臺灣博碩士論文加值系統

CD45是ㄧ個典型的受體型酪胺酸去磷酸酶，主要扮演調節免疫T細胞活化與死亡的角色。靈芝蛋白(Ling Zhi-8)其結構類似凝集素，最初是由Ganoderma lucidum靈芝屬中萃取出來並藉由酵母菌複製合成重組蛋白(rLZ-8)。首先我們發現LZ-8會和CD45或T細胞抗原受器結合並快速的促使T細胞走向程序性凋亡。具體來說，經由rLZ-8刺激之後可觀察到caspase-8的活化與cytochrome c 釋放到細胞質的現象，接著我們使用caspase-3抑制劑(Z-DEVE-fmk)來探討LZ-8促使T細胞走向程序性凋亡的機制，發現Fas-FasL的訊息傳遞與粒線體調節的路徑皆有參與在此程序性凋亡的機制中。除此之外，在LZ-8促使T細胞走向程序性凋亡的機制中也發現一條經由CD45參與的路徑。實驗上，藉由CD45缺陷的細胞株(J45.01)我們證明了相較於一般正常細胞株(Jurkat)而言，在CD45缺陷的情況下可大幅降低LZ-8所引發的細胞程序性凋亡，並且CD45的去磷酸酶活性並不參與在此細胞程序性凋亡的機制中。而在另一方面，我們發現CD45與其去磷酸酶的活性也參與在LZ-8刺激細胞活化產生介白素-2的過程中。總結我們目前的發現證明了CD45在LZ-8刺激細胞死亡與活化中扮演了多重的角色。

CD45, a prototypic receptor-like protein tyrosine phosphatase plays a critical role in the control point for T cell life and death. A lectin-like protein Ling Zhi-8, originally extracted from Ganoderma lucidum is been cloned as a yeast recombinant protein. Initially, we found binding of LZ-8 to CD45 and/or T cell antigen receptor quickly induces T cell apoptosis. Specifically, upon LZ-8 stimulation, we detected caspase 8 was activated and cytochrome c was released to cytosol. To dissect the LZ-8 mediated T cell apoptosis, using caspase-3 inhibitor (Z-DEVE-fmk), we found both Fas-FasL interaction and mitochondria-mediated pathways involved in the apoptosis. In addition, a CD45-dependent pathway participating LZ-8-induced apoptosis was explored. In essence, using a CD45 deficient cell line (J45.01), we demonstrated a distant decreasing of LZ-8 induced apoptosis compared to Jurkat cell, though independing on CD45 activity. On the other hand, we also found CD45 involved in LZ-8-stimulated interleukin-2 (IL-2) production. In conclusion, our current findings demonstrate that CD45 involved in LZ-8-mediated apoptosis with multi-pathways fashion.

Contents
�� Contents 1
�� Abstract (in Chinese) 6
�� Abstract (in English) 7
�� Introduction 8
�� Materials and Methods 12
�� Cell lines 12
�� Cell preparation 12
�� CD4+ T cells culture 12
�� Antibodies 12
�� Reagents 13
�� Preparation of membrane protein 13
�� Western blotting analysis 13
�� Apoptosis analysis 14
�� MTT assay 14
�� Measure intracellular ROS releasing 15
�� Measurement of the mitochondrial membrane potential 15
�� Mitochondrial isolation 15
�� siRNA transfections 16
�� Statistical analysis 16
�� Results 17
�� Recombinant Ling-Zhi-8 induces apoptosis of human primary T cells and human Jurkat T cells 17
�� Fas-FasL interaction is involved in rLZ-8-induced Jurkat T cells apoptosis 18
�� Mitochondria-involved pathway is one of rLZ-8-induced Jurkat T cells apoptosis mechanisms 19
�� rLZ-8 induces a caspase-independent activation in Jurkat T cell apoptosis 20
�� Receptors are involved in rLZ-8-induced Jurkat T cell apoptosis, especially CD45 21
�� The rLZ-8-induce Jurkat T cell apoptosis mechanism is not dependant on CD45 activity 22
�� CD45 not only involved in apoptosis but also play a role in Jurkat T cell activation 23
�� Discussion 24
�� References 30
�� Figure legend 36

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