- 學位論文
探討氯化三苯錫對於正常大鼠上皮肝臟細胞間隙交流影響
Effect on Gap Junctional Intercellular Communication by TPT in WB-F344 Rat Liver Epithelial Cells
摘要
自從1853年第一個有機錫被合成出來之後，有機錫現在廣泛的被應用在工業及農業上，不論是PVC穩定劑、船舶防污劑、殺菌劑、木材防腐都有一定的功效。由於與荷爾蒙結構相近因此屬於環境荷爾蒙的一種，進入生物體之後會干擾正常體內代謝作用造成各種危害。本實驗目的為探討氯化三苯錫（TPTC）對於大鼠正常上皮肝臟細胞（WB-F344）細胞間隙交流影響。 由MTT Assay結果可得知TPTC作用於WB-F344中一天濃度介於10~60 ppb存活率會隨著濃度的增加而減少，具有Time and Dose dependent，IC50為59 ppb。細胞聚落形成效率結果得到，在TPTC作用於WB-F344細胞約9天濃度在0~20 ppb之間聚落形成效率會隨著濃度增加而變少。TPTC對於細胞的細胞間隙交流，在作用30分鐘濃度為1.5 ppm時會阻斷細胞間隙的交流，在以相同濃度1.5 ppm作TPTC用時間分別為15、30、45、60分鐘之後可以得到細胞間隙交流長度隨著時間增加而減少。加入PD98059（ERK inhibitor）與LY294002（PI 3-kinase inhibitor）測試後會使已經阻斷的間隙交流恢復，加入GF109203X hydrochloride（Protein Kinase C inhibitor）則無法使間隙交流恢復。測定細胞內外鈣離子濃度可以發現TPTC會使WB細胞鈣離子增加。由西方墨點與免疫染色得到TPTC對於細胞膜上E-cadherin螢光表現有增強的結果但是不改變含量、狀態，β-caternin螢光表現沒有差異的結果但是不改變含量、狀態，α-tublin在Western blot中沒有差異，在螢光免疫中Cx43螢光強度有減弱，在Western blot中發現隨著暴露的時間越長磷酸化的表現越強烈。 根據以上的結果，TPTC在低時間作用下對於WB-F344細胞膜上的蛋白功能影響導致GJIC間隙交流被阻斷，實驗證明GJIC功能的障礙是細胞內鈣離子量的變化使得PI 3-kinase與MEK活化促使Cx43磷酸化而造成。
並列摘要
Organotin compounds (OTCs) are used extensively as stabilizers in the production of plastic,agricultural pesticides, antifoulant paints and wood preservation. Owing to their structure are similar to hormone. OTCs are category Environmental Hormone induce toxicity in animals and human such as interrupt normal metabolism and neurotoxicity. There are a few studies about Gap Junctional Intercellular Communication (GJIC) of cells by OTCs. Therefore, this study discussed the effect on GJIC in WB-F344 rat liver epithelial cells by TPT. According to the MTT assay, cell availabilities of WB-F344 cell were degression when treaded with high concentration TPTC (IC50=59 ppb) and had Dose & Time dependent. And WB-F344 cells Colony Forming Efficiency were reversed of TPTC dose when exposured 9 days. During the GJIC experimentation,WB-F344 cells GJIC were block when treated 1.5 ppm TPTC in 30 minutes. The diffusion length were decreased gradually when exposured 1.5 ppm TPTC 15、30、45、60 minutes. The GJIC were recover when WB-F344 cells treated with PD98059(ERK inhibitor ) and LY294002（PI 3-kinase inhibitor） previously, but treated with GF109203X hydrochloride (Protein Kinase C inhibitor)were not. TPTC increased extra- and intra-cellular calcium in WB-F344 cells In the Immunostain analysis & Western Blot experimentation, the expression of GAPDH、β-caternin were the same, but E-cadherin in WB-F344 cells with TPTC were strong than control. The fluorescence of Cx43 in WB-F344 cells with TPTC was decrease and Cx43 phosphoration be found in Western Blot experimentation . According to those experimentations, the mechanism of WB-F344 cells GJIC block was TPTC destroyed the function of protein on the cell. Because of membranes that intracellular calcium concentration change, PI3-kinase and MEK pathway were be activate and caused Cx43 phosphoration.