內質網主要為合成與修飾蛋白質的重要胞器，當其受到壓力時會引起一些基因表現及生理新陳代謝的改變，導致內分泌失調及神經方面等自體免疫疾病的產生。 目的： 利用數種引起內質網壓力的藥物 (thapsigargin、ionomycin、tunicamycin、brefeldin 和DTT) 刺激HL-60細胞，觀察其基因表現、細胞存活及對細胞週期的影響。 方法： 藥物處理過後之細胞以PI (propidium iodide) 及Hoechst 33342 螢光染色，以正立螢光顯微鏡分析上述五種藥物對內質網所造成的細胞凋亡，且探討引起之細胞的生存能力；以reverse transcription polymerase chain reaction (RT-PCR) 分析基因表現；利用Western blot 分析蛋白質表現；使用流式細胞儀 (flow cytometry) 觀察細胞週期型態的變化。 結果： (1)在HL-60細胞中，加入thapsigargin、ionomycin和tunicamycin造成細胞死亡數低於brefeldin A 和DTT所引起的結果。 (2)Thapsigargin和ionomycin會引起細胞週期抑制因子p21表達。 (3)Thapsigargin、ionomycin和tunicamycin在HL-60細胞中會引起細胞週期循環暫停。 (4)在HL-60細胞中，除了ionomycin外其他的藥物皆會引起CHOP、GRP78、sXBP-1 mRNA表達。 (5)在低濃度的tunicamycin能夠造成G1期細胞生長暫停後，恢復原有的細胞週期循環。 結論： (1)內質網壓力為快速的反應。 (2)thapsigargin和tunicamycin可能活化細胞內某些回饋路徑，防止內質網壓力反應持續擴大；brefeldin A和DTT可能缺乏此機制，所以會使內質網壓力反應會持續擴大。 (3)Thapsigargin、ionomycin和tunicamycin會引起G1期細胞週期暫停，但只有tunicamycin不是經由p21的表達。 (4)在病理機制中，受損的細胞若有內質網壓力反應模式，不但可以增加細胞的存活，更可避免細胞演變成官能障礙、持續性高血糖，最後導致糖尿病。 Background: ER (endoplasmic reticulum) is an organelle for the biosynthesis and medificate of secretory and plasma membrane proteins. when they are accept ER stress and the result will cause some changes of gene displaying, physiological metabolism change and autoimmue disease to lead to secretion imbalance and neural respect ,etc.. Aims of study: We used different ER stress drugs (thapsigargin, ionomycin, tunicamycin, brefeldin A and DTT) to study the cellular responses, including gene expression, cell viability and cell cycle arrest in HL-60 cells. Methods: Different drugs were used as ER stresses induces. Cell viability was analyzed by fluorescent microscopy. Messager RNA levels were analyzed by RT-PCR. Westren blot were used to analyzed by protein expression. Flow cytometry were analyzed by cell cycle. Results: (1)Treatment thapsigargin, ionomycin and tunicamycin caused lower cell death than brefeldin A and DTT in HL-60 cells. (2)Cell cycle inhibitor p21 was induced by thapsigargin and ionomycin. (3)Treatment thapsigargin, ionomycin and tunicamycin caused cell cycle arrest in HL-60 cells. (4)Induction of CHOP, GRP78 mRNA, and splicing of XBP-1 were detected in HL-60 cells treated with all drugs used expcept ionomycin. (5)Cells were return cell cycle after lower concentrate tunicamycin caused G1 arrest at 24hr. Conclusions: (1)ER stress genes expression are quick. (2)Thapsigargin and tunicamycin may activate some feedback pathways in cells, and prevent to prolonged ER stress responses but brefeldin A and DTT may lack this mechanism. (3)Thapsigargin, ionomycin and tunicamycin caused G1 arrest but only tunicamycin did not by the expression of p21 in HL-60 cells. (4)In the pathology mechanism, if damaged cells have ER stress response mode, cells can increase survival and avoided to dysfunction, casued prolonged hyperglycemia and diabetes finally.
