出版年:

研究生:

林士弘

研究生(英文姓名):

Shyh-Horng Lin

論文名稱:

分析鼻咽癌患者體內alphabeta T細胞受器的基因表現

英文論文名稱:

Analysis of alphabeta T-cell receptor gene expression in patients with nasopharyngeal carcinoma

指導教授:

楊倍昌

指導教授(英文姓名):

Bei-Chang Yang

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物及免疫學研究所

學年度:

90

語文別:

英文

論文頁數:

68

關鍵詞:

鼻咽癌 ; 腫瘤浸潤淋巴球 ; T細胞受器

英文關鍵詞:

nasopharyngeal carcinoma (NPC) ; tumor infiltrating lymphocyte (TIL) ;
T-cell receptor (TCR)

被引用次數:

0

[ 摘要 ]

鼻咽癌(nasopharyngeal carcinoma, NPC)是東南亞族群特有的疾病。此疾病的一個特徵是NPC腫塊之中有許多T細胞浸潤，但腫瘤仍繼續生長。這群T細胞的特性仍不清楚。為了研究腫瘤浸潤淋巴球(tumor infiltrating lymphocyte, TIL)當中T細胞受器(T-cell receptor, TCR)的基因使用情形(TCR usage)，所以用反轉錄聚合脢鏈反應(reverse transcriptase polymerase chain reaction, RT-PCR)，從TIL中分離TCR基因。此RT-PCR使用的2組退化引子(degenerate primer)，是依照多種TCR V基因片段(gene segment)之間的高度相似序列所設計，可以分別增幅多種V基因片段的alpha或beta基因，並且將RT-PCR產物直接植入TA-cloning載體。在20株TCRalpha和25株TCRbeta的質體之中，由其序列可確認有5種Valpha片段、15種Jalpha片段、10種Vbeta片段和8種Jbeta片段存在，此RT-PCR產物的組成涵蓋了TCRalpha和TCRbeta的V到C區段，及其中的3段互補決定區段(complementarity determining region, CDR)。每一條alpha片段或每一條beta片段之間，在DNA序列上及胺基酸序列上皆不相同。序列的資料指出，在NPC腫瘤組織之間浸潤的T細胞，其來源具有高度變異性(polyclonal)。本實驗利用微陣列(microarray)技術，將85株TCRalpha及93株TCRbeta的質體DNA，分別點在尼龍膜(nylon membrane)上，然後用質體或cDNA進行PCR放大TCR基因，其產物標記上[32P]作為TCR探針。使用單一株TCR質體製備TCR探針進行DNA雜交(hybridization)，結果顯示，探針上特定V或J片段之間的差異，可以造成截然不同的訊號。分離TIL及NPC患者、正常成人和氣喘患者的周邊血液單核細胞(PBMC)，以其cDNA製備TCR探針進行DNA雜交，得到的訊號樣式均很相似。這些結果指出，NPC患者體內的T細胞，不論位於周邊血液或是TIL之內，都沒有改變TCR的使用。

[ 英文摘要 ]

Nasopharyngeal carcinoma (NPC) is a characteristic disease in population in South-eastern Asia. Although there are many T cells infiltrating in NPC mass, tumor still develops. The property of these T cells is not clear yet. To study T-cell receptor (TCR) repertoire usage in these tumor infiltrating lymphocytes (TIL), the TCR genes in the TIL were isolated by reverse transcriptase polymerase chain reaction (RT-PCR) using 2 sets of degenerate primers based on the conserved TCR V genes, that can amplify alpha and beta genes of wide range V gene segments, respectively. The PCR product was directly cloned in TA-cloning vector. 5 Valpha segments, 15 Jalpha segments, 10 Vbeta segments, and 8 Jbeta segments have be identified in sequences of 20 TCRalpha and 25 TCRbeta clones, and the PCR product spanned V to C region of alpha and beta chain containing 3 complementarity determining regions. Each alpha fragment was different in DNA sequence and in amino acid sequence, so was beta. Sequence data indicated that polyclonal T cells infiltrated into tumor tissue of NPC. Plasmid DNA of 85 TCRalpha clones and 93 TCRbeta clones were arrayed onto nylon membrane with microarray technique. TCR genes were amplified with PCR from plasmid DNA or cDNA, and the PCR products were labeled with [32P] as TCR probes. The TCR probes were used in DNA hybridization. With TCR probes from plasmid DNA with single TCR clone, hybridization results showed distinct signals for probes containing particular V or J segment. Using TCR probes derived from cDNA of TIL, peripheral blood mononuclear cells (PBMC) of NPC patients, normal adults, and asthma patients, similar signal patterns in hybridization were observed among these groups. These results indicated that TCR repertoire was not changed in PBMC and TIL in NPC patients.