篩選治療乳癌的標的基因及藥物

Screening of therapeutic target genes and drugs for breast cancer

阮晉弘(Chin-Hung Juan)

指導教授 ： 鄭添祿

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

本研究以功能性基因體學研發治療乳癌的蛋白質藥物。研究分為兩部分：1. 藉微矩陣列技術及生物資訊篩選出乳癌差異性表現之膜蛋白2. 以噬菌體胜肽呈現技術篩選治療乳癌之胜肽藥物。
關於第一部分的研究，主要是挑選出於乳癌細胞上可以作為藥物標靶的基因，膜蛋白為細胞接觸外來刺激的第一道關卡，若能控制細胞膜蛋白的功能，即可以直接控制細胞的生理。因此我們選定的標的蛋白是為乳癌專一性過度表現且位於細胞膜上的蛋白質；首先我們取三對乳癌組織檢體進行Microarray刪減性分析並從中篩選出乳癌高表現基因，進而以生物資訊軟體整合性地分析，我們找到18個候選膜蛋白基因，再以高醫基因體中心開發之呈色型尼龍膜陣列(colorimetric membrane array)，利用40個臨床檢體對此18個膜蛋白基因確認其在乳癌組織的高表現；目前我們已篩選出5個可作為藥物標靶的候選膜蛋白基因標的。為了更進一步證實這些基因可調控細胞生長，我們針對FLJ10525以及FLJ14624兩個未知功能的基因進行基因功能試驗，以RNAi基因默化技術，研究基因與癌細胞生長之相關性，發現基因在被默化後確實導致癌細胞生長速率明顯降低，推測此兩基因與腫瘤增生有密切相關，未來相當有潛力作為設計乳癌藥物的標靶膜蛋白。
第二部分以隨機性篩選的方式尋找治療乳癌之胜肽藥物，利用12mer phage display library技術對乳癌細胞篩選可抑制其生長的peptide drugs；從龐大的胜肽分子庫中，以逐次縮減族群的方式，篩選出選擇性地結合到乳癌細胞，並可進而抑制癌細胞生長的單株phage-peptide；利用此方式我們已成功地篩選出3株phage-peptide clone並定序，其中一株序列為HHEWTHHWPPPP，此12個胺基酸的小胜肽分子未來在測試polypeptides的選擇性及毒殺性後，未來相當有潛力作為抗乳癌之胜肽類藥物。

關鍵字

噬菌體 ； 乳癌 ； 噬菌體展現技術 ； 膜蛋白 ； 藥物 ； 胜?

並列摘要

This research is to develop protein drugs for breast cancer by the functional genomic study. The study divides into two parts: 1. Screening the beast cancer differentially expressed membrane proteins by microarray and bio-informatic method. 2. Screening Peptide drugs against breast cancer by phage-peptide display library.
The most important thing under the first part is to select the drug target membrane protein genes of breast cancer cell. The membrane protein is the first gate of receptor to outer stimulation. If we can manipulate the membrane protein, we can control cell physiology directly. For this reason, we choose membrane proteins specifically over-expressed in breast cancer. At first, we took 3 pairs of normal and breast cancer samples for microarray and subtraction analysis to screening over-expression genes, and by combining bio-informatic methods we select 18 candidate genes. And by processing colorimetric membrane array developed by Kaohsiung Medicine School Genomic Center, we confirm the eighteen genes over-expression probability in 40 breast cancer samples. For the moment we select 5 target genes that could be as drugs target. To prove these genes are associated to cancer cell growth, we proceeded gene function assays for the FLJ10525 and FLJ14624 two novel genes by RNAi technique and to study the correlation of gene and cancer cell proliferation. We discovered the cancer cell growth rate decline after gene silencing. And we conclude these genes are associated to tumor proliferation. For the reason, they will be as drugs target membrane protein in the future.
The second part of the study is to discover therapeutic peptide drugs for breast cancer by randomly selection method. We use 12mer phage display library to screen peptide drugs against breast cancer cell and to inhibit the cancer cell growth. From the huge variants of peptide library, we screened phages specifically bind to cancer cell and have anti-cancer cell proliferation ability. By this method, now we successfully screened 3 clone phages and sequenced, one of the discovered polypeptide sequence is HHEWTHHWPPPP, the 12mer polypeptide future may will potentially be a kind of peptide drug after processing selectivity and toxicity functional assays.

並列關鍵字

peptide ； drugs ； drug ； membrane protein ； breast cancer ； phage display ； phage

參考文獻

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