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作 者:李永宁[1] 熊小婧 李青松[2] 刘莹[2] 胡铮[3] 曹文武[3]
机构地区:[1]大连医科大学附属第一医院急诊科,大连116011 [2]大连医科大学附属第一医院心内科,大连116011 [3]哈尔滨工业大学声光诊疗实验室,哈尔滨150001
出 处:《上海交通大学学报(医学版)》2016年第5期648-654,共7页Journal of Shanghai Jiao tong University:Medical Science
基 金:国家自然科学基金(81400247)~~
摘 要:目的研究5-氨基乙酰丙酸(5-ALA)介导的声动力疗法(SDT)对人肝癌Hep G2细胞的促凋亡作用及机制。方法取对数期生长Hep G2细胞,通过CCK-8法检测5-ALA和超声干预对细胞存活率的影响,优化出声动力治疗最佳5-ALA药物浓度及超声辐照强度。取对照组(未处理细胞)、5-ALA组、超声组和SDT组Hep G2细胞,流式细胞仪检测细胞凋亡率、坏死率及线粒体膜电位(MMP)的改变,荧光探针DCFH-DA检测活性氧类(ROS)的产生,Western blotting检测凋亡蛋白caspase-3、caspase-9和细胞色素C(Cyt-C)的表达,透射电镜观察细胞超微结构的改变。结果与对照组、5-ALA组和超声组比较,SDT组Hep G2细胞存活率较低,细胞凋亡率升高,ROS的产生增加,MMP降低,caspase-3、caspase-9和Cyt-C高表达。透射电镜观察发现SDT组Hep G2细胞表面微绒毛减少、内质网和线粒体肿胀、凋亡小体产生。结论 5-ALA介导的SDT诱导Hep G2细胞凋亡以内源性线粒体凋亡为主要途径。Objective To investigate the underlying mechanisms of cell apoptosis induced by 5-aminolevulinic acid-m ediated sonodynam ic therapy( 5-ALA-SD T) in hum an hepatocellular carcinom a H ep G2 cells. Methods The CCK-8 assays were used to exam ine the effects of 5-ALA and ultrasound intervention on the viability of HepG2 at logarithm ic grow th phase. The 5-ALA level and intensity of ultrasonic irradiation with best efficacy were identified. The apoptosis rate, mortality rate, and changes of mitochondrial membrane potential( MM P) in HepG2 cells in the control group( untreated cells), 5-ALA group, ultrasonic group, and SD Tgroup were detected with flow cytometry assays. The production of reactive oxygen species( ROSs) were detected with the fluorescent probes. W estern blotting w as used to exam ine expressions of cytochrome C( Cyt-C) and apoptosisassociated proteins caspase-3 and caspase-9. The ultrastructural changes in cells were observed with the transm ission electron microscope. Results The SD Tgroup had low er survival rate, higher apoptosis rate,increased ROS production, decreased MMP, and elevated expressions of caspase-3, caspase-9, and Cyt-C as com pared with the control group, 5-ALA group, and ultrasonic group. The transm ission electron m icroscope revealed changes such as reduced cell microvilli, sw elling endoplasm ic reticulum and m itochondria, and the production of apoptotic bodies in H ep G2 cells in the SD Tgroup. Conclusion Endogenous mitochondrial apoptosis is the m ain m echanism of 5-ALA-SD T-induced cell apoptosis in Hep G2 cells.
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