內質網壓力及calpain誘發類風濕性關節炎自體抗原之研究

前言︰內質網的功能是合成及修飾分泌性蛋白，並儲存大量鈣離子，細胞質的鈣離子濃度上升時可以活化訊息傳遞路徑及其他細胞功能。當內質網功能受到干擾時，內質網會產生內質網壓力 (ER stress) 反應。calpain 是中性蛋白酶家族，其活性受鈣離子濃度調控，其中calpain 2的活化需要高濃度 (mM) 的鈣離子，和細胞凋亡有關。本實驗室之前研究發現，內質網壓力加上細胞質鈣離子濃度上升會誘發HL-60細胞的calpain 2表現，而且和細胞凋亡有關。研究目的：類風濕性關節炎 (rheumatoid arthritis, RA) 是一種自體免疫疾病，有自體抗體的產生，免疫細胞過度活化，可能誘發內質網壓力，及calpain 2的表現，活化的calpain 2可能切割蛋白，形成新的自體抗原；calpain 2也可能參與細胞凋亡，當其活性受到干擾時，可能造成細胞凋亡反應不完全，因而曝露自體抗原。本實驗研究目的是利用HL-60細胞，模擬上述情形，以細胞的蛋白萃取物，來篩選RA病人的自體抗體。實驗方法︰HL-60細胞培養的條件如下：1. 對照組：基本條件 (不加任何藥物) 36小時；2. 實驗組1：加thapsigargin (TG) 36小時，誘發calpain 2表現及細胞凋亡；3. 實驗組2：加入thapsigargin (TG) 18小時後，加入calpain抑制劑PD150606 18小時，干擾細胞凋亡。之後將這三組細胞製備成蛋白質樣品以Westen blot分析，濾紙切成長條狀，分別和健康的人與病人的血清進行抗原抗體反應，篩選出可能的自體抗體。結果：我們發現健康組沒有而RA病患才有的自體抗原。實驗結果有以下四種情況： (1) 對照組、實驗組1、實驗組2皆有出現band，例如84 KDa (12/42)。(2) 實驗組1與實驗組2出現分子量相同的band，在42位病人只有65 KDa出現2次。其他52 KDa、56 KDa、71 KDa只出現一次。(3) 實驗組1有出現，而實驗組2沒有的band，在RA病人中並沒有重複出現的bands，只有140 KDa、83 KDa、50 KDa、63 KDa、40 KDa各出現一次。(4) 實驗組1沒有，而實驗組2有出現的band，例如47 KDa (20/42)。結論：本篇研究發現，在RA發病過程中，免疫細胞活化可能產生ER stress及鈣離子失衡，這過程會誘發calpain 2表現及細胞凋亡，細胞凋亡過程當calpain 2的活性受干擾時可能會產生自體抗原，這顯示calpain 2的表現與活性與類風濕性關節炎的發展是具有關聯性，未來也可以進一步研究calpain 2與其它自體免疫疾病的相關性。

關鍵字

內質網壓力

並列摘要

Background: The function of the endoplasmic reticulum (ER) is the synthesis and modification of secretory proteins, as well as storeage of large amounts of calcium ions. Elevated cytosolic [Ca2+] can activate the signal transduction pathways and other cellular functions. Disturbance of ER function may elicit unfoled protein responses (UPRs). Calpain is a neutral protease family whose activity is regulated by the concentration of calcium ions. Calpain 2 activity requires a high concentration (mM) of calcium and is associated with apoptosis. Previous studies in our lab showed that in HL-60 cells ER stress plus elevated cytosolic [Ca2+] induced calpain 2 expression and was associated with apoptosis. Aims of study: Rheumatoid arthritis (RA) is an autoimmune disease with the production of autoantibodies. Over-activation of immune cells may induce ER stresses and calpain 2 expression. Activated calpain 2 may cleave proteins to form new autoantigens and may also be involved in apoptosis. Disturbance of calpain activity may cause incomplete apoptosis and formation of autoantigens. The purpose of this study is to use cell culture of HL-60 cells to simulate the above situation, and to use the cell protein extract to screen autoantibodies in RA patients. Methods: HL-60 cell culture conditions are as follows: 1. Control group: the basic conditions (without any drugs for 36 hours); 2. Experimental group 1: thapsigargin (TG) treatment for 36 hours, induced calpain 2 expression and apoptosis; 3. Experimental group 2, TG treatment for 18h followed by treatment of the calpain inhibitor PD150606 for another 18h. Protein samples were separated on SDS-PAGE and then transferred to nitrocellose membranes. The membranes were cut into strips and incubated with sera from healthy people and patients to screen potential autoantibodies. Results: In the screeing of autoantibodies, we found 4 band patterns: (1) Bands appeared in control, experimental 1 and experimental 2, eg. 84 KDa (12/42). (2) Bands appeared in experimental 1 and experimental 2, eg. 65 KDa (2/42); 52 KDa, 56 KDa, 71 KDa only appeared once. (3) Bands appeared in experimental 1 but not experimental 2, eg. 140 KDa, 83 KDa, 50 KDa, 63 KDa, 40 KDa all appeared only once. (4) Bands did not appear in experimental 1 but appeared in experimental 2, eg. 47 KDa (20/42). Conclusions: In this study, we found that during the pathogenesis of RA overactivation of immune cells may produce ER stresses and loss of Ca2+ homeostasis, which induced calpain 2 expression and apoptosis. Disturbance of apoptosis pathway or the activity of calpain 2 may elicit autoantigens. The data showed that calpain 2 is highly correlated with rheumatoid arthritis. Further studies can be performed to investigate the association of calpain 2 with other autoimmune diseases.

並列關鍵字

calpain

參考文獻

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延伸閱讀

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內質網壓力及calpain誘發類風濕性關節炎自體抗原之研究

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