白鲜碱对小鼠脾淋巴细胞活力的体外抑制作用及机制研究  

Study on the Viability Inhibitory Effects and Mechanism of Dictamnine on Mice Spleen Lymphocyte in vitro

作　　者：丛欢[1] 杨莹[1] 包亚男[1] 洪博[1] 郭丽娜[1] CONG Huan;YANG Ying;BAO Yanan;HONG Bo;GUO Lina(College of Pharmacy,Qiqihar Medical University,Heilongjiang Qiqihar 161006,China)

机构地区：[1]齐齐哈尔医学院药学院

出　　处：《中国药房》2019年第21期2963-2967,共5页China Pharmacy

基　　金：黑龙江省教育厅基本科研业务费项目（No.2016-KYYWF-0862）

摘　　要：目的:研究白鲜碱对小鼠脾淋巴细胞活力的体外抑制作用并探讨其可能的作用机制。方法:分离并培养小鼠原代脾淋巴细胞,以0(空白对照)、50、100、150μmol/L白鲜碱作用细胞24 h后,采用MTT法检测细胞活力,乳酸脱氢酶(LDH)法检测细胞LDH释放率,流式细胞术检测细胞早期凋亡率,Hoechst 33342和碘化丙啶(PI)双染法检测细胞坏死率,Western blot法检测细胞中胱天蛋白酶3(Caspase 3)、剪切体Caspase 3(Cleaved-Caspase 3)蛋白表达水平以及彗星试验法检测细胞DNA损伤(以DNA拖尾区域比例反映)。结果:与空白对照比较,100、150μmol/L白鲜碱可显著抑制细胞活力(P<0.01);150μmol/L白鲜碱可显著增加细胞LDH的释放(P<0.05),释放率达到79.37%;50、100、150μmol/L白鲜碱均可提高细胞的早期凋亡率,但差异均无统计学意义(P>0.05);150μmol/L白鲜碱可显著增加细胞坏死率(P<0.05),坏死率达到78.64%;50、100、150μmol/L白鲜碱均可升高Caspase 3蛋白表达水平,但差异均无统计学意义(P>0.05),然而50、100μmol/L白鲜碱可显著提高Cleaved-Caspase 3蛋白表达水平(P<0.05);白鲜碱可呈剂量依赖性地引起DNA损伤,其中100、150μmol/L白鲜碱可显著增加DNA拖尾区域比例(P<0.01)。结论:白鲜碱可以抑制脾淋巴细胞活力,其作用机制可能与诱导脾淋巴细胞坏死和造成DNA损伤有关。OBJECTIVE:To study the inhibitory effect of dictamnine on the viability of mice spleen lymphocyte in vitro and explore its potential mechanism.METHODS:The primary spleen lymphocytes of mice were isolated and cultured.The cells were treated with 0(blank control),50,100,150μmol/L dictamnine for 24 h.MTT assay was used to determine the cell viability;Lactic dehydrogenase(LDH)assay was used to determine release rate of LDH.Early apoptosis rate was detected by flow cytometry.Necrosis rate was detected by Hoechst 33342 and PI double staining;Western blot assay was used to detect the protein expressions of Caspase 3 and Cleaved-Caspase 3 in cells.Comet assay was used to detect DNA damage in cells(reflected in the proportion of DNA tail area).RESULTS:Compared with blank control,100,150μmol/L dictamnine could significantly inhibit the viability of lymphocytes(P<0.01).150μmol/L dictamnine could significantly increase the release of LDH(P<0.05),and release rate reached 79.37%.50,100,150μmol/L dictamnine could improve the early apoptotic rate of lymphocyte,but there was no statistical significance(P>0.05).150μmol/L dictamnine could significantly increase the necrosis rate(P<0.05),and necrosis rate reached 78.64%.50,100,150μmol/L dictamnine could increase the protein expression of Caspase 3,but there was no statistical significance(P>0.05),while 50,100μmol/L dictamnine could improve the protein expression of Cleaved-Caspase 3 significantly(P<0.05).DNA damage was induced in a dose-dependent manner by dictamnine,in which 100 and 150μmol/L dictamnine could significantly increase DNA tail area(P<0.01).CONCLUSIONS:Dictamnine can inhibit spleen lymphocyte viability,and the mechanism may be related to inducing spleen lymphocyte necrosis and DNA damage.

关 键 词：白鲜碱 脾淋巴细胞 凋亡 坏死 DNA损伤 体外试验 

分 类 号：R285[医药卫生—中药学] R992[医药卫生—中医学]