discussed.     

低剂量阿糖胞苷对Jurkat T淋巴细胞CD147表达的影响

目的: 探讨低剂量阿糖胞苷(Ara-C)对Jurkat T淋巴细胞CD147表达的影响。方法: 实验细胞分6组,为对照组和1、5、10、20、30 ng/ml Ara-C组。采用RT-PCR、Western blot和流式细胞术分别检测Ara-C干预前后Jurkat细胞CD147基因和蛋白的表达。用倒置显微镜观察、比较Ara-C干预前后Jurkat细胞集落形成情况。结果: Ara-C各浓度组中细胞CD147 mRNA和蛋白的表达均明显高于对照组(P < 0.01),Ara-C干预组细胞集落形成明显增加。结论: 低剂量Ara-C可上调Jurkat T淋巴细胞CD147的表达,增强细胞间黏附力。     

雷公藤甲素对白血病HL-60和Jurkat细胞增殖及凋亡的影响

目的:探讨雷公藤甲素对人急性髓系白血病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。方法:采用MTT法、Annexin V-FITC/PI双染法、PI染色法、透射电镜观察不同浓度雷公藤甲素作用于两种细胞,对其生长增殖、凋亡、细胞周期及形态等方面的影响。结果:雷公藤甲素能显著抑制HL-60、Jurkat细胞的增殖,降低其存活率。给药24 h后,半数细胞抑制剂量(IC50)分别为(42.50±9.38)nmol/L和(60.91±9.77)nmol/L。能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。结论:雷公藤甲素能显著抑制HL-60、Jurkat细胞的生长增殖,并诱导细胞凋亡。     

Isolation of two CD50 (ICAM-3)-negative Jurkat T-cell clones and their application for analysis of CD50 function

Abstract: The leukocyte differentiation antigen CD50 (intercellular adhesion molecule-3, ICAM-3), mediates cell-cell adhesion through its ligand LFA-1 and is a transducting receptor molecule during T-cell activation. Since CD50 homologues in other species have not yet been identified, the role of this molecule can only be analyzed in human cell models. Thus, to better study CD50 function in T cells, we have obtained two CD50-negative T-cell clones, named CAMY.l and CAMY.2. These clones were derived from the Jurkat T-cell variant PPL.l. Data from analysis of protein expression, specific mRNA content and calcium mobilization assays have confirmed the absence of functional CD50 molecules on these two clones. Thus, CAMY.l and CAMY.2 show no CD50 expression by phenotypical and immunoprecipit-ation analysis. CD50-sperific mRNA content is undetectable by Northern blot analysis in these clones and, only, when RT-PCR was performed could specific mRNA be detected. Additionally, CD50 cross-linking on theses clones shows no increase in intracellular calcium. Transfection of CD50 cDNA on CAMY cells restores not only CD50 surface expression, but its functional ability to induce calcium mobilization, CD69 upregulation and cell morphological changes. The CAMY.l and CAMY.2 clones provide useful model systems to analyze CD50 function in T cells.     

Different effects of two cyclic chalcone analogues on cell cycle of Jurkat T cells

It has been previously shown that the cyclic chalcone analogues E-2-(4′-methoxybenzylidene)- (2) and E-2-(4′-methylbenzylidene)-1-benzusuberone (3) inhibited proliferation of various murine and human tumor cells. In order to gain new insights into the cytotoxic mechanism of the two compounds detection of apoptosis and necrosis of Jurkat T cells exposed to 2 and 3 were performed by flow cytometry using the Annexin V-FITC and propidium iodide double staining method. Analysis of the DNA histograms at 8, 24 and 48 h exposure times showed that near equitoxic doses of 2 and 3 had different effects on the cell cycle of the exposed cells. The immediate (8 h) effect of 2 was a remarkable decrease of cells in the G₀/G₁ phase and increase in the G₂/M phase. This effect could also be seen in the histogram of cells at the 24 h time point. On the contrary, such an effect of 3 could not be observed. At the 24 and 48 h time points accumulation of sub-G₀ (apoptotic and necrotic) and hyperdiploid cells could be detected after both treatments. Incubation of 2 and 3 with reduced glutathione under cell-free conditions indicated spontaneous conjugation (non-redox) reaction at pH 7.4 and pH 9.0. Analyzing the mechanism of action the total thiol content of the cells exposed to compounds 2 and 3 was determined. Compound 2 showed to reduce the total cellular thiol level both under nutrient-free and nutrient-supplemented conditions. Under the latter conditions an increase of the total thiol level of the cells exposed to 3 for 4 h could be observed. The different effect of the two compounds on the cellular thiol status might contribute to the different tumor cytotoxicity of the cyclic chalcone analogues 2 and 3. Investigation of antioxidant capacity of the compounds by monitoring time course of the Fenton-reaction initiated in vitro degradation of 2-deoxyribose indicated that both compounds displayed hydroxyl radical scavenger activity. The experiments provide further details of dual – cytotoxic and cytoprotective (chemopreventive) – effects of the compounds.