摘要
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。
To investigate the activity of recombinant Vibrio vulnificus hemolysin （rVvhA） on the apoptosis of human umbilical vein endothelial cell （HUVEC） and the related mechanism, the cytotoxic effect of rVvhA on the growth of HUVEC was identified by MTT; cellular morphology was observed by inverted microscope, ultrastructure of cell and mitochondria were observed by transmission electron microscopy （TEM）; apoptosis or necrosis in HUVEC was measured using flow cytometry; activities of caspase-3, caspase-8, caspase-9 were detected by spectrophotometry. We found that the viability of HUVEC exposed to rVvhA was inhibited. Cellular and mitochondrial morphology both occurred to change obviously observed by TEM in HUVEC treated by 3.0 HU/ml and 4.0 HU/ mi rVvhA after 8 hours, and 4.0 HU/ml rVvhA （51.73± 1.96）% had a better cytotoxic effect on HUVEC than that of the normal （3.53±0.64）% and 3.0 HU/ml rVvhA group （12.83±5.00）%. Besides, caspase-3 and caspase-9 activities of HUVEC treated with 4.0 HU/ml rVvhA both raise, but caspase-8 remained unchanging. The apoptosis of HUVEC treated by 4.0 HU/ml rVvhA＋caspase-3 inhibitor （Ac-DEVD-FMK） or caspase-9 inhibitor （Ac-LEHD-FMK） was relatively reduced to （14.2±3.47）%, （24.93±3.93）%, respectively. At the same times, the caspase-3/caspase-9 ac- tivity reduced gradually by Ac-DEVD-FMK/Ac-LEHD-FMK. Our results discovery that the rVvhA has cytotoxic effect on HUVEC; Mitochondria-mediated apoptosis pathway which is dependent on caspase may be related to apoptosis induced by rVvhA in HUVEC.
出处
《中国细胞生物学学报》
CAS
CSCD
2010年第1期103-108,共6页
Chinese Journal of Cell Biology
基金
浙江省自然科学基金(No.Y2090468)
浙江省科技计划(No.84008012)资助项目~~
