- 學位論文
探討登革病毒蛋白酶造成微血管內皮細胞凋亡的分子機制
To investigate the molecular mechanism of DENV protease-induced microvascular endothelial cell apoptosis
摘要
登革是重要的傳染疾病，先前指出引發出血的原因為登革病毒與內皮細胞交互作用，進而導致內皮細胞死亡。已知登革病毒蛋白酶可引發內皮細胞凋亡，故本研究目的為探討登革病毒 NS3 蛋白酶造成人類微血管內皮細胞 (HMEC-1) 凋亡的分子機制。首先我建構帶有登革病毒 NS2B-蛋白酶、蛋白酶、NS2B 與失去活性 H51A 點突變 NS2B-蛋白酶之慢病毒載體，再分別導入 HMEC-1 中。Annexin V 與 propidium iodide 染色結果顯示，登革病毒 NS2B-蛋白酶或蛋白酶會引發 HMEC-1 細胞凋亡，但 NS2B 與 H51A 點突變 NS2B-蛋白酶則無法引發凋亡，顯示蛋白酶活性為 NS3 蛋白酶造成之 HMEC-1 細胞凋亡所必需。 藉由使用 L-NAME 與 apocynin，證明活性氧與活性氮並未參與蛋白酶造成之細胞凋亡。同時發現登革病毒 NS2B-蛋白酶會引發 HMEC-1 caspase-8、caspase-3 但非 caspase-9 活化，給予細胞 caspase-8、caspase-3 或 pan-caspase 抑制劑後可有效減少蛋白酶造成之細胞凋亡。而利用 BAY 11-7082 抑制 NF-κB 活化即可阻斷 NS2B-蛋白酶造成之 HMEC-1 細胞凋亡，證明 NF-κB 訊息傳遞路徑與 NS2B-蛋白酶造成之細胞凋亡有關。 本篇研究結論為登革病毒 NS3 蛋白酶透過活化細胞內 NF-κB，造成 caspase-8 與 caspase-3 活化，進而引發 HMEC-1 凋亡。
並列摘要
Dengue is an important infectious disease. We reported previously that dengue virus interaction with endothelial cells resulting in endothelial cell death is key to hemorrhage development. Dengue viral protease has been shown to cause endothelial cell apoptosis. The goal of this study is to investigate the mechanism of how DENV NS3 protease mediates human microvascular endothelial cell line (HMEC-1) apoptosis. First of all, I generated lentiviral constructs that carry dengue viral components NS2B-protease, protease, 2B and protease activity-dead mutant 2B-protease H51A. HMEC-1 cells were transduced by these components separately. The results of Annexin V and propidium iodide staining showed that transduction of 2B-protease or protease but not 2B or 2B-protease H51A induced HMEC-1 apoptosis, indicating that NS3 protease mediated-HMEC-1 apoptosis is protease activity-dependent. By use of L-NAME and apocynin, I ruled out the involvement of ROS/RNS production in protease-induced apoptosis. In the mean time, I found that DENV 2B-protease induced caspase-8 and caspase-3 but not caspase-9 activation in HMEC-1 cells. Treatment by caspase-8, caspase-3 and pan-caspase but not caspase-9 inhibitors ameliorated protease-induced apoptosis. Interestingly, inhibition of NF-κB activation by BAY 11-7082 blocked 2B-protease-induced HMEC-1 apoptosis, indicating that NF-κB signaling is involved in 2B-protease-induced apoptosis. In summary, these findings revealed that DENV NS3 protease induces cellular caspase-8 and caspase-3 activation, triggering HMEC-1 apoptosis, and NF-κB signaling is required for the triggering of the caspase pathway.