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系統識別號 U0026-2508201102175800
論文名稱(中文) 探討磷酸酶PTPRR在口腔癌當中所扮演的角色
論文名稱(英文) The Study of Protein Tyrosine Phosphatase Receptor type R (PTPRR) in Oral Cancer
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 99
學期 2
出版年 100
研究生(中文) 陳明暉
研究生(英文) Ming-Hui Chen
學號 T16981106
學位類別 碩士
語文別 中文
論文頁數 40頁
口試委員 指導教授-吳梨華
口試委員-張玲
口試委員-蔣輯武
中文關鍵字 酪胺酸磷酸酶  酪胺酸去磷酸酶  口腔癌  去磷酸化 
英文關鍵字 oral cancer  protein tyrosine phosphorylation  phosphatase  PTPRR 
學科別分類
中文摘要 酪胺酸磷酸酶(protein tyrosine kinase, RTK)及酪胺酸去磷酸酶(protein tyrosine phosphatase, PTP)所調控的酪胺酸的磷酸化、去磷酸化參與許多細胞內的生理反應,如細胞生長、分化、生存、細胞間的訊息傳遞、細胞的移動等。Protein Tyrosine Phosphatase Receptor type R (PTPRR)為PTPs的其中一種,據研究已經有四個不同的異構物 (isoform):具有跨膜結構 (transmembrane domain) 的PTPRR (membrane form,mPTPRR variant 1、 variant 3、variant 4) 以及不具跨膜結構的PTPRR(cytosolic form, cPTPRR variant 2),這樣的差別可能會造就不同的蛋白質被去磷酸化。PTPRR一開始發現於神經細胞中,並且在動物小鼠實驗中發現PTPRR剔除的小鼠其平衡及運動能力會喪失。實驗室先前藉由削弱PTPRR的表現,發現可以有效降低內皮細胞的生長與移動;加上近年來研究指出PTPRR在結腸癌、卵巢癌、及胰臟癌中有不正常的表現。因此本研究遂探討PTPRR的表現量失調是否和口腔癌細胞生理活性相關。在口腔癌細胞中過度表現mPTPRR後,癌細胞的增生、移動、及Matrigel侵犯的能力增加;反之,削弱PTPRR的表現則降低細胞生理活性。同時我們將PTPRR substrate trapping mutant (mPTPRR-C343S和 mPTPRR-D309A/C343S)送入293T細胞中,以免疫沈澱、一維電泳及蛋白質鑑定的方式來尋找PTPRR可能的下游受質蛋白。我們發現到三個可能的下游受質蛋白: GnT-V、Selectin P及CELSR-2,未來我們會進一步針對這三個蛋白質做研究。這些結果將使我們瞭解PTPRR在口腔癌細胞的癌症生成及血管新生中所扮演的角色,並且提供一個可以當作臨床診斷、治療的方向。
英文摘要 Protein tyrosine phosphorylation is a reversible process controlled by protein tyrosine kinases and phosphatases, and controls many physiological processes growth, differentiation, cell cycle progression, cell-cell communications, cell migration, and survival. Protein tyrosine phosphatase receptor type R (PTPRR) is a member of the protein tyrosine phosphatase (PTP) family. This gene encodes four isoforms, including both membrane-bound (mPTPRR: variant 1, variant 3 and variant 4) and cytosolic PTPRR (cPTPRR: variant 2), which may reflect differential roles in the mediation of protein tyrosine phosphorylation. PTPRR was originally found in neuronal cells. PTPRR knockout mice displayed significant defects in their fine motor coordination and balance skills that are reminiscent of a mild ataxia. We previously found that the proliferation and migration abilities of PTPRR knockout endothelial cells were decreased. The expression of PTPRR was recently reported to be deregulated in colon, ovarian and pancreatic cancer types. Consistent with the notion, we hypothesized that PTPRR may play a role in oral carcinogenesis. Both overexpression and knockdown approaches were used to address the function of PTPRR in the proliferation, migration, and invasion of oral cancer cells. Ectopic mPTPRR overexpression promoted oral cancer cell proliferation, migration and Matrigel invasion. By contrast, PTPRR knockdown decreased oral cancer cell proliferation, migration and invasion. Co-immunoprecipitation and SDS-PAGE were used to identify the potential interaction protein of PTPRR following ectopic expression of PTPRR substrate mutants (mPTPRR-C343S or mPTPRR-D309A/C343S) in 293T cells. GnT-V, Selectin P and CELSR-2 were identified in the immune-complex. More studies are needed to confirmed if any of these 3 proteins is a substrate of PTPRR and to study the functional implication of their interaction. Taken together, this study underscores the previously unknown involvement of PTPRR in oral carcinogenesis and may provide alternative therapeutic target for oral cancer treatment.
論文目次 中文摘要 I
Abstract III
致謝 V
圖目錄 IX
表目錄 X
英文縮寫對照表 XI
壹、緒論 1
1. 口腔癌及其發生率 2
2. PTP介紹 3
3. PTPRR的介紹 4
4. PTPRR和疾病的關係 5
5. 研究目的、動機及策略 6
貳、材料與方法 7
1. 抗體及使用質體 8
2. 細胞來源及資料 8
3. 細胞培養 8
4. 建構mPTPRR穩定轉染細胞株 9
5. 點突變 10
6. 免疫組織染色 (Immunohistochemistry) 10
7. 免疫螢光染色 11
8. 免疫沉澱法 12
9. 西方墨點法 12
10. In Vitro phosphatase assay 13
11. 一維電泳、銀染及蛋白質身份鑑定 13
12. 細胞生長速率 (Cell growth rate) 14
13. 傷口癒合實驗 (Wound healing assay) 14
14. 侵襲能力測試 (Invasion ability assay) 14
參、結果 15
一、 口腔癌細胞及口腔癌病人中PTPRR蛋白質表現的型態 16
二、 PTPRR的過度表現對口腔癌細胞所造成的生長、移動的影響 16
三、 PTPRR的削弱表現降低口腔癌細胞所造成的生長、移動與侵犯能力 17
四、 外生性PTPRR的表現降低ERK1/ERK2及mTOR-p70-S6 17
五、 鑑別可能的PTPRR下游作用蛋白及受質 18
肆、討論 19
伍、參考文獻 24
陸、圖與表 29
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