基于红细胞免疫功能调节的石蒜碱体内抗肿瘤作用机制研究  

Anti-tumor mechanism of lycoline in vivo based on regulation of red blood cell immune function

作　　者：辛国松 杨燚[1] 张晶 于淼 王福玲 刘斌[1,2] 李文兰 季宇彬 XIN Guo-song;YANG Yi;ZHANG Jing;YU Miao;WANG Fu-ling;LIU Bin;LI Wen-lan;JI Yu-bin(Engineering research center for medicine,Harbin University of Commerce,Harbin 150076,China;Anti-tumor Natural Drug Engineering Research Center,Ministry of Education,Harbin 150076,China)

机构地区：[1]哈尔滨商业大学药物工程技术研究中心,黑龙江哈尔滨150076 [2]国家教育部抗肿瘤天然药物工程研究中心,黑龙江哈尔滨150076

出　　处：《中草药》2022年第2期432-440,共9页Chinese Traditional and Herbal Drugs

基　　金：中国博士后面上项目(2019M651296);黑龙江省自然科学基金联合引导项目(LH2019H066);哈尔滨商业大学青年创新人才项目(18XN024,2019CX37);中央支持地方高校改革发展基金优秀青年人才项目(2020YQ12);黑龙江省博士后科研启动资助项目(LBHQ20026);2021年哈尔滨商业大学教师“创新”项目支持计划项目。

摘　　要：目的研究石蒜碱对肝癌H22细胞荷瘤小鼠红细胞膜结构功能的影响。方法通过抑瘤率实验、生命延长率实验、流式细胞仪检测肿瘤细胞凋亡率实验,验证石蒜碱的体内抗肿瘤活性;采用试剂盒测定红细胞膜总蛋白、胆固醇和唾液酸含量,考察石蒜碱对H22荷瘤小鼠红细胞膜主要组分的影响;采用荧光偏振法检测红细胞膜流动性,荧光染色法检测红细胞膜封闭度,验证石蒜碱对H22荷瘤小鼠红细胞膜结构功能的影响;采用三磷酸腺苷(adeno sinetripho sphate,ATP)酶活性检测试剂盒、荧光分光光度计、激光共聚焦扫描显微镜、Western blotting法,检测石蒜碱对H22荷瘤小鼠红细胞膜离子通道相关因子活性的影响;采用Western blotting法检测石蒜碱对H22荷瘤小鼠肿瘤细胞内活化的半胱氨酸蛋白酶-3 (cleaved Caspase-3)、cleaved Caspase-9、B淋巴细胞瘤2 (B-cell lymphoma 2,Bcl-2)和Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)表达的影响。结果石蒜碱能够显著抑制H22荷瘤小鼠肿瘤生长、有效延长H22荷瘤小鼠生存时间,并且能够诱导H22荷瘤小鼠肿瘤细胞凋亡(P<0.05、0.01);石蒜碱能够增加H22荷瘤小鼠红细胞膜总蛋白、唾液酸和胆固醇含量,同时提高红细胞膜流动性和封闭度,增强H22荷瘤小鼠红细胞膜Na^(+),K^(+)-ATP、Ca^(2+),Mg^(2+)-ATP酶活性,升高H22荷瘤小鼠红细胞内pH值,降低红细胞内H+、Ca^(2+)、Cl^(-)浓度,增强H_(22)荷瘤小鼠红细胞对HCO^(3-)/Cl^(-)转运活性,升高Band3蛋白表达水平(P<0.01);石蒜碱能够升高H22荷瘤小鼠肿瘤细胞内cleaved Caspase-3、cleaved Caspase-9蛋白表达水平,升高促凋亡蛋白Bax表达、降低抑凋亡蛋白Bcl-2表达,使Bcl-2/Bax降低(P<0.01)。结论石蒜碱对H22荷瘤小鼠具有抗肿瘤作用,能够通过调控H22荷瘤小鼠红细胞膜组成结构,提高细胞膜离子通道的活性,在离子通道之间建立联系相互促进,通过这种相互促进作用来调节胞内酸Objective To study the effect of lycorine on structure and function of red blood cell membranes in H;tumor-bearing mice.Methods Anti-tumor activity of lycorine in vivo was verified by tumor inhibition rate experiment, life extension rate experiment, and tumor cells apoptosis rate test by flow cytometry. Contents of total protein, cholesterol and sialic acid of erythrocyte membrane were determined by assay kit to detect the effect of lycorine on content of main components of erythrocyte membrane in H;tumor-bearing mice. The erythrocyte membrane fluidity was detected by fluorescence polarization method, and erythrocyte membrane sealing degree was determined by Zamudio method to verify the effect of lycorine on erythrocyte membrane structure function of H_(22)tumor-bearing mice. Adenosine triphosphate(ATP) activity assay kit, fluorescence spectrophotometer, laser confocal scanning microscopy and Western blotting method were used to detect the effect of lycorine on erythrocyte membrane ion channel activity of H;tumor-bearing mice. Western blotting was used to detect the effects of lycorine on protein expressions of cleaved Caspase-3, cleaved Caspase-9, B-cell lymphoma 2(Bcl-2) and Bcl-2 associated X protein(Bax) in tumor cells of H;tumor-bearing mice. Results Lycorine inhibited tumor growth in H;tumor-bearing mice,prolonged the survival time of H;tumor-bearing mice, and induced tumor cell apoptosis in H;tumor-bearing mice(P < 0.05, 0.01);Lycorine increased erythrocyte membrane total protein, sialic acid and cholesterol contents in H;tumor-bearing mice, improved the fluidity and tightness of erythrocyte membrane, enhanced the erythrocyte membrane Na^(+), K^(+)-ATPase and Ca^(2+), Mg^(2+)-ATPase activity of H^(+)tumorbearing mice, increased the intracellular pH of H;tumor-bearing mice, decreased H;, Ca^(2+), Cl^(-)concentrations in red blood cells, enhanced the HCO^(3-)/Cl^(-)-transport activity of H;tumor-bearing mice, and increased Band 3 protein expression(P < 0.01). Lycorine increased the protein expressions of cle

关 键 词：石蒜碱 H_(22)荷瘤小鼠 红细胞免疫 细胞凋亡 抗肿瘤 

分 类 号：R285.5[医药卫生—中药学]