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研究生:賴國筑
研究生(外文):Kuo - Chu L ai
論文名稱:頭頸部癌症病人放射治療前後HPRT基因突變頻率之研究
論文名稱(外文):The Study of Mutant Frequency at HPRT Locus in Patients with Head and Neck Cancers Before and After Radiotherapy
指導教授:黃正仲黃正仲引用關係
指導教授(外文):Jeng - Jong Hwang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:放射醫學科學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:60
中文關鍵詞:淋巴球細胞頭頸部癌
外文關鍵詞:HPRTlymphocyteHead and Neck Cancers
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摘要
頭頸部癌症在亞洲地區的發生率很高,幾乎佔所有癌症一半的趨勢,在台灣罹患癌症死亡人數中佔約8%(每年有約2,400左右的患者死亡)。所謂頭頸部癌症包括發生在副鼻竇、鼻咽、鼻腔、口腔、口咽、下咽、喉、及唾液腺等部位的癌症。其中以口腔癌為最多,其次為鼻咽癌,而頭頸部癌的治療方式以放射治療為主。因此本研究主要是比較頭頸部癌病人單獨接受放射治療或放射治療合併化學治療前後其周邊血液淋巴球之HPRT基因的突變頻率是否有差異。 本研究利用細胞群落(colony)形成法及免疫螢光染色法(immunofluorescence staining)來偵測癌症病人接受放射治療前後HPRT基因的突變頻率。
實驗結果發現不論是以細胞群落形成法或以免疫螢光法所得到的HPRT 基因的突變頻率,兩種不同技術所得的結果相似[(2.32±1.21) ×10-5及(2.51±1.94) ×10-5]。癌症病人在接受放射治療前免疫螢光法求得HPRT 基因突變頻率平均值為(3.37± 2.14)× 10-5。比較癌症病患在治療前、治療期間的HPRT 基因突變頻率發現,頭頸部癌症病患在接受放射治療期間或治療後45%(9人中有5人)病患其HPRT基因突變頻率在接受治療期間HPRT基因突變頻率比治療前有顯著增加(P<0.01),78%(9人中有7人)接受治療後HPRT基因突變頻率比治療前有顯著增加。而在整個療程結束後,除了6號病患其HPRT 基因突變頻率並沒有增加,其餘病患在治療結束後其HPRT 基因突變頻率增加倍數從3倍至13倍,其平均增加約有6倍。

Abstract
The head and neck cancers, include tongue, larynx, pharynx, tonsils and nasopharyngeal carcinoma, are common diseases in Asia. In Taiwan, about 8% of cancer mortality, i.e. 2400 people each year, belongs to the head and neck cancer. In this study, colony and immunoflurescent staining assays were used to detect mutant frequency and molecular characterization of the HPRT gene in lymphocytes of patients with head and neck cancers before and after radiotherapy.
No significant difference was found for HPRT variant frequency (Vf) measurement between the colony assay and the immunofluorescence staining assay (P=0.754). A mean variant frequency of (3.37±2.14) ×10-5 of the HPRT gene in patients before radiotherapy. The variant frequency of HPRT gene is significantly increased in five patients (45%) during treatment, and in seven patients (78%) after treatment. The HPRT Vfs of patients, were elevated 3 folds to 13 folds, respectively, (p<0.01) at the end of radiotherapy. These data suggest that the HPRT variant frequency of head and neck cancer patients was increased about 6 folds after radiotherapy.

目錄
致謝辭………………………………………………………..I
中文摘要………………………………………………………....II
英文摘要……………………………………………………….....IV
縮寫表………………………………………………………....V
壹、前言…………………………………………………….1-18
一、頭頸部癌…….…………………….……………………1
二、化療藥物—cisplatin在頭頸部癌症治療的角色…………2
三、游離輻射引起的基因變異………….……………………3
四、T淋巴球細胞…………………………………………5
五、放射治療對細胞的傷害……………………….……5
六、次黃嘌呤-鳥糞嘌呤磷酸核醣轉化(HPRT)……………….7
七、HPRT基因突變頻率分析之應用………………………….9
(一)職業暴露引起細胞HPRT基因傷害………………..10
(二)年齡、抽煙習慣對正常人的影響………………..……11
(三)受到輻射意外暴露後生還者的基因傷害………………12
(四)癌症患者接受放射治療或化學治療前後HPRT基因突變頻率的差異…………………………...…………………14
六、鹼基變異分類………………………………………………16
七、總結…………………………………………………………17
貳、研究目的………………………………………………………………19
參、材料與方法…………………………..………………………….…20-25
一、研究對象……………………………………...…………….20
二、分離淋巴球及體外培養…………………………………20
三、HPRT基因突變頻率分析……………………………………..21
四、免疫螢光染色法……………………………………….22
五、統計方法……………………………………………………….25
肆、研究結果……………………………………………………………26-33
一、放射治療前HPRT基因突變頻率………………………………26
二、放射治療前、期間、後HPRT基因突變頻率的差異……….…26
三、單獨接受放射治療及放射治療合併化學治療HPRT基因突變頻率的比較…………………………………………………………31
四、細胞群落形成法所得HPRT基因突變頻率結果………………32
五、細胞群落形成法與免疫螢光標定染色法所得HPRT基因突變頻率的差異………………………………………………32
伍、討論……………………………………………………..…34-40
一、放射治療後淋巴球細胞所接受照射的劑量……...……………34
二、放射治療前的HPRT基因突變頻率……………………………34
三、將本實驗中所得的結果和之前發表過的報告作一比較……35
四、HPRT基因突變頻率與白血球數目、淋巴球比率之關係…….36
五、體內及體外照射1.5Gy HPRT基因突變頻率………………….37
六、活體內細胞HPRT基因突變所需要的時間表現………………37
七、細胞群落形成法及免疫螢光染色標定法之比較……………38
八、結論……………………………………………………………39
陸、圖表說明
圖一、HPRT基因突變頻率 (mutant frequency) 分析之流圖...………..41
圖二、免疫螢光染色法分析之流程圖……………..……….…….42
圖三、10位癌症病人病人接受放射治療前、期間及結束HPRT 基因的突變頻率…….…………………………………………………..…..43
圖四、BrdU、一次抗體及二次抗體之間的原理及在螢光顯微鏡下觀察的情況……………………………………………………………….....44
表一、十位頭頸部癌症病患的資料及放射治療前HPRT基因突變頻率…………………………………………………………………….45
表二、十位頭頸部癌症病患接受放射治療前、治療期間及治療後HPRT基因突變頻率……………………………………………...………..46
表三、單獨放射治療(RT)或放射治療合併化學治療(RT + CT)之HPRT基因突變頻率………………………..……………………………...47
表四、利用細胞群落形成法所得到HPRT基因突變頻率……………..…48
表五、比較Colony assay與Immunofluorescence staining assay 所得HPRT基因突變頻率的差異…...………………………………………..…49
表六、本研究結果和之前發表過的結果作一比較……………….………50
表七、二號病患在接受放射治療前、期間及結束後HPRT基因突變頻率與白血球數之關係…………………………….………………….51
柒、參考文獻…………………………………………………..…….52-60
捌、附錄

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