Mer在Jurkat细胞中的表达和抗凋亡作用

背景与目的:Mer是Axl受体酪氨酸激酶家族中的一员,其配体Gas6和Mer结合后可以激活Mer的酪氨酸激酶活性以及下游信号转导途径,在细胞炎症、凋亡等方面发挥作用.近年来对于Mer及其家族成员功能的研究不断完善,但关于其在恶性疾病方面的研究报道甚少.本实验探讨Mer在恶性T淋巴细胞白血病细胞株Jurkat中的表达及其对Jurkat细胞凋亡过程的影响,并对其抗凋亡机制进行初步探究.方法:利用流式细胞仪分别检测正常外周血、骨髓中T淋巴细胞以及Jurkat细胞中Mer的表达.利用RNAi技术敲除Jurkat细胞中Mer的表达后采用噻唑蓝(MTT)法检测Mer对于Jurkat细胞增殖的影响,Annexin V/PI双染流式细胞术检测Mer对于Jurkat细胞血清撤离诱导凋亡过程的影响.最后利用荧光定量PCR检测RNA干扰后凋亡相关基因Bcl-2和Caspase-3以探讨Mer在Jurkat细胞中抗凋亡机制.结果:Mer在正常外周血和骨髓的T细胞中均未见表达,而在Jurkat细胞中高表达(51.1%).特异性的siRNA(第1403位点)可以抑制86.0%Mer在Jurkat细胞中的表达.在血清撤离48 h导致的凋亡实验中正常Jurkat细胞仅有1.5%的凋亡率,而敲除Mer后的Jurkat细胞凋亡率达到15.3%,而MTT增殖实验中阻断Mer前后差异无统计学意义(p＞0.05).定量PCR结果显示敲除Mer基因后Jurkat细胞中Bcl-2明显下调,为对照组的42.7%,而Caspase-3变化不显著.结论:Mer在Jurkat细胞中异常高表达,并可能通过Bel-2途径影响细胞凋亡过程.     

重组人可溶性DR5蛋白的分离纯化及其生物活性检测

目的:提纯毕赤酵母GS115菌株表达的重组人可溶性死亡受体5(sDR5)蛋白,并研究其对肿瘤坏死因子相关凋亡诱导配体(TRAIL)所诱导细胞的阻断效应。方法:大量扩增可溶性DR5酵母表达载体转化阳性的毕赤酵母GS115菌株,收集上清,利用Ni-NTA Agarose亲和层析柱纯化获得重组可溶性DR5,用SDS-PAGE检测纯化产物的纯度。用流式细胞仪检测纯化的可溶性DR5对TRAIL诱导Jurkat细胞凋亡作用的阻断效应。结果:转化阳性的毕赤酵母GS115菌株可以成功表达分子量为23kD的可溶性DR5蛋白,经过Ni-NTAAgarose亲和层析柱纯化,SDS-PAGE鉴定显示获得了高纯度的重组人可溶性DR5蛋白,培养上清产量达28.69mg/L;体外活性检测结果显示,纯化的可溶性DR5蛋白几乎完全可以阻断TRAIL诱导的Jurkat细胞凋亡作用。结论:经纯化后的重组人可溶性DR5蛋白可以有效阻断TRAIL诱导的细胞凋亡作用,显示DR5在TRAIL诱导Jurkat细胞凋亡作用中起着十分关键的作用。     

鸦胆子油乳诱导Jurkat细胞凋亡及与柔红霉素的协同作用

目的：观察鸦胆子油乳对诱导Jurkat细胞凋亡的影响及联合柔红霉素的协同作用。方法：采用MTT比色法、DNA凝胶电泳、流式细胞仪检测鸦胆子油乳及联合柔红霉素诱导Jurkat细胞凋亡情况。结果：鸦胆子油乳（0.25-1.25g/L）对Jurkat细胞有诱导凋亡作用,并具有一定时间剂量-效应关系。0.25g/鸦胆子油乳与0.5μmol/L柔红霉素联合作用对Jurkat细胞凋亡率较单用时的凋亡率高。结论：鸦胆子油乳对Jurkat细胞有诱导凋亡作用,呈剂量相关性,并与柔红霉素有协同作用。     

TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning

The identification of novel tumor antigens is of extreme importance for effective immunotherapy against cancer. A major obstacle in this field is the limited life span of tumor-specific cytotoxic T lymphocytes (CTLs) in vitro. Therefore we searched for a method to isolate the tumor specificity of these CTLs, i.e., their T-cell receptors (TCRs) and transfer it to an immortalized T-cell line. For this purpose, a TCR-negative Jurkat T-cell line was equipped with a nuclear factor of activated T cells (NFAT)-luciferase reporter construct to allow measurement of TCR-mediated activation. To establish the feasibility of this tumor-specific TCR transduction, we cloned the TCR genes of a known T-cell clone specific for the tumor antigen CAMEL (CTL-recognized antigen on melanoma) into a retroviral construct. Jurkat reporter cells transduced with this construct, Jrt-TCRalpha3beta5, were tested for their reactivity against CAMEL-expressing melanoma cells, peptide-loaded T2 cells and CAMEL-transfected COS-1 cells. The melanoma cell lines were poorly recognized, but peptide-pulsed and -transfected cells effectively stimulated NFAT signaling. The activation of TCR(+) Jurkat reporter cells was shown to be dependent on the antigen density on the target cells and the expression level of the coreceptor CD8 on the Jurkat cells. To verify the benefit of this TCR reconstitution method for identification of novel antigens, pools of the cDNA library from which CAMEL was originally cloned were transfected in COS-1 cells and screened with Jrt-TCRalpha3beta5. Identical cDNA pools were found that were positive with these cells and with the CAMEL-specific CTL clone. Our results illustrate that TCR-reconstituted Jurkat reporter cells are a useful tool in the identification of novel tumor antigens by cDNA expression cloning.     

