探討Plexin A2 和 Plexin D1之間的關聯性

Abstract

Semaphorin 3A (Sema3A) 為Semaphorins 家族中的一個成員，是一種分泌蛋白，其生物作用包括指引軸突逐退的線索 (axonal repulsive guidance cue) 與正常或腫瘤細胞的附著、 游移等有關。過去研究已知穿膜蛋白Plexins和Neuropilins共同組成受體蛋白複合體 (receptor protein complex)，來傳導Sema3A在細胞內的訊號。但不同種類的Plexins是否可形成受體蛋白複合體，則尚未被報導。 本文論文皆在探討 (一) Plexin A2和Plexin D1是否會形成蛋白複合體來傳遞Sema3A的生物效應；以及 (二) Plexin A2和Plexin D1是否會共同或各別表現在特定的組織腫瘤中？若其然，則與腫瘤生成或病人病程有否統計上的關聯。針對探討主旨一，採用之實驗方法包括 1.利用西方墨點法及免疫沉澱法來偵測Plexin A2 和Plexin D1是否會形成蛋白複合體。方法2.利用免疫螢光法來觀察Plexin A2和 Plexin D1是否會在同一細胞的相同位置中表現 (co-localized)。方法3.為了更進一步確認Plexin A2和Plexin D1之間是否會直接形成蛋白複合體，於是設計了一連串的融合蛋白：將Plexin A2轉形到帶有螢光蛋白ECFP的ECFP-N1的載體中；將Plexin D1轉形到帶有螢光蛋白EYFP 的 EYFP-N1的載體中，利用螢光共振能量轉移的方法來研究Plexin A2和Plexin D1之間的關係。針對研究主旨二，為了得知Plexin A2和Plexin D1是否會共同或各別表現在特定的組織腫瘤中，採用本實驗室已架構的腫瘤tissue array，以免疫組織化學 (immunohistochemistry) 的方法，先初步篩選Plexin A2和Plexin D1會在哪些腫瘤組織中表現，再進一步擷取更多病人的檢體及組織切片，進行免疫組織化學染色，以生物統計方法來觀察Plexin A2和Plexin D1與腫瘤的生成或病人的存活率是否有關聯性。

Semaphorin 3A, as one member of Semaphorin superfamily, is a secreted protein. Sema3A, functions as negative mediator of axonal guidance and is involved in the attachment and migration of cells during embryonic development and tumor formation /progression. The transmembrane protein Plexins and Neuropilins can form receptor protein complexes to transduce the signalings of Sema3A in cells whether different kinds of Plexins can compose receptor protein complex remains unknown. The specific aim of my study is to explore whether Plexin A2 and Plexin D1 can form protein complexes to transduce Sema3A signaling (specific aim-1).Moreover, we explore whether Plexin A2 and Plexin D1 were co-expressed or differentially expressed in certain types of tumor (specific aim-2).For specific aim-1, we used Western blotting, immunoprecipitation, and FRET to detect whether Plexin A2 and Plexin D1 can form protein complexes. For specific aim-2, we used the tumor tissue array paired with normal tissue to screen the expression of Plexin A2 and Plexin D1 by immunohistochemistry. For those tissue-types of tumor, more cases were collected for immunohistochemistry and statistical analysis.