HL-60 細胞分化及感染時基因表現，及氯喹所引發其細胞凋亡之研究

The study of HL-60 cell gene expression during differentiation and infection, and the apoptosis induced by chloroquine

莊千純(Chien-Chun Chuang)

指導教授 ： 陳孟琦

中山醫學大學/醫學院/免疫學研究所/碩士(2005年)

摘要

細菌及病毒感染時會活化巨噬細胞，細菌的產物脂多醣體 (lipopolysaccharide; LPS)、及RNA病毒的產物雙股RNA (double-stranded RNA; dsRNA) 可分別和其接受器toll-like receptor (TLR) 結合，經由訊號傳遞活化基因表現。抗瘧疾藥物-氯喹 (chloroquine; CQ)，目前用於治療類風濕性關節炎病人。其副作用是血球數目減少造成貧血，氯喹對血球細胞是否有細胞毒性，其作用機制目前仍然未知。

實驗目的
我們探討人類前骨髓性白血病細胞 (HL-60細胞) 細胞分化之基因表現，模擬病毒或細菌感染及細胞激素刺激細胞時的基因表現，比較其基因表現的差異。並探討氯喹刺激HL-60細胞，造成血球細胞死亡的機制。

方法
以reverse transcription polymerase chain reaction (RT-PCR) 分析HL-60細胞經all-trans retinoic acid (ATRA)、12-O-tetradecanoyl phorbol-13-acetate (TPA)、alpha 1,25-dihydroxyvitamin D3 (Vit-D3) 刺激分化HL-60細胞，並以LPS、雙股RNA的類似物poly inosinic-polycytidylic acid sodium salt (poly IC)、干擾素interferon-r (IFN-r) 刺激時之基因表現。利用Western blot分析氯喹刺激HL-60細胞之蛋白質表現。以PI (propidium iodide) 及Hoechst 33342螢光染色分析氯喹造成的細胞死亡，並以cathepsin、calpains的抑制劑分析細胞凋亡路徑。

結果
以ATRA、TPA、Vit-D3分化細胞，細胞間質的蛋白酶matrix metalloproteinase-2 (MMP-2) 皆會表現。當TPA、Vit-D3分化HL-60細胞，共同誘發細胞激素Interleukin-1b (IL-1β)、噬中性球的趨化因子Interleukin-8 (IL-8)、MMP-2基因表現。HL-60細胞刺激LPS後，IFN-γ會加強整個反應。未分化的HL-60細胞不表現TLR3，所以poly IC無法引發其發炎基因的表現。我們以75 mM氯喹刺激HL-60細胞，約有50%細胞死亡並測到caspase-3、Bid蛋白質的切割。cathepsin B, L的抑制劑z-FA-fmk及cathepsin D的抑制劑pepstatine A，可以減少由氯喹引起的細胞凋亡，而calpains抑制劑PD150606只能部分抑制由氯喹引起的細胞凋亡。

結論
(1).HL-60細胞分化時會表現細胞激素或趨化因子及和型態改變有關之酵素。
(2).氯喹會造成溶酶體膜的不穩定，造成溶酶體的蛋白酶cathepsin B, L‚ D釋放至細胞質，可能因此啟動了Bid切割及活化下游粒腺體路徑，而活化caspase-3造成細胞死亡。

關鍵字

分化 ； 感染 ； 基因 ； 氯喹細胞凋亡

並列摘要

Background: Macrophages are activated during infection by bacteria or virus. The processes include binding of ligands lipopolysaccharide (LPS‚from bacteria) or double-stranded RNA (dsRNA‚ from RNA virus) respectively‚ to their the toll-like receptor (TLR) 3 and 4 and activate gene expression through signal transduction pathways. Anti-malaria drug chloroquine (CQ) is used to treat patients with rheumatoid arthritis. The mechanism for its cytotoxic effect on blood cells is still unknown.

Aims of study:
We investigated the HL-60 cell gene expression during differentiation and infection of bacteria and virus, and compare their gene expression profiles. In addition, we study the possible apoptotic pathway in HL-60 cells treated with CQ.

Methods: HL-60 cells were treated with all-trans retinoic acid (ATRA)、12-O-tetradecanoyl phorbol-13-acetate (TPA)、alpha 1,25-dihydroxyvitamin D3 (Vit-D3) to differentiate and the gene expression in response to LPS and poly IC (mimics dsRNA) were analyzed by reverse transcription polymerase chain reaction (RT-PCR). CQ-induced HL-60 cell apoptosis was evaluated by fluorescent microscopy using propidium iodide and Hoechst 33342 staining method. The possible CQ-induced apoptosis pathways were using chemical inhibitors for protease cathepsins and calpain.

Results: Cytokine or chemokine genes Interleukin-1b (IL-1β)、IL-8 or ECM protease matrix metalloproteinase-2 (MMP-2) can be induced by either ATRA、TPA、or Vit-D3. Stimulatory effect of LPS on HL-60 cells can be enhanced by IFN-γ. poly IC could not induce these effects because we could not detect TLR3 gene expression on HL-60. We observed that at 24 h, 75 microM CQ caused about 50% of cell death in HL-60 cells. We also detected cleavage of caspase-3 and Bid in CQ-treated cells. Cell death was decreased by either cathepsin B, L inhibitor z-FA-fmk or cathepsin D inhibitors pepstatine A. Calpain inhibitor PD150606 only provided partial protection against CQ-induced apoptosis.

Conclusions: (1). HL-60 cells produce cytokines or chemokines during differentiation which are important in the regulation of inflammatory responses. (2). The lysosomotropic anti-malaria drug CQ may cause lysosomal membrane instability, and lysosomal proteases cathepsin B, L, D are possible initiators which cause Bid cleavage and activate the downstream mitochondrial apoptotic pathway leading to caspase-3 cleavage and cell death.

並列關鍵字

gene ； differentiation ； infection ； apoptosis ； chloroquine

參考文獻

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