金丝桃素对小鼠巨噬细胞内刚地弓形虫速殖子的增殖抑制作用  

Inhibitory effect of hypericin on intracellular proliferation of Toxoplasma gondii in macrophages

作　　者：崔洁 王旗[4] 杨小迪 孙希萌[5] 汪瑞[2] 姜辉[2] 王守祥 张敏 魏明 夏惠 陈兴智[2] 沈继龙[1] CUI Jie1,2,3, WANG Qi4, YANG Xiao-di2, SUN Xi-meng5, WANG Rui2, JIANG Hui2, WANG Shou-xiang2, ZHANG Min2, WEI Ming2, XIA Hui2, CHEN Xing-zhi2, SHEN Ji-long1(1. Department of Parasitology, School of Basic Medicine, Anhui Medical University, Hefei 230032, China; 2. Department of Microbiology and Parasitology, Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu 233030, China; 3. Microscopic Morphology Integrated Laboratory, Bengbu Medical College, Bengbu 233030, China; 4. Anhui Provincial Institute of Schistosomiasis Control, Hefei 230061, China; 5. Department of Parasitology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China)

机构地区：[1]安徽医科大学基础医学院病原生物学教研室病原生物学安徽省重点实验室安徽省人兽共患病重点实验室(安徽医科大学),合肥230032 [2]蚌埠医学院病原生物学教研室安徽省感染与免疫重点实验室,蚌埠233030 [3]蚌埠医学院显微形态综合实验室,蚌埠233030 [4]安徽省血吸虫病防治研究所,合肥230061 [5]首都医科大学基础医学院人体寄生虫学教研室,北京100069

出　　处：《中国人兽共患病学报》2018年第7期583-587,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.81471983)资助;蚌埠医学院自然科学基金面上项目(No.BYKY1653)资助;安徽省大学生创新创业训练计划项目(No.201610367025)资助;安徽省血吸虫病防治研究所科研基金项目(No.1603)资助~~

摘　　要：目的观察金丝桃素(Hypericin,Hy)对小鼠巨噬细胞内弓形虫的增殖抑制作用,为抗弓形虫天然药物的筛选提供线索。方法分离小鼠腹腔巨噬细胞(Macrophage,Mφ),建立体外Mφ感染稳定表达绿色荧光蛋白的弓形虫(RH-green fluorescent protein,RH-GFP)速殖子模型,观察不同剂量Hy的最佳作用效果。实验分4组:(1)A组(对照组),不加Hy;(2)B组(Mφ感染RH-GFP+100μg/mL Hy);(3)C组(Mφ感染RH-GFP+200μg/mL Hy);(4)D组(Mφ感染RH-GFP+400μg/mL Hy),分别在培养1、2和3h后利用荧光显微镜和流式细胞术观察计数感染的Mφ和游离速殖子在各药物浓度及各时间点的变化;透射电镜观察虫体超微结构的变化。结果与对照组相比,随着Hy浓度的增加和用药时间的延长,Mφ内速殖子数量明显减少,游离速殖子数量也显著减少,荧光强度逐渐降低;经药物组不同浓度Hy作用后,游离/Mφ内弓形虫的比值逐渐升高,至400μg/mL剂量作用3h后开始下降,游离与胞内速殖子比例的差异在同一时间段内均有统计学意义(P<0.05),但药物作用1h或2h各实验组之间无统计学差异(P>0.05),作用3h各浓度组之间差异有统计学意义(P<0.05),各实验组同一药物浓度下在孵育1、2和3h后效果增强(P<0.05)。透射电镜观察显示,随Hy作用时间延长,虫体逐渐出现肿胀,胞膜与基质之间空隙明显,空泡形成且增多、变大,胞膜断裂,内部结构溶解溢出。结论体外实验初步显示Hy呈剂量和时间依赖性地增强对Mφ内弓形虫速殖子增殖的抑制作用,可降低宿主细胞感染率,且对胞外速殖子有一定杀伤作用。The suppressive effect of hypericin on the intracellular proliferation of Toxoplasma gondii （T.gondii） tachyzoites in mouse peritoneal macrophages was explored in this study. Macrophages were isolated and infected with T. gondii tachyzoites （type I, RH strain） expressing green fluorescent protein （GFP）. There were four groups： the control group （macrophages infected with RH-GFP without treatment, group A） and the other three groups （macrophages infected with RH-GFP treated with different concentrations of hypericin： group B, with 100 μg/mL; group C, with 200 μg/mL; and group D with 400 μg/mL）. Tachyzoites were collected after 1 hour, 2 hours, and 3 hours of treatment. The infected macrophages and the free parasites were observed under different concentrations of hypericin in various time intervals by fluorescence microscopy and flow cytometry. The ultrastucture of tachyzoites was also observed by transmission electron microscopy. Compared with the control group, the number and fluorescence intensity of tachyzoites were gradually decreased in a concentration and time dependent manner under the experimental condition. The ratio of free parasites to those within macrophages increased and reached the peak at the dosage of 400 μg/mL on 3 hours after treatment. With the extension of treatment duration, it was found that tachyzoites showed swelling and deformed; apparent gap appeared between cell membrane and matrix; more and bigger vacuoles were formed; the cell membrane became dissolved and disrupt, the inner structure overflowed. It’s suggested that the inhibition of hypericin on intracellular proliferation of T. gondii tachyzoites in macrophages is presented in a concentration and time dependent manner in vitro, meanwhile, a remarkable killing effect on free tachyzoites is also found.

关 键 词：金丝桃素 刚地弓形虫 巨噬细胞 抑制 

分 类 号：R382.5[医药卫生—医学寄生虫学]