一、利用蛋白質體學探討氧化壓力、抑制Src激酶和榭黃素對大鼠心肌細胞的影響:建立心臟缺血再灌流以及治療的細胞模組 二、利用蛋白質體學分析可利用於疾病中的生物標記蛋白

PART І. 利用蛋白質體學研究大鼠心肌細胞在氧化壓力、酪氨酸激酶抑制子以及榭黃素的影響: 建立心臟缺血再灌流和治療的細胞模型心肌缺血再灌流的氧化壓力傷害，容易藉由蛋白質磷酸化的變化來調控基因之表現和代謝，進而影響細胞貼附與存活能力。在本研究中，受到過氧化氫刺激後的大鼠心肌細胞是用來當作心臟缺血再灌流所導致傷害的細胞模組，且經由氧化壓力刺激細胞後，細胞內的蛋白質上產生大量磷酸化的酪胺酸。所以，為了純化氧化壓力所誘導的酪胺酸磷酸化蛋白，我們使用親和性樹脂來得到酪胺酸磷酸化蛋白質，進而利用液相串聯式質譜儀來鑑定磷酸化酪胺酸蛋白質。根據質譜儀分析結果發現約23種蛋白質其功能參與細胞間傳遞、維持細胞骨架以及細胞貼附能力；由此可知，氧化壓力對於細胞貼附能力，型態和存活影響甚鉅，經由STRING網站分析得知Src激酶為這些蛋白質的上游調控者。進一步研究發現，我們利用免疫螢光染色法、螢光偵測流式細胞儀和細胞貼附計數法來證實過氧化氫致使細胞不具貼附能力並走向凋亡，然而前處理固定量的PP1或榭黃素後可保護過氧化氫對細胞所造成的傷害。而榭黃素透過抑制STAT3活性來阻礙過氧化氫所導致細胞發炎反應，藉此有效避免心肌細胞受到缺血再灌流的傷害。綜合以上結果，在氧化壓力下所誘導蛋白質磷酸化以及細胞傷害中Src激酶扮演著重要角色，並且使用Src激酶的抑制劑以及榭黃素可有效避免心臟因缺血再灌流所導致的傷害。 PART П. 利用蛋白質體學分析可利用於疾病中的生物標記蛋白在現今社會裡，糖尿病和癌症的發生是相當普遍且為國人十大死因。早期疾病的發現、手術治療與術後治療可以提升疾病患者存活率。在本論文中，收集代謝性疾病裡包含糖尿病引起的肢體末梢缺血、第一型糖尿病、或移行性膀胱癌和子宮肌瘤病患的血清，進而利用螢光標定二維電泳結合基質輔助雷射脫附游離飛行質譜儀來分析鑑定，並找出相關生物標記蛋白質。從本研究中，特定疾病血清中具有其高度表現的特有生物標記蛋白，例如糖尿病引法末梢肢體缺血病患中所引發的DAPP1、第一型糖尿病患中的血紅素、膀胱癌病患中的硒半胱胺酸專一的延長因子和子宮肌瘤患者中的維生素D鍵結蛋白；特別的是，在第一型糖尿病患中，血紅素呈現氧化狀態。但是大部分鑑定出來的血清蛋白幾乎都和免疫反應以及血液凝結相關的常見蛋白質。為了避免這些血清在血清中大量蛋白質的干擾，所以我們利用細胞來找尋生物標記蛋白。在本論文中，使用胰臟癌細胞和乳癌細胞來尋找關於引發抗藥性和癌化的生物標記蛋白。在gemcitabine所誘導的抗藥性胰臟癌細胞裡含有大量核糖核苷二磷酸還原酶而且胰臟癌抗藥性的發生可能和腫瘤抑制子p53有關，而在高度癌化的乳癌細胞中，粒腺體大量表現出鈣結合線粒體載體蛋白SCaMC-1，進一步，SCaMC-1被證實在乳癌病患血清中也有較高含量。從此論文得知，從血清中找尋可偵測或治療疾病的生物標記蛋白仍是相當困難，但另一方面，先從細胞平台上獲得具疾病專一性的標記蛋白質後，再進一步利用病患血清來確認哪些生物標記蛋白是可被用於疾病篩檢與治療。

關鍵字

蛋白質體學；蛋白質磷酸化；缺血再灌流傷害；榭黃素；大鼠心肌細胞；氧化壓力；肢體末梢缺血；糖尿病；移行性膀胱癌；氧化還原蛋白質體學；子宮肌瘤癌；健擇；胰臟癌；乳癌；粒線體

