根據之前的研究指出Bcl2L12在神經膠母細胞瘤中普遍過度表現，且有抵抗細胞凋亡的功能，這部分的作用主要藉由抑制p53、caspase-3/7等分子之作用，是一種抗後粒線體凋亡作用的機制。然而，已知Bcl2L12被分類在Bcl2家族蛋白中，此一家族的蛋白大多透過與其他的Bcl2家族蛋白交互作用進而參與細胞凋亡或抗細胞凋亡。因此本研究也想探討Bcl2L12是否也和Bcl2家族成員交互作用而產生抗細胞凋亡的功能。首先，利用酵母菌雙雜交法驗證Bcl2L12會與BclxL以及Bcl2作用。為了找出Bcl2L12上負責與Bcl2家族蛋白交互作用的區域，以縮小的Bcl2L12片段經由酵母菌雜交法進行進一步測試，並使用ISIS蛋白質交互作用預測程式，發現Bcl2L12212-216是與BclxL交互作用的重要區域。之後將此一區域與不同的Bcl2家族蛋白成員進行多序列比對、比較Bax分子BH3區段不同作用模式以及不同Bcl2蛋白的3D構形。另外將此一區域五個忌水性或帶電荷的胺基酸殘基加以突變去驗證它們是否與Bcl2L12和Bax對其他Bcl2家族蛋白的作用有關，結果顯示h2(L213) 胺基酸對於Bcl2L12與Bax對BclxL的作用相當重要。綜合以上，從實驗結果顯示Bcl2L12的確存在BH3-like的區段且作用與Bax的BH3區段相似。在神經膠母細胞瘤中外源性地表達Bcl2L12野生型以及BH3相似區段的突變蛋白的確造成抗細胞凋亡蛋白表現上的差異。根據以上資料結論，Bcl2L12的BH3相似區段的確存在且對其本身之抗細胞凋亡作用有重要的意義。

Previous reports have suggested that Bcl2L12 is overexpressed in glioblastoma (GBM) and directly contributies anti-apoptotic effect by inhibiting caspase-3/7 and p53, a mechanism of inhibition of post-mitochondria apoptosis induction. Bcl2L12 is known as a member of Bcl2 family, (generally, Bcl2 family members imply their anti-/pro-apoptotic function by interacting with others Bcl2 family members). This study identify whether Bcl2L12 also confers an anti-apoptotic role by interacting with others Bcl2 family members. First, used yeast two-hybrid assay to identify Bcl2L12 has the ability to interact with BclxL and Bcl2. To explore the region responsible for interaction with Bcl2 family members on Bcl2L12, Bcl2L12 fragments have been analyzed by using yeast two-hybrid assay and combined ISIS program to predict the interacting region for BclxL at Bcl2L12212-216. Thereafter, aligned Bcl2L12 with Bcl2 family members, compared interacting pattern of Bax BH3 domain and 3D structure models with Bcl2 members. Five site-directed mutants corresponding to hydrophobic and charge residues within this region were generated to examine whether these residues are critical for Bcl2L12 or Bax-mediated binding. Our data did show the h2 residue (L213) is essential for the Bcl2L12 and Bax interaction with BclxL. Altogether, the data revealed that a novel BH3-like domain on Bcl2L12 may be responsible for its interaction as similar as BH3 domain in Bax. Moreover, ectopic overexpressed Bcl2L12wt and BH3-like domain mutant in GBM cell lines resulted in variations on expression levels of apoptotic markers. We concluded that BH3-like domain does exist and is also critical for Bcl2L12’s anti-apoptotic role.

中文摘要………………………………………………………….…….….i

English Abstract……………………...………………..…………….….….ii

Introduction…………………………………………………...……………1

Specific Aims….……………………………………………...……………4

Materials and Methods……………………………………...…...…....……5

Results..………………………………………………...……………..…..15

Discussion…………………………………………….…………….….....18

Conclusion………………………………………….…………………..…19

References..……………………………………..……………….………..20

Figures and Tables………………………………..……………….………24

Appendix………………...……………………………………………......33

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