CAJ | 학술논문

目的研究重组创伤弧菌溶细胞素(rVvhA)诱导小鼠单核巨噬细胞(J774A.1)凋亡的作用及机制.方法 MTT法、电子透射电镜、流式细胞仪结合Annexin V-PI标记法、线粒体膜电位和cagpase活性检测等方法测定rVvhA诱导J774A.1凋亡的作用.结果 MTT结果显示rVvhA能够抑制J774A.1生长.2.0溶血单位(HU)/ml和3.0 HU/ml的rVvhA作用J774A.18 h后,透射电镜观察细胞和线粒体形态发生凋亡改变;流式细胞仪检测凋亡率分别为(7.80±0.62)%、(12.33±0.12)%,均高于正常组(3.07±0.67)%;线粒体膜电位也下降,流式细胞仪结果显示绿色荧光率分别是9.8%、39.2%.其中3.0 HU/ml rVvhA作用组,caspage-3、9活性增加,并且具有时间依赖性,而cagpage-8活性没有明显改变.3.0HU/ml rVvhA加caspage-3抑制剂(Ac-DEVD-FMK)或caspase-9抑制剂(Ac-LEHD-FMK)的凋亡率与3.0 HU/ml rVvhA作用组相比都降低,分别为(6.23±3.95)%、(9.60±3.14)%;同时cagpage-3、9活性也下降.结论 rVvhA具有诱导J774A.1凋亡的生物学活性;其机制可能与依赖cagpase的线粒体途径有关.
목적 연구중조창상호균용세포소(rVvhA)유도소서단핵거서세포(J774A.1)조망적작용급궤제.방법 MTT법、전자투사전경、류식세포의결합Annexin V-PI표기법、선립체막전위화cagpase활성검측등방법측정rVvhA유도J774A.1조망적작용.결과 MTT결과현시rVvhA능구억제J774A.1생장.2.0용혈단위(HU)/ml화3.0 HU/ml적rVvhA작용J774A.18 h후,투사전경관찰세포화선립체형태발생조망개변;류식세포의검측조망솔분별위(7.80±0.62)%、(12.33±0.12)%,균고우정상조(3.07±0.67)%;선립체막전위야하강,류식세포의결과현시록색형광솔분별시9.8%、39.2%.기중3.0 HU/ml rVvhA작용조,caspage-3、9활성증가,병차구유시간의뢰성,이cagpage-8활성몰유명현개변.3.0HU/ml rVvhA가caspage-3억제제(Ac-DEVD-FMK)혹caspase-9억제제(Ac-LEHD-FMK)적조망솔여3.0 HU/ml rVvhA작용조상비도강저,분별위(6.23±3.95)%、(9.60±3.14)%;동시cagpage-3、9활성야하강.결론 rVvhA구유유도J774A.1조망적생물학활성;기궤제가능여의뢰cagpase적선립체도경유관.
Objective To investigate the activity of recombinant Vibrio vulnificus hemolysin (rVvhA) on the apoptosis of J774A.1 cells and the related mechanism. Methods The cytotoxic effect of rVvhA on the growth of J774A.1 cells was identified by MTT, celluar and mitochondrial morphology were observed by transmission electron microscopy, apoptosis or necrosis and mitochondrial membrane potential in J774A.1 cells were measured by flow cytometry, activities of caspase-3 ,-8,-9 were detected by spectrophotometry. Results The viability of J774A.1 cells exposed to rVvhA was inhibited, and it is dependent on dose. Celluar and mitochondrial uhrastructure both occurred to change obviously observed by transmission electron microscopy in J774A.1 treated by 2.0 HU/ml and 3.0 HU/ml rVvhA after 8 hours; and 3.0 HU/ml rVvhA group had a better cytotoxic effect on J774A.1 than that of 3.0 HU/ml rVvhA group. The percentage of apoptosis is (7.80±0.62)%, (12.33±0.12)%, respectively. Besides, the mitochondriai membrane potential also reduced, because the rate of fluorescence which is green increase 1.0% (normal) to 9.8% (2.0 HU/ml rVvhA) and 39.2% (3.0 HU/ml rVvhA). At the same time, the caspase-3, -9 activity increased gradually, but caspase-8 remained unchanging. In J774A.1 cells treated by 3.0 HU/ml rV-vhA + caspase-3 inhibitor(Ac-DEVD-FMK) or caspase-9 inhibitor(Ac-LEHD-FMK), The apoptosis of was reduced to(6.23±3.95)% ,(9.60±3.14)%, and the activity of caspase-3, -9 reduced, too. Conclusion The rVvhA has cytotoxic effect on J774A.1. Mitochondria-mediated apoptosis pathway which is dependent on caspase may be related to apoptosis induced by rVvhA in J774A.1.