論文名稱:

實驗性口腔癌生成之分子變異

 

Molecular Alterations in Experimental Oral Carcinogenesis

研究生:

蔡珮漪  Pei-I Tsai

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

  Kuo-Wei Chang

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

860H04

          學年度:

87

          語文別:

中文

          出版年:

88

關鍵字:

口腔鱗狀上皮癌  oral squamous cell carcinoma

 

p53抑癌基因  p53

 

Ha-ras致癌基因  Ha-ras

 

端粒  telomere

 

倉鼠  hamster

        論文頁數:

69

摘要:

過去的初步研究口腔癌發現Ha-rasp53有明顯的過度表現,而端粒長度變化與基因穩定性有關,均在癌生成扮演重要角色。經22週塗抹致癌物MBN所誘發於倉鼠頰囊內之鱗狀細胞癌,利用(a)免疫組織化學染色法觀察p53表現的改變,(b)聚合A鏈鎖反應/單鏈構形多型性偵測Ha-rasp53的突變,以及(c)端粒複序擴大法測試端粒A活性的異常。倉鼠分別於最後一次MBN誘導後停頓3(A組:89個腫瘤)及停頓7(B組:105個腫瘤)後犧牲,介於3天與7週的等待期,在免疫組織化學染色法測定腫瘤p53的表現從91 % 降至50 %;而分析兩組腫瘤,顯現p53表現子58序列改變之頻率亦非常相似(A5/15 = 33 % vs. B14/45 = 31 %)A組腫瘤p53免疫染色顯現出高度活性,可能是誘導後期因DNA受損而造成野生型p53的高度表現,且由於硝氨的特性,使p53突變接近80 %G:C → A:T的轉位突變。証實了p53基因大部份的點突變皆發生或接近在人類口腔鱗狀細胞癌熱點密碼子175245248273附近。A組和B組腫瘤Ha-ras基因的突變率分別為1/15 (7 %) 7/45 (16 %),同樣地,G:C → A:T的轉位突變亦佔了75 %。此外,端粒複序擴大法分析顯示,11/13 (85 %) A組腫瘤及23/24 (96 %) B組腫瘤有高度端粒A活性。在MBN誘發倉鼠頰囊癌模式下所觀察之p53Ha-ras 和端粒A活性的改變,與跟煙草誘發之人類口腔鱗狀細胞癌所得之觀察相似,此動物模式對於發展口腔癌化學治療和基因療法應可提供重要之參考策略。

   
 

High levels of p53 and Ha-ras expression are the most common molecular abnormaltis in oral squamous cell carcinomas (SCCa) and a large proportion of their precancerous lesions (PL). The length of telomere is critical for maintenance of genetic stability that could play an important role in carcinogenesis. In this study, squamous cell carcinomas induced in hamster buccal pouch (HBP) by 22 weeks of topical methylbenzylnitrosamine (MBN) treatment were evaluated for (a) altered expression of p53 using immunohistochemistry (IHC), (b) mutations in Ha-ras and p53 using PCR/single strand conformation polymorphism (SSCP), and (c) abnormal telomerase activity using telomerase repeat amplification protocol (TRAP). Hamsters were sacrificed for tumor analysis at either 3 days (Group A, 89 tumors) or 7 weeks (Group B, 105 tumors) following the final MBN application.  Between 3 days and 7 weeks post treatment, the proportion of tumors exhibiting p53 IHC activity (at least 10 % of nuclei stained using DO7 Abs for detection of both mutant and wild type p53) fell from 91 % to 50 %. However, during this same post-treatment period the frequency of analyzed tumors exhibiting confirmed sequence alterations in the conserved exons (E5E8) of p53 remained constant (5/15 = 33 % in Group A vs. 14/45 = 31 % in Group B). Heightened expression of wild type p53 resulting from DNA damage in the immediate post-treatment period, is likely to have contributed to the high proportion of group A tumors exhibiting p53 IHC activity. As is characteristic of this nitrosamine, nearly 80 % of the identified p53 mutations were G → A and C → T transitions. The identified p53 point mutations occurred at or near (within 3 codons) corresponding hot spot codons (175, 245, 248 and 273) of human oral SCCa. The proportion of analyzed Group A and Group B tumors exhibiting Ha-ras mutations was 1/15 (7 %) and 7/45 (16 %), respectively, with G → A transitions predominating (75 %). Telomerase activation was demonstrated in 11 of 13 Group A tumors (85 %) and 23 of 24 (96 %) Group B tumors analyzed. The alterations in p53, Ha-ras and telomerase activity observed in the HBP-MBN model, are similar in many respects to those observed in tobacco-associated human oral SCCa. This model may be useful for development of oral cancer gene therapy and chemoprevention strategies.