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脂筏在TRAIL引起的细胞凋亡早期事件中的作用研究
【作者】 闵一帆；
【导师】 郑德先；
【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2007， 博士
【摘要】 TRAIL是TNF家族的成员之一,与其他成员不同的是TRAIL和其受体在绝大多数细胞和组织中均有表达,但TRAIL可选择性地杀伤肿瘤细胞,对大多数正常细胞没有细胞毒作用。由于这种特性的存在,TRAIL成为一种极具治疗前景的抗癌药物。然而,随着研究的深入,人们发现有许多肿瘤细胞对TRAIL并不敏感,这在一定程度上限制了TRAIL的应用。我们利用实验室保存的两株对TRAIL敏感性不同的Jurkat T淋巴细胞为模型,探讨其敏感性不同的原因,重点关注TRAIL诱导细胞凋亡早期阶段的差别。脂筏是细胞膜上的特殊微区,其中含有大量的胆固醇和鞘磷脂,因为这两种脂类的稳定性较强,可以使脂筏微区与其它磷脂双层分相,并为许多膜蛋白和病原体提供进出细胞和下传信号的平台。由于脂筏是接受大多数外界信号刺激的门户,我们假定两株Jurkat T淋巴细胞对rsTRAIL敏感性不同的原因可能与脂筏有关。对于TRAIL的敏感性与脂筏微区关系的研究,目前还是空白。我们首先以自己制备的重组可溶性TRAIL(rsTRAIL)处理两株Jurkat T淋巴细胞白血病细胞,发现二者对TRAIL细胞毒作用的敏感性存在明显差别。对TRAIL敏感的称JKS细胞,对TRAIL不敏感的称为JKR细胞。Western Blot分析显示,JKS细胞经rsTRAIL处理后,其细胞浆中的促凋亡分子procaspase-8和RIP表达减少,而在JKR细胞中检测不到这种变化。而且未经rsTRAIL处理的JKS细胞内procaspase-8的表达量也比JKR细胞为多。rsTRAIL处理使JKS细胞膜中的procaspase-8、RIP和FADD发生相应的募集或减少,而JKR细胞膜中的FADD和RIP未发生明显变化,procaspase-8则检测不到,提示procaspase-8表达量低可能是JKR细胞对TRAIL不敏感的原因。但是caspase—8抑制剂能全部抑制rsTRAIL引起的JKR细胞凋亡,但只能部分抑制JKS细胞凋亡,抑制率约为50%,表明procaspase-8的表达量差别不能完全解释这两株细胞对TRAIL敏感性的不同,procaspase-8的上游因素可能也影响细胞对TRAIL的敏感性。我们进一步研究了两株细胞脂筏成分的变化,结果表明两株细胞受到TRAIL刺激后,募集促凋亡分子的能力不同。DR5、FADD、procaspase-8、RIP和PI3K-p85亚基在JKS细胞中均是脂筏预装蛋白,TRAIL刺激20分钟内DR5、FADD、procaspase-8和PI3K-p85亚基均增加,而RIP减少。尽管DR5和RIP在JKR细胞中是脂筏预装分子,但随TRAIL刺激的延长,并没有检测到两种分子的变化,而DISC复合物中的FADD和procaspase-8没有出现在脂筏样品中,PI3K-p85亚基只在20分钟时有轻微增加。进一步研究显示,脂筏募集蛋白能力的这种差别与酸性鞘磷脂酶有关。JKR细胞中的酸性鞘磷脂酶较少,而JKS细胞中较多。该酶的活性分析表明,JKR细胞酸性鞘磷脂酶的活性较低,神经酰胺的增加和鞘磷脂的减少都不及JKS细胞明显,JKS细胞的酸性鞘磷脂酶的本底活性较JKR细胞为高,4小时时神经酰胺增加的倍数达到6.3倍以上。酸性鞘磷脂酶与脂筏的共定位研究显示,JKR细胞接受TRAIL刺激后,酸性鞘磷脂酶与脂筏逐渐共定位,有时空的变化。JKS细胞的酸性鞘磷脂酶与脂筏始终是密切共定位,不存在时空的变化。说明JKS细胞中的酸性鞘磷脂酶能够在脂筏原位发挥更强大的作用,利于脂筏中促凋亡分子的募集。综上所述,两株Jurkat T淋巴细胞中酸性鞘磷脂酶的含量、活性和定位的变化是其对TRAIL敏感性不同的重要原因。本项研究可为急性T淋巴细胞白血病的治疗和预后判断提供新的线索。
【Abstract】 TRAIL(TNF-related apoptosis-inducing ligand) is a novel member of the TNF super-family.Unlike other members of TNF family,whose expression is restricted to some cells and tissues such as activated T-cells,natural killer(NK) cells,and immtme-privileged sites,TRAIL and TRAIL receptors are widely expressed in many cell types and tissues,suggesting that most tissues and cell types are potential targets for TRAIL.TRAIL selectively trigges apoptosis of tumor cells and is not toxic to most normal cells,suggesting that this unique molecule is a promising anti-cancer agent.However,reports on the resistance to TRAIL have been increased in various tumor cell lines,which might limit the utility of this molecule in clinical application. In the present study,recombinant soluble TRAIL(rsTRAIL) was used as apoptosis-inducer,and two Jurkat T lymphocyte cell lines(JKR and JKS) with different sensitivity to rsTRAIL were used to explore the molecular mechanism of apoptosis regulation,especially in the early stage of apoptosis induction.Lipid rafts,as defined by Simoms and Ikonen are unique membrane