2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38040Apoptosis is an important mechanism to remove self-reactive and unwanted cells. Defects in apoptotic processes or in phagocytotic clearance of apoptotic cells will result in either immune assault of target tissues by self-reactive lymphocytes or exposure of modified proteins which are potential autoantigens from apoptotoic cells. Both are possible mechanisms that lead to the development of autoimmune diseases. Apoptosis is stimulated by extracellular and intracellular stimuli, and regulated by different caspase pathways: (1) in cell membrane via receptor-associated caspase 8, (2) in nucleus via p53 and its downstream Bax, (3) in mitochondria via caspase 9, and recently identified (4) in endoplasmic reticulum (ER) via caspase 12. ER is an important site for biosynthesis and modification of serectory and membrane proteins. In cells that actively produce secretory proteins, e.g. in pancreatic β-cells producing insulin and in activated lymphocyte producing cytokines and chemokines, accumulation of unfold proteins will stimulate unfolded protein responses (UPRs), including (a) increased expression of chaperon and proteasome proteins to assist folding and degradation of malfolded proteins (b) induction of caspase 12 gene expression and activation of ER-related apoptotic pathways (c) attenuation of protein synthesis. In the present study we plan to investigate expression of genes and proteins (including modified proteins) that are induced during ER stress-mediated apoptosis. For this we will use tunicamycin (inhibits protein glycosylation) and brefeldin (inhibits disulfide bond formation) to treat differentiated HL60 cells and use oligonucleotide microarray to screen ER stress-related genes in these cells. Using specific inhibitors or small interference RNA (siRNA) we will study the signal transduction pathway that regulate gene expression, and study the outcomes of treated cells in the absence of certain genes e.g. CHOP or caspase 12. The findings from in vitro study will be further extended to study in RA and SLE patients. We will analyze mRNA level of ER stress-related gene CHOP, GRP78 and PDI by using real time PCR and search possible DNA polymorphisms that are associated with these diseases. In in vitro protein studies, we will use 2D PAGE to study protein expression and modification profile in cultured HL60 cells. These proteins will be identified and their function studied. The possibility of these modified proteins as potential autoantiges will be proved in the screening the sera from RA and SLE. The data of in vitro study (gene and protein expression profiles), and human studies (autoantibodies, DNA polymorphisms) are valuable information for study the autoimmune diseases. They will be stored in the database of the core center (Autoiimmune Disease Center) for systemic comparison. Our collected DNA, sera, and plasma samples from RA and SLE patients will be valuable tools that can be utilized by other projects.免疫耐受性的調控出現問題是自體免疫疾病重要的致病機轉之㆒，細胞凋亡(apoptosis)則是維 持免疫耐受性的方式之㆒，細胞凋亡不完全使得殘留的self-reactive 免疫細胞可能對組織細胞 進行的殺傷（例如第㆒型糖尿病T1DM），而細胞凋亡過程㆗引起蛋白質修飾變化，堆積結合 形成具有抗原性的分子，若是不及時由吞噬細胞清除可能引起死亡細胞崩解並釋放出抗原分 子，免疫複合物會造成全身性堆積（例如紅斑性狼瘡SLE）。如果能清楚的了解在不同自體免 疫疾病㆗引起細胞凋亡的原因及調控，將有利我們更進㆒步了解致病機轉，以及藥物的研發。 細胞凋亡可經由不同的藥物刺激，可引發不同的死亡路徑，包括（1）細胞膜接受器-caspase 8 （2）細胞核-p53 及其㆘游Bax，（3）粒線體-caspase 9，以及最近幾年發現的（4）內質網 -caspase12。內質網是細胞合成及修飾分泌性蛋白例如 β細胞之胰島素、免疫細胞之細胞激 素、趨化因子或細胞膜蛋白例如接受器、MHC 分子，以及調控鈣離子的重要胞器。當這些功 能調控失常就會引發內質網病變反應（ER stress responses）包括（a）引發基因表現，（b）蛋 白質合成降低，（c）加速異常蛋白的分解，及（d）細胞凋亡（ER stress-mediated apoptosis）。 內質網病變引起的細胞凋亡主要靠caspase 12 及 CHOP 所調控。 本研究的兩大方向﹕ （A） 探討內質網病變所引起的基因表現﹕ • 利用基因晶片來大量篩選培養之HL60 細胞在內質網病變所造成基因的改變，並利用抑制 劑（p53, NFκB, MAP kinases）或專㆒性干擾RNA（small interference RNA，簡稱siRNA） 來分析基因表現訊號傳遞路徑。 • 比較內質網病變及其他刺激所引發的caspase 路徑及其調控的機制，及研究缺少特定基因 表現時（利用siRNA 抑制caspase 12 及CHOP 等）對細胞凋亡以及基因表現所造成的影 響。 • 利用Real time PCR 分析在SLE 及類風濕性關節炎（RA）病患和內質網病變有關的基因 表現（包括CHOP、GRP78、 PDI、 caspase 12 等）。 • 尋找和內質網病變有關的基因之多型性，並探討和疾病的相關性 （B） 探討內質網病變所引起的蛋白質表現及修飾 • 利用2D PAGE 蛋白質電泳研究培養細胞的蛋白質表現及修飾，鑑定及分析這些蛋白質 • 利用ELISA 研究病患血清和內質網病變有關的蛋白質表現量 • 利用ELISA、Western Blot 技術及病患血清研究這些修飾後的蛋白質是否具有抗原性。 這兩大方向均包含in vitro 細胞培養及臨床病患基因及生化分析，這些資料將存放在總計劃的 資料庫，供其他子計劃成員利用，並可作系統性的比較。本研究也包含病患檢體之收集（DNA、 血清、血漿），對總計劃的樣品庫的建立也有顯著的貢獻，這些樣品也可應用在其他子計劃的 研究。內質網細胞凋亡自體免疫疾病基因晶片autoimmunityER stressapoptosisoligonucleotide microarrayProject