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Title:	Pencarian penanda biologi berpotensi untuk kanser kolorektum menggunakan pendekatan proteomik serum
Authors:	Lim Lay Cheng (P53449)
Supervisor:	A Rahman A Jamal, Profesor Datuk Dr.
Keywords:	Penanda biologi Kanser kolorektum Proteomik serum Dissertations, Academic -- Malaysia
Issue Date:	7-Jan-2015
Description:	Pengesanan awal kanser kolorektum (CRC) adalah sangat penting untuk memperbaiki prognosis penyakit. Walaubagaimanapun sehingga kini tiada penanda biologi dalam darah yang sensitif dan spesifik untuk diagnosis awal penyakit ini. Dalam kajian ini, pendekatan berasaskan-gel dan bukan berasaskan-gel digunakan untuk mengenalpasti penanda biologi berpotensi dalam CRC. Perbezaan pengekspresan protein dalam serum antara pesakit CRC peringkat awal (Dukes' A dan B) (n=8), CRC peringkat lewat (Dukes' C dan D) (n=8) dan kawalan normal (n=8) dikenalpasti dengan menggunakan kaedah elektroforesis gel pembezaan fluoresens dua dimensi (2D-DIGE). Analisis peta gel 2D dan kromatografi cecair spektrometer jisim bertandem (LC-MS/MS) menunjukkan lapan protein mengalami pengawalaturan meningkat dan dua protein mengalami pengawalaturan menurun pada CRC peringkat awal berbanding kawalan normal; manakala sebanyak 14 protein mengalami pengawalaturan meningkat dan empat protein mengalami pengawalaturan menurun pada CRC peringkat lewat berbanding kawalan normal (p≤0.05). Daripada analisis tapak jalan Ingenuity Pathway Analysis (IPA), lima protein iaitu apolipoprotein A1 (APOA1), apolipoprotein E (APOE), faktor komplemen H (CFH), galektin-7 (GAL7) dan sinaptojanin-2 (SYNJ2) telah dipilih untuk divalidasi menggunakan asai imunojerapan berpaut enzim (ELISA). Hasil menunjukkan APOA1, GAL7, APOE dan CFH menunjukkan keputusan yang konsisten dengan 2D-DIGE. Dengan berpandukan kepada fungsi dan keputusan kajian lepas, APOA1 dan GAL7 dipilih untuk divalidasi seterusnya pada tisu FFPE dengan kaedah imunohistokimia (IHC). Keputusan IHC menunjukkan imunoreaktiviti negatif untuk GAL7 pada tisu CRC. APOA1 menunjukkan imunoreaktiviti positif tetapi corak pengekspresannya tidak berkorelasi dengan peringkat Dukes' (χ2=4.86, p=0.30), gred tumor (χ2=1.19, p=0.88) dan status nodus limfa (χ2=4.46, p=0.11). Pengekspresan berbeza protein serum pada CRC telah dikenalpasti dengan menggunakan LC-MS/MS dan sequential window acquisition of all theoretic mass spectra-mass spectrometer (SWATH-MS) dengan set sampel yang berlainan di mana masing-masing telah menemui 21 dan 22 calon penanda biologi yang berpotensi untuk CRC (p≤0.05). Daripada keputusan yang diperolehi melalui 2D-DIGE, LC-MS/MS dan SWATH-MS, terdapat 11 protein yang sama telah diperolehi sekurang-kurangnya melalui dua kaedah pendekatan yang berbeza di mana ia adalah tergolong sebagai protein fasa akut. Corak autoantibodi (AAB) pada CRC telah dikenalpasti dengan sampel serum subjek CRC peringkat awal (n=5) dan kawalan normal (n=5). Didapati AAB terhadap osteoprotegerin (OPG), molekul adhesi sel leukosit teraktif (ALCAM/CD166) dan faktor pertumbuhan fibroblas asas (FGF-b) menunjukkan peningkatan aras pada CRC peringkat awal (p≤0.05). Kesimpulannya, perubahan pengekspresan APOA1 dan GAL7 pada sampel serum CRC berbanding kawalan normal dan keputusan imunoreaktiviti positif APOA1 pada tisu CRC menunjukkan potensi penggunaan APOA1 dan GAL7 sebagai penanda biologi untuk CRC. Perubahan corak pengekspresan panel 11 protein fasa akut dan peningkatan AAB terhadap OPG, ALCAM dan FGF-b dalam sampel serum CRC menunjukkan potensi penggunaanya sebagai penanda biologi komplementari untuk CRC.,Early detection of colorectal cancer (CRC) is vital for the improvement of disease prognosis. However to date there are no blood-based biomarkers sensitive and specific enough for early diagnosis. In this study, we used gel-based and non-gel-based approaches to identify the potential biomarkers in CRC. We analyzed the differences in serum protein expression between early stage CRC (Dukes' A and B) (n=8), late stage CRC (Dukes' C and D) (n=8) and normal controls (n=8) using the 2D Fluorescence Difference Gel Electrophoresis (2D-DIGE). Two dimensional gel maps and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis showed that eight proteins were up-regulated and two were down-regulated in early CRC when compared to normal controls; whereas 14 proteins were up-regulated and four proteins were down-regulated in late CRC when compared to normal controls (p≤0.05). From the pathway analysis using Ingenuity Pathway Analysis (IPA), five proteins, namely apolipoprotein A1 (APOA1), apolipoprotein E (APOE), complement factor H (CFH), galectin-7 (GAL7) and synaptojanin-2 (SYNJ2) were chosen and validated using enzyme-linked immunosorbent assay (ELISA). It was found APOA1, GAL7, APOE and CFH showed consistent findings with 2D-DIGE. Based on the protein functions and previous findings, APOA1 and GAL7 were chosen for further validation in FFPE tissues using immunohistochemical staining (IHC). Results showed negative immunoreactivity for GAL7 in CRC tissues. APOA1 showed positive immunoreactivity but its expression did not correlate with Dukes' staging (χ2=4.86, p=0.30), tumour grading (χ2=1.19, p=0.88) and lymph node involvement (χ2=4.46, p=0.11). We have analyzed differential expression of serum proteins in CRC with LC-MS/MS and sequential window acquisition of all theoretic mass spectra-mass spectrometer (SWATH-MS) by using different set of samples in which 21 and 22 candidate biomarkers were identified, respectively (p<0.05). From the 2D-DIGE, LC-MS/MS and SWATH-MS results, 11 similar proteins were identified with at least two of the different methods used for identification in which most of them comprised of acute phase proteins. In addition, autoantibody (AAB) signatures in CRC were identified by analyzing serum samples from early CRC (n=5) and normal controls (n=5). It was found AAB against osteoprotegerin (OPG), activated leukocyte cell adhesion molecule (ALCAM/CD166) and basic fibroblast growth factor (FGF-b) showed increased expression in early stage of CRC (p≤0.05). In conclusion, changes of APOA1 and GAL7 expression in the serum samples of CRC compared to normal controls and positive immunoreactivity of APOA1 in CRC tissues showed its potential as candidate biomarkers for CRC. Alterations of proteomic expression patterns of the 11 identified acute phase proteins and increased expression of AAB against the OPG, ALCAM and FGF-b in the serum samples of CRC showed its potential to be used as CRC complementary biomarkers.,PhD
Pages:	209
Publisher:	UKM, Kuala Lumpur
Appears in Collections:	Faculty of Medicine / Fakulti Perubatan

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