摘要
目的:探讨创伤弧菌溶细胞素融合蛋白(rVvhA)对人Jurkat T淋巴细胞凋亡的作用及凋亡过程中Caspase-3酶活性的变化。方法:利用基因工程方法体外表达、制备和纯化rVvhA;MTT法检测rVvhA对Jurkat T淋巴细胞的细胞毒活性影响;Annexin-V/PI双染流式细胞仪检测rVvhA诱导Jurkat T淋巴细胞凋亡情况;Hochest33258荧光染色激光共聚焦显微镜观察rVvhA诱导Jurkat T细胞核形态的改变;分光光度法检测凋亡过程中Caspase-3酶活性的变化。结果:纯化复性后rVvhA的纯度达到92%以上;MTT结果显示rVvhA活性蛋白能显著抑制Jurkat T淋巴细胞的生长,有效浓度为1.5 HU/mL;流式细胞仪和激光共聚焦检测发现rVvhA作用后Jurkat T淋巴细胞核固缩,产生亮蓝荧光,1.5 HU/mL rVvhA,作用4 h后即可有效诱导其凋亡,凋亡率为(39.13±3.33)%,对照组为(3.43±0.34)%,且能被Caspase全酶抑制剂一定程度抑制。1.5 HU/mL rVvhA作用Jurkat T淋巴细胞3 h,Caspase-3酶活性达到高峰。结论:rVvhA能损伤人Jurkat T淋巴细胞,诱导其发生细胞凋亡,Caspase-3可能在rVvhA诱导的Jurkat T细胞凋亡过程中起重要作用。
Objective： To study the effect of recombinant Vibrio vulnificus cytolysin（rVvhA）on the activity of Caspase-3 and apoptosis in Jurkat T-cells.Method： The growth inhibition of Jurkat T-lymphocytes was measured by MTT assay.The effect of rVvhA on induction of apoptosis of Jurkat T-lymphocytes was studied by Hochest33258 stain and flow cytometry（FCM）,respectively.Commercially available colorimetric assay kit was used to measure the activity of caspase-3 based on the spectrophotometric method.Results： Refolded rVvhA was purified with high purity up to 92%;the viability of Jurkat T-cells treated with 1.5 HU/mL rVvhA was inhibited significantly;1.5 HU/mL rVvhA could significantly induce apoptosis on Jurkat T-cells.After treatment with 1.5 HU/mL rVvhA at various times,the activity of Caspase-3 in Jurkat T-cells reached the peak at 3 h.Conclusion： The apoptosis of Jurkat T-cells can be induced by rVvhA,Caspase-3 may play a major role in the apoptosis of rVvhA-exposed cells.
出处
《温州医学院学报》
CAS
2011年第4期332-336,共5页
Journal of Wenzhou Medical College
基金
浙江省科技计划资助项目(2008C33059)
