探討 Pyrrolidine Dithiocarbamate 影響人類臍帶靜脈內皮細胞株與初級細胞對基質金屬蛋白酵素活化的作用與機轉差異

1. 探討Pyrrolidine Dithiocarbamate影響人類臍帶靜脈內皮細胞株與初級細胞對基質金屬蛋白酵素活化的作用與機轉差異 • 基質金屬蛋白酵素(matrix metalloproteinases, MMPs)為一種能夠分解細胞外基質與結締纖維的蛋白水解酵素，因而對於組織之結構重組、修補與破壞都扮演相當重要之角色。同時MMPs的含量與活性表現受到許多方式嚴密地調節控制。許多文獻指出，類風濕性關節炎的軟骨組織不正常破壞或粥狀動脈血管斑塊組織的剝離以及癌細胞的生長與惡性轉移皆與異常基質崩解作用有關，其主要原因源自相關細胞產生及釋放大量MMPs所致，諸如血管內皮細胞、癌細胞、單核球或巨噬細胞等。一般而言，發炎性細胞激素以及生長因子等，均會刺激細胞表現MMPs基因及其酵素蛋白之生合成。本實驗選取兩種內皮細胞以探討模擬體內在發炎成分作用下，刺激內皮細胞產生基質金屬蛋白酵素的機轉及比較初級細胞與細胞株之間的特性差異與藥物反應。首先在大規模中藥材萃取物及化學合成等藥物成分篩選實驗下，本實驗室發現Pyrrolidine dithiocarbamate (簡稱 PDTC)具有影響MMPs活性的作用，此藥物目前被認定為是一種抗氧化劑、金屬螯合劑和Nuclear factor-B (NF-B)抑制劑。於是利用人類臍帶靜脈內皮細胞之細胞株ECV304與初代培養的人類臍帶靜脈內皮細胞(human umibilical vein endothelial cell, HUVEC)為實驗細胞，先以tumor necrosis factor- (TNF-)與phorbol-12-myristate-13-acetate (PMA)為刺激劑處理。在ECV304細胞中，利用電泳酵素分析法(gelatin zymography)，發現TNF-和PMA皆可明顯刺激誘導出細胞MMP-9的活性表現，而不同濃度PDTC會有意義地促進以TNF-或PMA誘導的MMP-9活性，尤以濃度20 M時促進效果最佳。同樣地在西方點墨法(Western blot)與反轉錄-聚合酵素鏈鎖反應(Reverse transcription-polymerase chain reaction, RT-PCR)實驗中，得知以TNF-誘導細胞內的MMP-9蛋白質與mRNA均有增加，故證實藥物作用在細胞內MMP-9蛋白質表現，且更深入到影響細胞轉錄(transcriprion)之層面。於是進一步來探討PDTC在訊息傳遞中作用機轉的方式。根據實驗的結果得知，此PDTC藥物可能經由c-Jun-NH2-terminal kinase (JNK)和phosphatidylinositol 3-kinase (PI-3K)的訊息傳遞路徑來達到促進TNF-誘導的MMP-9蛋白質表現。然而在HUVEC細胞方面，在電泳酵素分析法(gelatin zymography)中，發現PMA可誘導MMP-2的活化態表現，TNF-則否，在加入不同濃度PDTC後，依藥物濃度效應有意義地抑制PMA誘導的MMP-2活性，且在西方點墨法(Western blot)實驗中也發現細胞內以PMA誘導的MMP-2有減少。PDTC在此初代細胞之訊息傳遞中的作用機轉，推測可能經由protein kinase C (PKC)此訊息傳遞路徑所導致的。綜合以上實驗結果顯示，ECV304和HUVEC細胞在MMPs的表現上有相當程度的差異，不論是對刺激劑或藥物PDTC的反應皆不相同，因其藉由不同的機轉而造成的。兩者細胞的特性不盡相同，此點可供日後研究時在選擇細胞與藥物處理之參考。

2. Investigation of the Different Effects and Mechanisms of Pyrrolidine Dithiocarbamate on Matrix Metalloproteinases Activation between ECV304 and HUVEC Cells • Matrix metalloproteinases (MMPs) can catalyze and degrade extracellular matrix (ECM), including ground substances and connecting fibers, which have their function to maintain tissue structure. Thus, MMPs play several important roles in tissue remodeling, repairing and destroys. The levels and activities of MMPs are strictly regulated and controlled in various ways. Many evidences indicate that human endothelial cells, cancer cells, monocyte and macrophages synthesize and secrete several MMPs which participate in the degradation of ECM components in rheumatoid arthritis tissues or atherosclerosis or during cancer growth and metastasis. In general, inflammatory cytokines and several growth factors can stimulate MMPs gene expression and biosynthesis. • In our study, we used two endothelial cells to investigate the mechanisms of MMPs production from stimulated endothelial cells which inflammatory mediators affect and look for the difference of characters and drug responses between the endothelial cell and endothelial cell line. However, according to previous experiments, we found Pyrrolidine dithiocarbamate (PDTC), regard as an antioxidant、a metal chelator and NF-kB inhibitor so far. Thus, we used spontaneously transformed human umbilical vein endothelial cell line (ECV304) and primary human umbilical vein endothelial cell (HUVEC) as our experimental cells and then treated tumor necrosis factor- (TNF-) and phorbol-12-myristate- 13-acetate (PMA) (10 ng/ml) as stimulators. By using gelatin zymography analysis, we found TNF- and PMA (10 ng/ml) both significantly stimulate the expression of MMP-9 on ECV304 cells and PDTC could significantly enhance TNF--induced or PMA-induced MMP-9 activities especially at 20M. By western bolt and reverse transcription-polymerase chain reaction (RT-PCR) method, we also found PDTC increased MMP-9 protein and mRNA on TNF--induced ECV304. This indicated that PDTC have effect on the protein expression of MMP-9 and deeper influence on the level of MMP-9 transcription. Further more, we investigated the mechanism of action of PDTC in various signaling pathways. According to results, we presume the mechanism that PDTC enhance the expression of MMP-9 on TNF-a-induced ECV304 through the JNK or PI-3 kinase signal pathways. On the other hand, we found PMA induce active form expression of MMP-2 on HUVEC by zymography analysis. Then, the expression of MMP-2 would be inhibited by PDTC on a concentration manner. By western blot method, we also found PDTC decrease MMP-2 protein on PMA-induced HUVEC. The mechanism that PDTC inhibit the expression of MMP-2 on PMA-induced HUVEC might through the protein kinase C (PKC) signal pathway . • According to our results, ECV304 and HUVEC cells have somehow difference in the expression of MMPs resulted from responding to stimulators or drugs by different mechanisms. The different characters between ECV304 and HUVEC would be a resource of research the future..