Apoptosis of Jurkat cells induced by serum of patients with acute severe brain injury

Objective To investigate the capacity of serum samples draining from the neuronal lesions to induce apoptosis of the lymphoid Jurkat cells in vitro and to analyze whether this effect is related to patient outcome.Design and setting Prospective clinical investigation in a 21-bed intensive care unit (ICU) in a university hospital.Patients Forty-two patients who had suffered from acute brain injury (traumatic brain injury or spontaneous intracranial hemorrhage) requiring intensive care.Interventions Blood samples were obtained simultaneously from jugular bulb vein (regional) and from central venous catheter (systemic) on admission to the ICU and after 24, 48, and 72 h. Jurkat cells were incubated in the presence of 10% of heat-inactivated patients sera. The percentages of apoptotic cultured cells was measured by staining with annexin V and propidium iodide.Measurements and results Regional serum draining from the lesions induced higher percentages of early and late apoptotic cells than systemic serum. The apoptotic effect was clearer with the sera from the patients who developed brain death. The apoptotic effect maintained a relationship with the mortality and the functional outcome at 6 months after the injury.Conclusions Despite being performed on lymphoid cells because of the easier technical handling, our data help to elucidate the role of apoptosis for brain damage in acute brain injury. This and other undergoing studies on neuronal cells will enhance the understanding and management of apoptotic cell death in patients with acute brain injury admitted to the ICU.This work was supported by grant PI020417 from the Fondo de Investigación Sanitaria, Spain, and by grants from the Fundación Marqués de Valdecilla, Spain.     

砷体外诱导人Jurkat T淋巴细胞的差异表达cDNA文库的构建

目的 了解三氧化二砷(As2O3)诱导人T淋巴细胞的差异表达基因,探讨砷的免疫毒性和免疫抑制的机制。方法 在体外用As2O3处理人Jurkat T淋巴细胞(5μmol／L,24h)后,应用抑制性消减杂交技术(SSH)和克隆技术构建差异表达的cDNA文库,用聚合酶链反应技术和测序技术鉴定阳性克隆。结果 用SSH技术成功地构建了由As2O3诱导的Jurkat T淋巴细胞的差异表达基因的正向消减cDNA文库。经测序分析该文库含有30个基因片段,其中仅有1个是重复基因片段。与基因库的序列比较显示,这些基因序列的同源性在95％～100％。该文库的基因有：氧化代谢基因(包括磷酸丙糖脱氢酶、酰胺腺嘌呤二核苷酸4亚单位、焦磷酸合成酶、16S核糖体、琥珀酰CoA合成酶和ATP合成酶6),转录和翻译基因(包括多聚腺苷结合蛋白、泛醌素特异性蛋白酶14、核糖体蛋白：[23、核糖体蛋白S15a、真核翻译起始因子3、Rab5相互作用蛋白、剪切因子和ADP-核糖体因子6样相互作用蛋白),与氧化应激相关的基因(铁蛋白重链和高泳动类蛋白2),与细胞分化或凋亡相关基因(髓细胞分化初期应答蛋白和凋亡相关蛋白),参与蛋白活化或信号传导的基因(酪氨酸激酶、丝氨酸激酶和磷脂酰4磷接头蛋白-1相关蛋白);5个功能不详的基因是KIAA0092、CGI-147蛋白、GCI-35、核磷蛋白Nopp34和与骨骼肌部分mRNA假设蛋白同源基因。1个在基因库中无同源序列的新基因。结论 应用SSH技术成功构建了As2O3诱导T淋巴细胞差异表达基因的正向消减cDNA文库。并且发现以往在砷的研究中从未涉及的基因。基因功能分析结果提示,As2O3在诱导T淋巴细胞凋亡的过程中不但有多种基因参与,而且还有一些未知的基因也参与细胞凋亡或(和)细胞抗砷毒性作用的应答过程。     

Effects of betulinic acid on proliferation and apoptosis in Jurkat cells and its in vitro mechanism

Summary  The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 μmol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G₀/G₁ phase and decrease in S phase. After treatment with 0, 20, 60, 100 μmol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00±1.25)% to (58.84±0.32)% in G₀/G₁ phase, whereas it was decreased from (61.45±1.04)% to (35.82±1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G₀/G₁ phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl. Zi CHEN, Female, born in 1980, Resident This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30500686).     

