【作者】 徐进；

【导师】 马宏；

【作者基本信息】 北京理工大学 ， 生物学， 2015， 硕士

【摘要】 本文对血清饥饿下神经胶质瘤细胞对TRAIL耐受性改变的效应进行了初步研究，实验中选取的神经胶质瘤细胞U251和神经胶质母瘤细胞U87，前者在正常培养下对TRAIL敏感，后者则不敏感。但是经过血清饥饿处理后，U251细胞的敏感性降低，而U87细胞的敏感性增加。为了研究其变化的机制，我们检测了两种细胞在正常培养、单纯TRAIL处理、血清饥饿、血清饥饿与TRAIL联合作用下的TRAIL受体在转录水平、蛋白水平和启动子活性的变化。结果表明：U251细胞株在正常培养下只高表达死亡受体DR5，这解释了U251细胞对TRAIL非常敏感的原因，但经过血清饥饿后，死亡受体DR5在表达水平下降，这初步解释了U251细胞经血清饥饿后对TRAIL耐受性增加的原因；而对于U87细胞，在高表达死亡受体DR5的同时，其假受体DcR2的表达也具有较高的表达水平，由此推测假受体DcR2的高表达是U87细胞对TRAIL不敏感的重要原因，同时经过血清饥饿后，假受体DcR2在转录水平和表达水平都有了显著性的降低，通过对其启动子活性的测定，我们也进一步证实了其DcR2水平的降低很可能是外界刺激导致的启动子活性降低，从而减少了mRNA的转录，进而使蛋白表达下降，最终导致经血清饥饿后的U87细胞对TRAIL的敏感性大大增加。除了对TRAIL受体变化的关注，我们还检测了血清饥饿后凋亡相关蛋白的变化，并发现p53野生型的U87细胞在饥饿与TRAIL联合处理下Bid切割不明显而促凋亡蛋白Bax有明显的升高，p53突变型的U251细胞Bid切割明显且在饥饿后有所上升，这可能提示了两种细胞内源凋亡途径激活的机制可能不尽相同。更多还原

【Abstract】 The mechanism of different resistance to TRAIL with or without serum wasinvestigated preliminary in this work, glioma cells U251and glial tumor cells U87has beenchosen to do it, U251is sensitive to TRAIL while U87is not.However, after serumstarvation, the sensitivity of U251cells decreased, and U87cells increased sensitivity. Inorder to strdy the mechanism, we tested the transcription level, protein level and the changeof promoter activity in different situavation like normal culture, pure TRAIL treatment,serum starvation, unit treatment of serum starvation and TRAIL. Results show thatU251cell lines only high expressed death receptor DR5, which explains the high sensitivityto TRAIL in normal culture, however, after serum starvation, death receptor DR5expression level decreased, it may be the reason for decrease of sensitivity of U251toTRAIL. On the contrary, U87cell is not only high expressing death receptor, but also has ahigh expression of false receptor DcR2, suggesting the high expression of false receptorDcR2is one of the important reasons is not sensitive to TRAIL U87cells, at the same timeafter serum starvation, false receptor DcR2in transcription and expression level havesignificantly reduced, through to the determination of its promoter activity, we also furtherconfirmed the DcR2level lower is likely to be caused by external stimuli promoter activityis reduced, thereby reducing the mRNA transcription, and then make protein expressiondecreased, resulting in U87cells after serum starvation sensitivity to the TRAIL. Inaddition to the attention of TRAIL receptors change, we also tested the change of apoptosisrelated proteins after serum starvation, and found that U87cells which is p53wild-typeincrease the level of pro-apoptotic protein Bax without the change of Bid cleavage,however, U251cells with the mutant p53decrease the cleavage of Bid after starvationcompared with TRAIL alone treatment, which suggest the mechianism with the two cellssource apoptotic pathway activation may different.更多还原