纳米氧化硅空心球负载光敏剂对肝癌体内外的杀伤作用研究

The Killing Effects of Photosensitizer-loaded Hollow Silica Nanoparticles on Hepatoma Cell in vitro and in vivo

目的:探讨空心纳米粒子光敏剂与普通光敏剂在不同实验条件下对人HepG2肝癌细胞增殖的抑制作用及其作用机制。方法:应用体内外培养的HepG2肝癌细胞,以纳米化的photosan及photosan为光敏剂,半导体激光器(波长630 nm)为光源进行光照射,在不同的浓度光敏剂经不同剂量的光照后,用MTT法测定光动力治疗后肝癌细胞的残存率,并用流式细胞分析检测光动力治疗对肝癌细胞凋亡的影响,采取Western blot检测光动力治疗肝癌后凋亡相关蛋白caspase-3和caspase-9的表达情况,并建立裸鼠动物模型,比较纳米粒子光敏剂与普通光敏剂在动物体内对肝癌抑制作用。结果:纳米粒子光敏剂介导的光动力治疗对肝癌有明显的抑制作用,在一定范围条件下,随着光敏剂浓度的增加和激光剂量的的增大,肝癌细胞的的残存率明显下降,且纳米粒子光敏剂在同等条件下要优于普通光敏剂,流式细胞分析显示,空心纳米粒子光敏剂光动力治疗肝癌出现了明显的凋亡,且比普通光敏剂更显著。Western blot检测结果显示,两种光敏剂均引起凋亡蛋白caspase-3和caspase-9的表达,在空心纳米粒子光敏剂光动力作用组该两种蛋白的表达水平要明显高于普通光敏剂组。体内实验显示:用纳米粒子光敏剂与普通光敏剂分别对裸鼠肝癌模型干预,在生存期,瘤体的体积大小,纳米粒子光敏剂组显然优越于普通光敏剂组。结论:空心纳米粒子光敏剂对肝癌细胞在体内外具有明确的抑制作用,这种抑制作用可能通过引起凋亡相关蛋白高水平的表达,从而促发凋亡通路的开放,引起癌细胞发生凋亡。

Objective：This study examined the inhibitory effects of conventional photosensitizers and photosensitizers delivered in hollow silica nanoparticles on the proliferation of HepG2 human hepatoma cells under different experimental conditions and the underlying mechanisms of these effects. Methods：Photosensitizers （conventional Photosan or nanoscale Photosan）were administered to in vivo and in vitro cultured HepG2 hepatoma cells,and a semiconductor laser was used as a light source to photoirradiate these cells.Photodynamic therapies（PDTs）involving different levels of light exposure and different photosensitizer concentrations were tested;To assess the effects of these PDTs,the cellular viabil-ity was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyl tetrazolium bromide （MTT）assay.Apoptotic and necrotic cells were measured by flow cytometry.The express of caspase-3 and caspase-9 caused in the photodynamic therapy by western blot.A nude mouse animal model was established to conduct in vivo experiments comparing the in-hibitory effects of nanoscale photosensitizers and conventional photosensitizers on liver cancer.Results：Nanoscale photo-sensitizer-mediated PDTs produced significant inhibitory effects on liver cancer cells.Within a certain range of condi-tions,hepatoma cell viability significantly decreased as photosensitizer concentrations and laser doses increased.Moreo-ver,under the same experimental conditions,the nanoscale photosensitizer performed better than the conventional photo-sensitizer（P＆lt;0.05）.The flow cytometry demonstrated that the laser induced cell death with PS-loaded HSNP was much more severe than that of free PS （P＆lt;0.05 ）.The activated forms the Caspase3 （Caspase3 20 kD）and caspase-9 （Caspase 9 35 kD）expression with PS-loaded HSNP were significantly higher than free PS（P＆lt;0.05 ）.In vivo experi-ments using either nanoscale photosensitizer or conventional photosensitizer in a nude mouse liver cancer model suggested that nanoscale photosensitizer treatments were superior to the corresponding conventional photosensitizer treatments with re-spect to survival time and tumor volume.Conclusions：Hollow nanoparticles containing photosensitizer clearly inhibited hepatoma cells both in vivo and in vitro.These inhibitory effects may result from the induction of high levels of apoptosis-re-lated protein expression,which trigger the activation of apoptotic pathways that cause the programmed death of cancer cells.