结肠癌细胞通过Fas/FasL诱导淋巴细胞发生凋亡

背景与目的:研究发现,肿瘤组织中Fas配体阳性表达区肿瘤浸润淋巴细胞的凋亡率比FasL阴性区高,推测肿瘤细胞可通过增加表达FasL来杀伤肿瘤浸润淋巴细胞。本研究拟在体外验证结肠癌细胞SW480是否可以诱导淋巴细胞发生凋亡。方法:结肠癌细胞系SW480(效应细胞)与对Fas介导凋亡敏感的Jurkat细胞(靶细胞)共培养,分为3种效靶比20∶1,10∶1,5∶1。生长曲线法、流式细胞术、荧光显微镜观察分别检测Jurkat细胞的凋亡情况。结果:流式细胞仪检测结果显示,SW480接种浓度为0(对照)、0.5×105/ml、1×105/ml、2×105/ml时,Jurkat细胞凋亡率为(1.8±0.21)%、(5.49±0.17)%、(11.18±0.14)%、(18.22±0.11)%。荧光显微镜观察结果表明,SW480接种浓度为0(对照)、0.5×105/ml、1×105/ml、2×105/ml时,Jurkat细胞凋亡率为(1.58±0.12)%、(5.22±0.13)%、(9.74±0.21)%、(19.33±0.18)%。3种效靶比条件下,SW480细胞均能诱导Jurkat细胞发生凋亡。随着SW480接种浓度的升高及共培养作用时间的延长,Jurkat细胞凋亡率逐渐增加。每个实验组与对照组相比,P值均小于0.01。结论:结肠癌细胞SW480可以通过Fas系统诱导淋巴细胞发生凋亡,这为结肠癌的免疫逃逸、反击机制提供了又一证据。     

脂氧素A4对Jurkat T细胞游离钙浓度变化及增殖的影响

目的 研究脂氧素A4(LXA4)对Jurkat T细胞内游离钙离子浓度变化及细胞增殖的影响.方法 钙离子荧光探针Fluo-3/AM负载细胞后,用激光共聚焦扫描显微镜动态检测活细胞内游离钙离子浓度的变化;CCK-8试剂盒检测细胞增殖情况,并结合流式细胞仪进行细胞周期分析.结果 ①采用含钙细胞外液时,LXA4降低正常Jurkat T细胞内游离钙离子浓度,而用无钙细胞外液时,细胞内游离钙离子浓度下降不明显;②LXA4呈剂量依赖性抑制钙池操纵钙通道(SOC)激活剂thapsigargin(TG)引起的Jurkat T细胞内游离钙离子浓度的增高,100 nmol/L LXA4也能抑制抗CD3单抗(a-CD3)引起的细胞内钙离子浓度的增高,SOC阻滞剂2-Aminoethoxydiphenylborate(2-APB)能显著抑制TG引起的钙内流;③LXA4呈剂量依赖性抑制a-CD3和抗CD28单抗(a-CD28)诱导的Jurkat T细胞增殖,细胞周期分析发现LXA4阻止JurkatT细胞由G1期进入S期,降低S期细胞比例、细胞增殖指数(PI)和DNA含量.结论 LXA4可能通过抑制Jurkat T细胞钙池操纵的钙通道引起的胞质内游离钙浓度的升高而抑制其增殖.     

衰变加速因子在Jurkat细胞增殖及分泌IL—2中的作用

目的 研究衰变加速因子对CD3阳性与阴性Jurkat细胞的增殖效应及对IL－2分泌的影响。方法 应用MTT比色实验观察交联DAF单抗DG3与固相化抗CD3联合作用对CD3阳性与CD3阴性Jurkat细胞增殖效应;IL－2依赖CTLL^3H-TdR掺入实验检测CD3阳性与CD3阴性Jurkat细胞IL－2的分泌;细胞ELISA检测CD3阳性与CD3阴性Jurkar细胞IL－2R的表达。结果 交联DG3可协助促进固相化抗CD3对CD3阳性Jurkat细胞的增殖效应和IL－2分泌,并可引起IL－2Rα链表达增加,此作用可被Src家族酪酸激酶抑制剂PP2所抑制,而对CD3阴性Jurkat细胞无此作用。结论 衰变加速因子可协同促进固相化抗CD3对CD3阳性Jurkat细胞的刺激增殖效应、IL－2分泌及IL－2R表达增加。     

