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为了研究重组创伤弧菌溶细胞素(rVvhA)诱导人脐静脉内皮细胞(HUVEC)凋亡的作用及其机制。采用MTT法、普通光学显微镜和电子透射电镜、流式细胞仪结合Annexin V-PI标记法、caspase活性检测等方法测定rVvhA诱导HUVEC凋亡的作用。结果显示rVvhA能够抑制HUVEC生长。3.0溶血单位(HU)/ml和4.0 HU/ml的rVvhA作用HUVEC 8 h后,细胞和线粒体的形态发生凋亡改变,其中4.0 HU/ml处理组的凋亡率(51.73±1.96)%高于对照组(3.53±0.64)%和3.0 HU/ml处理组(12.83±5.00)%,具有剂量依赖性;4.0 HU/ml rVvhA诱导HUVEC凋亡过程中,caspase-3、caspase-9活性增加,并且具有时间依赖性,而caspase-8活性没有明显改变;4.0 HU/ml rVvhA加caspase-3抑制剂Ac-DEVD-FMK或caspase-9抑制剂Ac-LEHD-FMK的凋亡率分别下降到(14.2±3.47)%、(24.93±3.93)%。同时caspase-3、caspase-9活性也下降。结果表明rVvhA具有诱导HUVEC凋亡的生物学活性;其机制可能与依赖caspase的线粒体途径有关。
In order to study the role of rVvhA in inducing apoptosis of human umbilical vein endothelial cells (HUVECs) and its mechanism. The apoptosis of HUVEC induced by rVvhA was measured by MTT assay, optical microscope and transmission electron microscopy, flow cytometry with Annexin V-PI, and caspase activity assay. The results show that rVvhA can inhibit HUVEC growth. The apoptotic rates of cells and mitochondria in HUVEC treated with 3.0 HU / ml and 4.0 HU / ml rVvhA for 4 h were significantly higher than those in 4.0 HU / ml control group (51.73 ± 1.96)% (3.53 ± 0.64)% and 3.0 HU / ml treatment group (12.83 ± 5.00)%, respectively. The activity of caspase-3 and caspase-9 increased in the apoptotic HUVEC induced by 4.0 HU / ml rVvhA The apoptosis rate of Ac-DEVD-FMK or caspase-9 inhibitor Ac-LEHD-FMK with 4.0 HU / ml rVvhA plus caspase-3 inhibitor decreased to (14.2 ± 3.47)%, (24.93 ± 3.93)%. At the same time, caspase-3, caspase-9 activity also decreased. The results showed that rVvhA has the biological activity of inducing HUVEC apoptosis; its mechanism may be related to caspase-dependent mitochondrial pathway.