micro-domains,which contain large amount of cholesterol and sphingomyelin. Cholesterol and spingomyelin are the most stable lipid in the membrane,and thus made lipid rafts separated from other phospholipids bilayer and a platform for membrane proteins signal transduction or virus to come in or out of the cell.We speculated that different sensitivity to rsTRAIL stimuli might be layed under the relationship between the pro-apoptosis molecules and the recruitment ability of lipid rafts.In our experiments,two different phenotype cell lines of Jurkat T lymphocytes showed different sensitivity to rsTRAIL stimuli,JKS cells were more sensitive than JKR cells.At cytosolic level,JKS cells showed a more rapid response to the rsTRAIL stimulation than JKR cells.The procaspase-8 and RIP were cleaved upon rsTRAIL stimuli for half an hour.The background concentrations of procaspase-8 were different in the two cell lines,JKS cells containd more procaspase-8 than JKR cells. At plasma-membrane level,JKS cells recruit more FADD,procaspase-8 molecules than JKR cells when stimulated by rsTRAIL for half an hour.The background concentration difference of procaspase-8 in the two cell lines did not fully explain the sensitivity difference.The caspase-8 inhibitor,Z-IETD-FMK,could block the early stage apoptosis of JKR cells,but the blocking ratio dropped to about 50%in the JKS cells.It might be some factors upstream of caspase-8 also affect the sensitivity.Then, we detected the changes happened to the two cell lines upon rsTRAIL treatment at the lipid rafts level.DR5 was a pre-located molecule in lipid rafts in both of the two cell lines.During a 0 to 20 min time course,JKS cells recruited more DR5,FADD, procaspase-8 into lipid rafts,PI3K also was increased in lipid rafts,while RIP was decreased;DR5 and RIP were unaffected in the lipid rafts of JKR cells.FADD, procaspase-8 were not detectable and PI3K had a slightly elevation.2DE image showed that about 33%dots emerging or were increased in the rafts sample of JKS cells after treated with rsTRAIL for half an hour.Most importantly,ASMase,which enable rafts come into being and expanding,was different in concentration,enzyme activity and co-location relationship with lipid rafts.JKS cells contained more ASMase than JKR cells.The enzyme activity of ASMase in JKS cells was higher than that of the JKR cells,with time went on,the enzyme activity of ASMase was even higher than that of the JKR cells.In JKR cells ASMase was gradually co-located with GM1,the symbol of lipid rafts,while in JKS cells ASMase was consecutively co-located with GM1.The results above indicated that concentration,activity and localization of ASMase may contribute to the different sensitivity to TRAIL cytotoxicity of the two cell lines.This study may through light on the therapy and prediction of the outcome of T lymphocyte leukemia.