內質網壓力對HL-60細胞之鈣離子平衡及細胞分化的影響

前言：內質網在細胞內部有合成蛋白、幫助正確蛋白的修飾，更重要的是儲存大量的鈣離子，並且利用鈣離子的調控來調整細胞的功能。當此調控受到干擾時，內質網會有一連串的壓力（ER stress）反應。我們利用會誘發ER stress相關藥物，tunicamycin（TM）、thapsigargin（TG）和引發鈣離子上升相關藥物ionomycin（Iono）來探討內質網壓力反應與細質鈣離子濃度的關聯性。我們同時也觀察內質網壓力對TPA誘發HL-60分化的影響。方法：HL-60細胞加入上述不同濃度的藥物在不同時間點下利用Ho342 （Hoechst 33342）和PI（propidium iodide）染劑以正立螢光顯微鏡觀察細胞存活；以RT-PCR分析基因表現；利用Fura-2AM（Fura-2 acetoxymethyl ester）測定鈣離子濃度的變化；用ELISA來測量IL-8蛋白分泌；ROS測量；以Western blot 來分析ERK磷酸化程度。在細胞分化研究方面，HL-60事先以TM誘發內質網壓力，再以 TPA刺激分化成巨噬細胞；以Liu’s stain分析分化程度；並觀察細胞存活率、鈣離子及IL-8的變化。結果：這三種藥物都會造成細胞死亡，TM主要引發細胞凋亡而TG和Iono會導致大量的細胞壞死。在基因表現層面也有所不同，只有TM和TG會誘發內質網壓力CHOP基因表現。TG和Iono會專一性的增加細胞質鈣離子濃度，ERK磷酸化，並且也會誘發IL-8和p21基因表現。內質網壓力下的細胞（以TM處理）經由TPA刺激，細胞分化程度下降並且細胞壞死數量增加。結論：我們證明不同的內質網壓力藥物會引發不同的細胞反應，其中細胞質鈣離子的失調會影響基因表現即造成細胞壞死，而IL-8可以作為有效的標記來分辨細胞的凋亡與壞死。另一方面，內質網壓力會干擾免疫細胞分化過程，增加細胞壞死。此一結果部份解釋在僵直性脊椎炎病人有長期病菌感染及持續發炎的現象。

關鍵字

內質網壓力；分化；鈣離子

並列摘要

Background: ER （endoplasmic reticulum）is an organelle for the the synthesis and modification of secretory proteins, as well as storage of calcium which plays important roles in the regulation of cellular function. Interference of ER functions may cause unfolded protein responses (UPRs). Aims of study: We used ER stress drugs tunicamycin（TM） and thapsigargin（TG）and calcium ionophore ionomycin （Iono）to investigate the effects of these drugs on TPA-stimulated cell responses and cytosolic [Ca2+] in HL-60 cells. We also investigated the effects of ER stress on TPA-stimulateed cell differentiation of this cell type. Methods: HL-60 cells were treated with either one of these drugs for different periods of time. Cells were then stained with Ho342 and PI, the survival rate was analyzed by fluorescent microscopy. Gene expression was analyzed by RT-PCR. Fura-2AM staining was used for cytosolic [Ca2+] measurement. IL-8 protein release was measured by ELISA. ROS amount was measured by H2DCFDA. ERK phosphrylation was analyzed by Western blot. In the study of cell differentiation, HL-60 cells were pretreated with TM for different periods of time followed by TPA stimulation. Liu’s stain was used to analyze the level of differentiation. Cytosolic [Ca2+], IL-8 secretion and gene expression were also analyzed. Results: All three drugs caused cell death. TM mainly caused apoptotic cell death, while TG and Iono caused a large portion of necrotic cell death. There was differential gene expression induced by these drugs. Only TM and TG induce ER stress gene CHOP expression. TG and Iono specifically increased cytosolic [Ca2+], ERK phosphorylation and expression of p21 and IL-8 genes. In ER stressed (TM treated) cells, there was decreased differentiation and increased necrotic death when cell were stimulated by TPA. Conclusions: In conclusion, we demonstrated that there were differential cellular responses to ER stress inducers. Deregulation of cytosolic [Ca2+] is one of the causes for gene expression and necrotic cell death. IL-8 can be a useful marker to distinguish apoptotic and necrotic death. We also found that ER stress can interfere with cell differentiation in HL-60, which may explain partly that in patients with ankylosing spondylitis, there is chronic bacterial infection and inflammation.

並列關鍵字

ER stress ； differentiation ； Ca2+

參考文獻

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內質網壓力對HL-60細胞之鈣離子平衡及細胞分化的影響

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