應用蛋白質體學技術鑑定肺癌新腫瘤標記

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應用蛋白質體學技術鑑定肺癌新腫瘤標記

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在數位參與台灣國家衛生院免疫治療法 ( Immunotherapy ) 之癌症末期病人中，有一位病人獲得顯著的緩解。本研究乃針對該免疫療法成功之肺癌末期患者案例，利用蛋白質體學配合西方墨點法來探測免疫療法引發的體液免疫所產生之特異性抗體的可能抗原。我們以患者在接受免疫法治療前與治療 77 天後所採得之血清做為免疫前及免疫後抗體，以 A549 腺肺癌細胞株培養後萃取細胞蛋白質做為癌細胞抗原，經過 2D-SDS-PAGE 蛋白質分析後，以西方墨點法研究肺癌患者治療前、治療後所產生的抗體在識別 A549 腺肺癌細胞所萃取之細胞蛋白質有何不同。我們發現肺癌患者在治療 77 天後所獲之恢復期血清可辨識四個 A549 腺肺癌細胞萃取液之特殊蛋白質點，經過一連串蛋白質塊切割，膠內酵素切割萃取蛋白質成分，再經高效能 MALDI- TOF 質譜儀分析，透過 NCBI 蛋白質資料庫比對得知此四個蛋白質點分別是 β-actin 、 Human Interferon-induced protein with tetratricopepti de repeats 2 (IFIT-2) 、 keratin 與 Wnt-8a 等四個蛋白質。本研究之結果顯示經進一步研究後，這四個蛋白質將可能當作新的癌症生物指標，亦可嘗試發展成為癌症疫苗之用。

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Proteomics-based Identification of Novel Biomarkers in Lung Cancer

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Several end stage lung cancer patient have been recruited to accept newly develope

d immunotherapy in a NHRI clinical trial project. One of the subjects revealed rem

arkable remission after immunotherapy. We hypothesized that the immunotherapy

has elicited both cellular and humeral immune responses. In the present study, we u

sed a proteomic approach to identify proteins that induce a humeral immune respon

se in lung cancer after immune therapy. Two-dimensional polyacrylamide gel electr

ophoresis ( 2-D PAGE ) was used to separate several thousand cellular proteins in

A549 lung cancer cells. Separated proteins are transferred onto membranes and im

muno blot with sera obtained from patient before immunotherapy and 77 days post

immunotherapy using Western blot analysis. Proteins that differentially react with t

he post-immune serum ( but not with pre-immnue sera ) were identified and excise

d. Four candidate protein spots were identified and subjected to in-gel digestion and

matrix assisted laser desorption ionization — time of flight ( MALDI-TOF ) analysi

s. Our data revealed that -actin, human interferon-induced tetratricopeptide repeats 

2 ( IFIT 2 ) , keratin and Wnt 8a were potential tumor antigens that elicited humeral

immune response in the clinical trial initiative. Thus, these proteins can be consider

ed as potential tumor markers that can be used in clinical laboratory. Furthermore, t

hese results may be used in future development of tumor-vaccine.