轉譯醫學生物標誌鑑定之研究：以大腸直腸癌和慢性阻塞性肺病為例

Abstract

鑑於疾病流行趨勢已由過去之急性與感染性疾病，轉變成需長期照護與衰弱失能性的慢性疾病為主。適當的生物標誌以運用於治療這些慢性疾病十分重要。本轉譯研究之主要目的，即為使用免疫蛋白質體學為基礎之相關實驗室技術以開發具臨床應用潛力的生物標誌，並利用臨床檢體來驗證其在大腸直腸癌及慢性阻塞性肺病之臨床處置的應用潛力。本研究的第一部分主要是探討 Phospholipid scramblase 1 (PLSCR1)蛋白於大腸直腸癌患者之組織與血漿中的表達狀況，並分析其表現量與各項病理參數的相關性。本研究使用免疫組織化學染色與西方墨點法等技術以分析PLSCR1在檢體中之濃度。結果顯示，相對於正常之大腸黏膜組織，PLSCR1 蛋白過度表達於惡性之腺癌組織 (p< 0.001)。此外，相較於健康對照組，大腸直腸癌患者血漿中之PLSCR1蛋白濃度亦明顯升高(p<0.001)。相同現象亦可發現存在於早期大腸直腸癌患者(p<0.001)。以PLSCR1作為偵測大腸直腸癌的指標，其靈敏度與專一性分別為80%與60%；ROC曲線下面積值為0.75。存活統計分析顯示PLSCR1表現量越高則大腸直腸癌患者之預後愈差。本論文的第二部份是有關慢性阻塞性肺病(COPD)的研究。先前研究指出自體免疫反應可能參與COPD的病理機轉，但此假說尚未充分獲得證明。因此，此部分研究主要目的是應用西方墨點法探討COPD患者血清中是否存在對抗肺臟細胞蛋白質的自體抗體，並利用免疫沉澱法、免疫螢光染色與質譜儀技術以鑑定出其相對應之自體抗原。同時也進ㄧ步分析了這些自體抗體的臨床應用性。研究結果顯示，76.0%的COPD患者與23.8%健康控制組的血清中存在對抗cytokeratin 18 (CK18) 蛋白之自體抗體 (p<0.001)。並且CK18自體抗體的濃度與COPD患者的FEV1 (L)值 (p=0.013)及FEV1 (%pred.)值(p=0.043)呈負相關。這些結果支持自體免疫作用參與COPD致病機轉的假說。結論：本研究結果顯示: (1) PLSCR1蛋白於惡性大腸腺癌組織中有過度表現的現象，而血漿中之PLSCR1蛋白具有發展成為大腸直腸癌之診斷與預後生物指標的潛力；(2)肺臟上皮細胞之CK18蛋白為COPD相關之自體抗原，且其相對應之自體抗體廣泛存在於COPD患者血清中。本論文研究有效地將實驗室所獲得有關疾病病理學的新知轉譯到臨床上，應用於開發有助於疾病控管之新的生物標誌。

The trend of disease is now shifting from more acute and lethal forms of disease to more long-term and debilitating chronic illness. The need of proper biomarkers for treatment of these chronic conditions is crucial. The aims of this translational study were to discover potential biomarkers by using immunoproteomic-based technologies in laboratory, then translated them into clinical by analyzing valid clinical samples to characterize their potential clinical benefit in the management of colorectal cancer (CRC) and chronic obstructive pulmonary disease (COPD). The first part of this study was aimed to examine the expression of phospholipid scramblase 1 (PLSCR1) in tumor tissues and plasma specimens of patients with CRC, as well as analyze its association with clinical parameters. To evaluate its potential clinical relevant, the levels of PLSCR1 were investigated by use of immunohistochemical and Western-blot analyses. Our results showed that PLSCR1 was overexpressed in malignant adenocarcinoma tissues compared with normal colorectal mucosa (p< 0.001). In addition, the plasma level of PLSCR1 was not only significantly elevated in CRC patients compared with healthy individuals (p<0.001), but it was also substantially increased in early stage CRC (p<0.001). Importantly, the overall sensitivity and specificity of PLSCR1 for CRC detection were 80% and 60%, respectively. The area under the receiver operating characteristic curve of PLSCR1 for CRC diagnosis is 0.75. The survival analysis revealed that elevated PLSCR1 expression indicated a poor prognosis for CRC. The second part of this study was focused on COPD. A mechanism involving the autoimmune reaction in the pathogenesis of COPD has been proposed but not confirmed. Therefore, we aimed to investigate whether serum autoantibodies against pulmonary cellular proteins are present in COPD patients by Western blot, and, if possible, to identify their counterpart autoantigens by use of immunoprecipitation, immunofluorescence staining and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry approaches. Meanwhile, the clinical potential of these autoantibodies were further examined as well. Results showed that autoantibodies against the cytokeratin 18 (CK18) protein was found in 76.0% of COPD patients and 23.8% of control subjects (p<0.001). Furthermore, the CK18 autoantibody level was significantly correlated with the FEV1 (L) (p=0.013) and FEV1 (%pred.) (p=0.043) values observed in COPD patients. These results support the hypothesis that humoral autoimmunity may be involved in the pathogenesis of COPD. Conclusion: Our results demonstrated that (1) PLSCR1 was overexpressed in malignant adenocarcinoma tissues and plasma PLSCR1 might be used as a noninvasive serological diagnostic and prognostic biomarker for CRC; (2) The pulmonary epithelial CK18 protein was identified as a COPD-associated autoantigen and its autoantibodies were prevalent in COPD patients. This study effectively translated the new knowledge of disease pathologies gained in the laboratory into the development of new biomarkers for management of disease.