並列摘要

PART І. Proteomics study of oxidative stress, Src kinase inhibition (PP1) and Quercetin in H9C2 cardiomyocytes: a cell model of heart ischemia reperfusion injury and treatment. Oxidative stress production of myocardial ischemia/reperfusion injury leads to protein phosphorylation in regulating gene expression, metabolism, cell adhesion and survival. In this thesis, we used hydrogen peroxide treatment of H9C2 rat cardiomyocytes as a model of oxidative stress in heart ischemia reperfusion injury. A proteomics approach using anti-phosphotyrosine affinity purification and LC-MS/MS was then used to identify the stress-induced protein phosphorylation. We showed that oxidative stress induces a robust tyrosine phosphorylation of multiple proteins in this cell type. Most of identified tyrosine phosphorylated proteins were relative to cell-cell junctions, the actin cytoskeleton and cell adhesion. This suggested that oxidative stress may have a profound effect on intercellular connections and the cytoskeleton to affect cell adhesion, morphology and survival. After stress-induced phosphotyrosine proteins were analyzed by STRING, Src kinase was shown to be a major upstream regulator of these events. Furthermore, immunofluorescence studies, fluorescent activated cell sorting and cell-based assays were used to demonstrate H2O2-induced modifications of cell adhesion structures and cytoskeleton, de-adhesion and apoptosis, which were reversed by treatment with the Src kinase inhibitor PP1 or quercetin. Moreover, quercetin likely blocked the H2O2-induced inflammatory response through STAT3 modulation, which also contributed in preventing ischemia/reperfusion injury in cardiomyocytes. These findings provide the critical role of Src kinase in oxidative stress-induced phosphorylation and cell damage in cardiomyocytes and suggested that targeting Src kinase or quercetin may be an effective strategy for preventing ischemia reperfusion injury in the heart. PART П. The application of Proteomics for disease biomarker discovery Cancer and diabetic are high incidence and mortality in worldwide; however, early detection, surgical resection and postoperative therapy can lead to survival improvement for cancer or diabetes. In recent study, body fluids of patient were used to screen markers, such as plasma, urine and cerebrospinal fluid. Here, plasma of critical limb ischemia (CLI), type 1 diabetic (T1DM), transition carcinoma cancer and uterine leiomyoma were collected and analyzed by 2D-DIGE and MALDI-TOF. Then, particular protein markers were found in specific diseases, such as dual adapter for phosphotyrosine and 3-phosphotyrosine and 3-phosphoinositide (DAPP1) in CLI, hemopexin in T1DM, selenocysteine-specific elongation factor in TCC and vitamin D-binding protein in uterine leiomyoma. Nevertheless, most identified plasma proteins are related to inflammatory responses and blood coagulation. Therefore, a cell-based platform was established to screen protein markers relating to gemcitabine (GEM)-induced drug resistance pancreatic cells and tumorigenic breast cells. In GEM-induced drug resistant pancreatic cells, ribonucleoside-diphosphate reductase large subunit significantly overexpressed and tumor suppressor protein p53 may interplay with GEM-induced pancreatic cell resistance. In addition, in breast cancer cells, the level of calcium-binding mitochondrial carrier protein SCaMC-1 in tumorigenetic breast cancer cells or breast cancer patients’ plasma was higher than that of normal cell or health donors’ plasma. These data demonstrate that plasma proteomics provides a lot of common proteins between various diseases, but a cell based strategy provides a good platform for specific protein markers discovery in particular disease and afterwards these protein markers are potential for disease screening.

並列關鍵字

Proteomics ； Protein phosphorylation ； Ischemia-repurfusion injury ； Quercetin ； H9C2 cardiomyocyte ； Oxidative stress ； Critical limb ischemia ； Diabetic ； Transition carcinoma cancer ； Redox proteomics ； Uterine leiomyoma ； Gemcitabine ； Pancreatic cancer ； Breast cancer ； Mitochondria

參考文獻

1. Takano, H., et al., Oxidative stress-induced signal transduction pathways in cardiac myocytes: involvement of ROS in heart diseases. Antioxid Redox Signal, 2003. 5(6): p. 789-94.

2. D'Autreaux, B. and M.B. Toledano, ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol, 2007. 8(10): p. 813-24.

3. Scandalios, J.G., The rise of ROS. Trends Biochem Sci, 2002. 27(9): p. 483-6.

4. Rhee, S.G., et al., Cellular regulation by hydrogen peroxide. J Am Soc Nephrol, 2003. 14(8 Suppl 3): p. S211-5.

5. Corcoran, A. and T.G. Cotter, Redox regulation of protein kinases. FEBS J, 2013. 280(9): p. 1944-65.

國際替代計量

一、利用蛋白質體學探討氧化壓力、抑制Src激酶和榭黃素對大鼠心肌細胞的影響:建立心臟缺血再灌流以及治療的細胞模組二、利用蛋白質體學分析可利用於疾病中的生物標記蛋白

全文下載

主題瀏覽