牛乳-紫草素纳米载药体系对肝癌细胞的杀伤作用研究

Antitumor effect of shikonin loaded milk derived exosomes on hepatoma cells

目的探讨牛乳-紫草素纳米载药体系对肝癌细胞的杀伤作用。方法采用实验研究方法。采用差速离心法分离出牛乳外泌体, 制备牛乳-紫草素纳米载药体系, 分光光度法测定并计算紫草素含量。采用磺酰罗丹明B比色法评价细胞毒性, Annexin V-FITC/碘化丙啶检测法测定细胞凋亡情况, Western blot法检测肝癌细胞中Bax和Bcl-2蛋白表达水平。单纯紫草素溶液处理人肝癌细胞系HepG2细胞为紫草素组, 牛乳-紫草素纳米载药体系处理人肝癌细胞系HepG2细胞为牛乳-紫草素纳米载药体系组。观察指标:(1)牛乳-紫草素纳米载药体系中紫草素载量百分比。(2)人肝癌细胞系HepG2细胞毒性评价。(3)人肝癌细胞系HepG2细胞凋亡情况分析。(4)人肝癌细胞系HepG2细胞Bax和Bcl-2蛋白表达水平检测。正态分布的计量资料以x±s表示, 组间比较采用t检查。结果 (1)牛乳-紫草素纳米载药体系中紫草素载量百分比:牛乳-紫草素纳米载药体系中紫草素载量百分比为22.8%。(2)人肝癌细胞系HepG2细胞毒性评价:紫草素组和牛乳-紫草素纳米载药体系组的肝癌细胞存活率分别为53.9%±2.9%和45.4%±1.9%, 两组比较, 差异有统计学意义(t=46.27, P<0.05)。(3)人肝癌细胞系HepG2细胞凋亡情况分析:紫草素组和牛乳-紫草素纳米载药体系组的肝癌细胞早期凋亡率分别为11.3%±1.5%和14.8%±2.2%, 两组比较, 差异无统计学意义(t=1.37, P>0.05)。紫草素组的肝癌细胞晚期凋亡率和总凋亡率分别为32.3%±1.3%和43.6%±4.3%, 牛乳-紫草素纳米载药体系组的肝癌细胞晚期凋亡率和总凋亡率分别为38.7%±3.2%和53.5%±4.4%, 两组比较, 差异均有统计学意义(t=37.39, 30.97, P<0.05)。(4)人肝癌细胞系HepG2细胞Bax和Bcl-2蛋白表达水平检测:紫草素组Bax和Bcl-2蛋白表达水平分别为232.0±2.6和32.0±1.6, 牛乳-紫草素纳米载药体系组中Bax和Bcl-2蛋白表达水平分别为286.0±3.8和17.0±1.5, 两组比较, 差异均有统计学意义(t=69.83, 53.32, P<0.05)。结论牛乳-紫草素纳米载药体系对人肝癌细胞系HepG2细胞有细胞毒性和凋亡诱导作用, 可抑制肝癌细胞生长。

Objective To investigate the antitumor effect of shikonin loaded milk derived exosomes on hepatoma cells.Methods The experimental study was conducted.Exosomes were isolated from milk by differential centrifugation and be loaded by shikonin to constructed a nano drug loading system.The shikonin content of this nano drug loading system was determined and calculated by spectrophotometry.The cytotoxicity of shikonin loaded milk derived exosomes was evaluated by sulfonyl rhodamine B colorimetry and cell apoptosis was determined by annexin V-FITC/propidium iodide assay.Western blotting was used to detect the Bax and Bcl-2 protein expression level in hepatoma cells.Human HepG2 hepatoma cells treated with shikonin were set as the shikonin treated group and human HepG2 hepatoma cells treated with shikonin loaded milk derived exosomes were set as the shikonin loaded milk derived exosomes treated group.Observa-tion indictors:(1)the loading percentage of shikonin in shikonin loaded milk derived exosomes;(2)the cytotoxicity of shikonin loaded milk derived exosomes on human HepG2 hepatoma cells;(3)cell apoptosis of human HepG2 hepatoma cells;(4)Bax and Bc-2 protein expression level in human HepG2 hepatoma cells.Measurement data with normal distribution were represented as MeantSD,and comparison between groups were analyzed using the t test.Results(1)The loading percentage of shikonin in shikonin loaded milk derived exosomes:the loading percentage of shikonin in shikonin loaded milk derived exosomes was 22.8%.(2)The cytotoxicity of shikonin loaded milk derived exosomes on human HepG2 hepatoma cells:the survival rates of hepatoma cells were 53.9%±2.9%and 45.4%±1.9%in the shikonin treated group and the shikonin loaded milk derived exosomes treated group,respectively,showing a significant difference(t=46.27,P<0.05).(3)Cell apoptosis of human HepG2 hepatoma cells:the early apoptosis rates of hepatoma cells were 11.3%±1.5%and 14.8%±2.2%in the shikonin treated group and the shikonin loaded milk derived.exosomes treated group,respectively,showing no significant difference(t=1.37,P>0.05).The late and overall apoptosis rates of hepatoma cells were 32.3%±1.3%and 43.6%±4.3%in the shikonin treated group,versus 38.7%±3.2%and 53.5%±4.4%in the shikonin loaded milk derived exosomes treated group,showing significant differences(t=37.39,30.97,P<0.05).(4)Bax and Bcl-2 protein expression level in human HepG2 hepatoma cells:Bax and Bcl-2 protein expre-ssion level were 232.0±2.6 and 32.0±1.6 in the shikonin treated group,versus 286.0±3.8 and 17.0±1.5 in the shikonin.loaded milk derived exosomes treated group,showing significant differences(t=69.83,53.32,P<0.05).Conclusion The shikonin loaded milk derived exosomes have cytotoxic and apoptosis inducing effects on human HepG2 hepatoma cells,which can inhibit the growth of hepatoma cells.