Inhibition of cell death by ribosomal protein L35a

In order to better understand how tumor cells develop resistance to chemotherapy drugs, we screened a human cDNA expression library in Jurkat cells for cDNA's that conferred resistance to doxorubicin-induced cell death. One of the cDNA's isolated in the screen codes for ribosomal protein L35a, a component of the large subunit of the ribosome. Jurkat cells engineered to overexpress L35a protein were more resistant not only to doxorubicin but also to UV-irradiation, anti-Fas antibody, and serum starvation compared to Jurkat cells expressing endogenous levels of L35a. Jurkat cells overexpressing L35a did not have increased levels of the anti-apoptotic proteins Bcl-2 or Bcl-xL, the drug efflux pump P-glycoprotein, nor altered cellular growth kinetics or total protein synthesis. Our results provide new insight into L35a function and suggest that it may have a role in the cellular response to cytotoxic damage. Since L35a RNA is overexpressed in a significant number of glioblastoma multiforme (GBM) brain tumors, our results may stimulate further investigation into the possible role of L35a in the resistance of GBM to cytotoxic therapy.     

离子辐射通过caspase途径而非p53途径诱导Jurkat细胞凋亡

目的:研究离子辐射诱导Jurkat细胞凋亡的动态趋势及其机制。方法:分别以5、10、20Gy的辐射量照射Jurkat细胞,其中一份样本在照射前加入zVAD.fmk,在照射后培养6、12、24、36和48h收获细胞,应用AnVPI双染和呈现subG1峰技术,在流式细胞仪上定量测定凋亡细胞。采用WesternBlot技术检测p53蛋白的表达。结果:细胞受照射后,凋亡细胞数随培养时间的延长和辐射量的加大而增加。在6h,只有20Gy才诱导细胞显著凋亡(P<0.005),5Gy的照射要到24h才出现明显的凋亡(P<0.05)。在48h,20Gy照射诱导的凋亡细胞数是所有时间点和剂量强度中最高的(P<0.001)。加有zVAD.fmk的细胞,尽管都是10Gy照射,比未加zVAD.fmk的细胞表现出凋亡细胞明显减少(P<0.005),但与未加zVAD.fmk、照射量是5Gy的细胞相比,凋亡细胞数仍显著增加(P<0.005)。Jurkat细胞在照射前后都没有p53表达。结论:离子辐射诱导细胞凋亡呈现出时间和剂量效应关系;capase抑制剂zVAD.fmk部分抑制离子辐射诱导细胞凋亡;离子辐射诱导细胞凋亡的机制独立于肿瘤抑制基因p53,caspase可能在其中起重要作用。     

Down-regulation of β-catenin Nuclear Localization by Aspirin Correlates with Growth Inhibition of Jurkat Cell Line

In this study, we examined the effects of aspirin on the growth rates, subcellar distribution of β-catenin protein, the expression of β-catenin/TCF signaling pathway target gene cyclinD1 mRNA, and cell cycle of Jurkat cell line （Human T-acute lymphoblastic leukemia）. Our results showed that the treatment with aspirin inhibited the growth of Jurkat cell line. Jurkat cells treated with 3 mmol/L of aspirin could significantly decrease nuclear localization of β-catenin, and at 5 mmol/L of aspirin, the nuclear localization of β-catenin was undetectable. QRT-PCR showed that the target gene cyclinDl mRNA expression was gradually decreased with the dosage of aspirin. Aspirin induced G0/G1 cell cycle arrest in Jurkat cells. We are led to conclude that aspirin acts through β-catenin-independent mechanisms. The effects of aspirin include down-regulation of β-catenin nuclear localization and G0/G1 cell cycle arrest, which might serve as a means of growth inhibition in aspirin-treated human Jurkat cell line.     

Tethered ligand-derived peptides of proteinase-activated receptor 3 (PAR3) activate PAR1 and PAR2 in Jurkat T cells

Proteinase-activated receptors (PARs) can activate a number of signalling events, including T-cell signal-transduction pathways. Recent data suggest that the activation of PARs 1, 2 and 3 in Jurkat T-leukaemic cells induces tyrosine phosphorylation of the haematopoietic signal transducer protein, VAV1. To activate the PARs, this study used the agonist peptides SFLLRNPNDK, SLIGKVDGTS and TFRGAPPNSF, which are based on the sequences of the tethered ligand sequences of human PARs 1, 2 and 3, respectively. Here, we show that peptides based on either the human or murine PAR(3)-derived tethered ligand sequences (TFRGAP-NH(2) or SFNGGP-NH(2)) do not activate PAR(3), but rather activate PARs 1 and 2, either in Jurkat or in other PAR-expressing cells. Furthermore, whilst thrombin activates only Jurkat PAR(1), trypsin activates both PARs 1 and 2 and also disarms Jurkat PAR(1) for thrombin activation. We conclude therefore that in Jurkat or related T cells, signalling via PARs that can affect VAV1 phosphorylation is mediated via PAR 1 or 2, or both, and that distinct serine proteinases may potentially differentially affect T-cell function in the settings of inflammation.