9.1C3胞内抗体基因的克隆及其对9.1C3 分子表达的抑制作用

目的 获得9．1C3胞内抗体基因的真核表达载体,观察胞内抗体对9．1C3分子表达的抑制作用,为进一步研究9．1C3分子对NK细胞功能的影响打下基础。方法 设计引物,以9．1C3 ScFv基因为模板进行PCR扩增,引入蛋白质内质网定位所需的信号肽、KDEL以及作为表达检测标记的Etag序列。回收纯化PCR产物,酶切后连接到pUC19载体上,并进行序列测定。序列正确后切下胞内抗体基因片段,重组到真核表达载体pRc/CMV中,酶切鉴定后用DEAE－dextran方法瞬时转染真核细胞COS7,用胞内染色和Western blot方法检测胞内抗体的表达。接着用Lipofectamine把胞内抗体基因转入9．1C3阳性的Jurkat细胞,用FCM观察胞内抗体对9．1C3分子表达的影响。结果 DNA序列测定证明,通过PCR成功地为9．1C3 ScFv引入了内质网定位所需的信号肽、KDEL及E－tag序列,成功地构建了胞内抗体真核表达载体,通过胞内染色方法和Western blot证明胞内抗体在COS7中得到表达。转入Jurkat细胞后发现胞内抗体能下调9．1C3分子的表达水平。结论 以9．1C3 ScFv基因为模板PCR扩增得到9．1C3胞内抗体基因,并构建了真核表达载体。     

白藜芦醇诱导急性T淋巴母细胞白血病Jurkat细胞凋亡

目的：探讨白藜芦醇抑制急性T淋巴母细胞白血病Jurkat细胞增殖、引发S期阻滞和诱导凋亡的作用。方法：白藜芦醇体外处理培养的Jurkat细胞后,采用MTT法测定细胞活力,Wrigh-Giemsa染色、Hoechest 33258/PI荧光染色和透射电镜观察Jurkat细胞的形态学改变;流式细胞术分析细胞周期。结果：白藜芦醇0.2 mmol·L^-1处理组的抑制Jurkat细胞增殖率达64.01%,并具有剂量和时间依赖性。白藜芦醇处理细胞24 h后可见凋亡形态学改变,Hoechest 33258/PI染色显示白藜芦醇处理组细胞部分细胞核呈亮蓝色,随培养时间延长,亮蓝色荧光染色细胞数量逐渐增多。Wrigh-Giemsa染色和透射电镜观察发现部分细胞体积缩小、染色质浓缩及边集等现象的出现。流式细胞术检测表明,0.05 mmol·L^-1白黎芦醇处理48 h组62.57％的细胞被阻滞于细胞周期S期,亚二倍体细胞数量12.01%,未加药对照组细胞亚二倍体细胞数量2.05%。结论：白藜芦醇可以抑制Jurkat细胞的增殖,引起细胞周期的S期阻滞,并诱导其发生凋亡     

Notch1信号途径参与低氧诱导的Jurkat细胞侵袭转移的作用机制研究

目的研究Notch1信号途径在低氧诱导的Jurkat细胞侵袭转移过程中的作用。方法分别在低氧和常氧环境下体外培养Jurkat细胞,观察细胞侵袭转移能力的变化,同时检测Notch1信号途径,基质金属蛋白酶(MMP)-2和MMP-9的表达变化。采用RNA干扰的方法阻断Notch1信号途径后,观察Jurkat细胞侵袭转移能力的改变以及MMP-2和MMP-9的变化情况。结果低氧能够促进Jurkat细胞的侵袭转移能力,同时在低氧环境下,Notch1信号途径能够被激活以及MMP-2和MMP-9的表达增加。阻断Notch1信号途径后,Jurkat细胞侵袭转移能力下降,而MMP-2和MMP-9的表达也下调。结论在低氧条件下,Jurkat细胞侵袭转移能力增加,其机制可能通过激活Notch1信号途径促使MMP-2和MMP-9的表达增加来实现的。