【作者】 李宪奎；

【导师】 武栋成；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 TDAG51基因是一个凋亡相关基因,最初在T细胞中被克隆。Fas受体在免疫系统中发挥重要作用,参与活化诱导的细胞凋亡（AICD,activation-induced cell death）;在活化的T细胞中Fas的表达有赖于TDAG51基因,TDAG51^?的T细胞Fas表达缺陷,因此认为TDAG51具有促进活化T细胞凋亡的作用,在免疫自稳中发挥重要作用。TDAG51基因在体内各组织都有表达,而TDAG51基因在这些组织中的功能仍然不清。近来有关TDAG51基因的研究引起人们的关注。TDAG51是PQR（proline-glutamine-rich）蛋白,在其C-端富含PQ即脯氨酸和谷氨酰胺。富含PQ模序（motif）的蛋白具有促进海马细胞、中隔细胞（septal cell）的凋亡的作用,而且这种凋亡是Fas非依赖的。甚至有的研究认为TDAG51基因是一个抑癌基因,由于其同TSSC3、Tih等基因的结构相似而将其归为一个家族。TDAG51基因在脑组织中表达比较丰富,目前有关其在神经系统中的作用研究的比较少。 神经细胞的凋亡可发生在很多种情况下,异常的神经元凋亡可以导致一些神经退行性疾病,如阿茨海默病（Alzheimer’s disease）、帕金森病（Parkinson’s disease）,这些疾病严重威胁着人们的健康。虽然人们已经意识到其危害,但在临床上仍没有有效的治疗方法。深入理解这些疾病的发生机制和神经细胞异常凋亡发生的过程,将有利于设计针对性的有效的预防和治疗策略。我们的实验设计以PC12细胞作为神经细胞的体外模型,探究TDAG51基因在神经系统中是否像在T细胞中那样,具有促进凋亡的作用,并初步探讨其机制。 方法:实验分为三组,实验组是用表达克隆载体绿色荧光蛋白质粒（pEGFP-C₁）载体转染外源性TDAG51的PC12细胞;空载体对照是转染空载体pEGFPC₁的PC12细胞;对照组是未转染的PC12细胞。在转染后24h、48h、72h收集PC12细胞,用Western blotting检测了DAG51蛋白,比较实验组细胞和各对照组细胞TDAG51蛋白的表达量;质粒转染后48h在相差显微镜和荧光显微镜下观察PC12细胞形态的差别;质粒转染后48h用Hoechst33258染色,观察三组细胞细胞核形态的差别;在高倍镜下,随机选取10个视野,计数凋亡细胞,做统计学分析;转染后18h,加入caspase-9抑制剂,观察细胞凋亡的变化。更多还原

【Abstract】 At first TDAG51 gene was cloned from T cells and considered as a apoptosis-associated gene and was essencial for the expression of Fas（CD95） in activated-T cells. In the TDAG51-/- T cells Fas （CD95） expression is deficient and T cells is resistant to activation-induced cell death（AICD）.So it is considered that TDAG51 promote the apoptosis of activated-T cells and plays a very important role in the immune homeostasis. Expression of TDAG51 gene almost exists in every tissue in our body, but how TDAG51 gene functions is still not very clear. Now study on TDAG51 gene catches more people’s attention. TDAG51 protein is rich in PQ（praline-glutamine） motif at the C terminal. It’s reported that protein rich in PQ motif probably promote the apoptosis of hippocampal and septal cells independently of Fas. Some study group sort TDAG51 gene as a anti-oncogene to a gene family including TSSC3 and Tih gene. And several studies have identified the abundant expression of TDAG51 gene in brain tissues. But it is not clear whether TDAG51 still acts as an apoptosis-promoting factor in brain tissues and as regard as this there are few study papers.Abnormal apoptosis of neurons occurs under many circumstances and causes many neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’ disease. Although we know the danger to our health , at present there are no treatments that can stop or reverse the inexorable neurodegenerative process. However, well understanding the cellular and molecular alternations that are responsible for the neuron’ s demise may soon help in developing effective preventative and therapeutic strategies.Our experiments are designed to study the functions of TDAG51 gene in neuronal system using PC12 cells as a model in vitro to see whether TDAG51 gene still functions as an apoptosis-associated gene in PC12 cells as in T cells.Method: Transfect PC12 with expression clone pEGFP-TDAG51 or pEGFP-C₁ or nothing using transfection kit. PC12 cells transfected with pEGFP-TDAG51 are setted as test cells; PC12 cells transfected with pEGFP-C₁ or nothing are seen ascontrol cells. Western blotting is employed to detect the expression of TDAG51 after transfection. Phase contrast microscope is used to see the change of the morphology of cultured PCI 2 between test group and control. Use Hoechst33258 for staining the nucleus at 48h after transfection and then observe the nuclear morphological difference between tests and control using fluorescent microscope. Under high-magnification fluorescent microscope randomly select 10 sight fields and count the apoptotic cells. Then analyze the data statistically. At 18h after transfection change the medium with medium containing lOuM caspase-9- specific inhibitor and continue to incubate to 48h. Then analyze the change of apoptotic cells number statistically between tests and control.Result: Our results indicate that constitutive expression of TDAG51 exists, but in the test cells expression of TDAG51 is higher than that in control. Under phase-contrast microscope the test PC12 cells become round and lose neurite growth compared with control. Under fluorescent microscope the test PC12 cells stained with Hoechst33258 exhibit nuclear condensation, chromatin aggregation and nuclear fragmentation characteristic of apoptosis which are not seen in control. Statistic analysis results show that at 48h after transfection the test PCI 2 cells go to apoptosis significantly in contrast with control. Caspase-9 inhibitor partly inhibits PC12 cells apoptosis induced by overexpression of TDAG51.Conclusion: PC12 cells express TDAG51 gene constitutively. Overexpression of TDAG51 induced PC12 cells apoptosis. Intrinsic apoptosis pathway mediated by caspase-9 takes part in the PC12 cells apoptosis induced by overexpression of TDAG51.更多还原