蒿甲醚脂质体体外抑制刚地弓形虫增殖作用的研究  

Study on the inhibitory effect of artemether liposome on Toxoplasma gondii proliferation in vitro

作　　者：赵紫琪 吕芳丽 ZHAO Ziqi;LV Fangli(Artemisinin Research Center,Guangzhou University of Chinese Medicine,Guangzhou 510405,Guang-dong,China;Department of Clinical Laboratory,The Seventh Affiliated Hospital,Sun Yat-sen Univer sity,Department of Parasitology,School of Medicine,Sun Yat-sen University,Shenzhen 518107,Guang-dong,China;Department of Parasitology,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,Guangdong,China;Key Laboratory of Tropical Disease of Ministry of Education,Sun Yat-sen University,Guangzhou 510080,Guangdong,China)

机构地区：[1]广州中医药大学青蒿研究中心,广东广州510405 [2]中山大学附属第七医院检验科,中山大学医学院寄生虫学教研室,广东深圳518107 [3]中山大学中山医学院寄生虫学教研室,广东广州510080 [4]中山大学热带病防治研究教育部重点实验室,广东广州510080

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第4期446-451,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金面上项目(82272366,81971955);广东省自然科学基金面上项目(2021A1515012115,2019A1515011667);深圳市自然科学基金面上项目(JCYJ20220530145002006);广东省研究生教育创新计划项目(2021SFKC003);中山大学校级本科教学质量工程项目(中大教务〔2022〕91号、中大教务〔2022〕93号)。

摘　　要：目的探讨蒿甲醚脂质体(ARM‑Lip)体外抑制刚地弓形虫(简称弓形虫)速殖子增殖的作用。方法将含有1×104个人宫颈癌细胞置于96孔平底细胞培养板中培养,每孔100μl,设置不同浓度的ARM‑Lip组(3.125、6.250、12.500、25.000、50.000、100.000、200.000、400.000和800.000μmol/L),对照组(仅含1×10^(4)个人宫颈癌细胞)和空白组(仅含培养液),24 h后ARM‑Lip组每孔分别加入对应药物100μl,48 h后各孔均加入10μl CCK‑8溶液,1 h后使用酶标仪检测450 nm波长处的吸光度(A值),计算细胞存活率和ARM‑Lip的半数抑制浓度(IC50)。进一步采用稳定表达绿色荧光的RH株(RH‑GFP)弓形虫速殖子感染人宫颈癌细胞,与不同浓度的ARMLip进行共培育,筛选细胞存活率大于50%的安全浓度的ARM‑Lip组,同时设置浓度为400μmol/L的磺胺嘧啶(SDZ)组和未加药组。荧光显微镜观察24、48和72 h后速殖子的增殖情况,测量共培养48 h时各组平均荧光灰度值,选取最佳浓度ARM‑Lip组。吉氏染色后,光学显微镜下观察各组细胞内的纳虫空泡和弓形虫速殖子的生长情况并计数。组间比较采用单因素方差分析,两两比较采用最小显著性差异法检验。结果ARM‑Lip在25μmol/L或以下浓度对人宫颈癌细胞几乎无毒性,IC50为285.1μmol/L。经100.000、200.000、400.000和800.000μmol/L的ARMLip处理后,平均细胞存活率分别为89.03%、76.30%、42.12%和27.85%,选取6.250、12.500、25.000、50.000和100.000μmol/L等5个浓度进行后续实验。荧光显微镜下可见未加药组弓形虫RH‑GFP株速殖子大量增殖,形态正常,呈现高强度的绿色荧光;随着ARM‑Lip浓度的增加,绿色荧光强度逐渐减弱。ARM‑Lip体外抑制弓形虫繁殖的最佳作用浓度为100.000μmol/L,最佳作用时间为48 h。未加药组、ARM‑Lip组(100.000μmol/L)和SDZ组作用48 h后,荧光灰度值分别为89.92±1.12、35.74±1.55和7.34±0.60(F=7916.301,P<0.01),ARM‑Lip组和SDZ组的平均�Objective To investigate the inhibitory effect of artemether liposome(ARM⁃Lip)on Toxoplasma gondii tachyzoite proliferation in vitro.Methods Human cervical cancer cells(Hela cells)were cultured in 96⁃well flat bottom cell culture plate,with 100μl cell culture medium in each well containing 1×104 Hela cells.Setting ARMLip groups with different concentrations(3.125,6.250,12.500,25.000,50.000,100.000,200.000,400.000 and 800.000μmol/L),a control group(only containing 1×104 human cervical cancer cells),and a blank group(only containing culture medium).A total of 100μl corresponding drugs were added to each well in the ARM‑Lip groups after 24 h,and 10μl CCK⁃8 solution was added to each well after 48 h.A microplate reader was used to detect the absorbance at 450 nm wavelength after 1 h(A‑value)to calculate the cell survival rate and 50%inhibitory concentration(IC50)of ARM⁃Lip.Hela cells were infected with T.gondii green fluorescence⁃expressing RH(RH⁃GFP)straintachyzoites,and co⁃cultivated with different concentrations of ARM‑Lip to screen safe concentrations of ARM⁃Lip those rendered higher than 50%cell survival rate.Through setting of 400μmol/L sulfadiazine(SDZ)group,and untreated group,the proliferations of tachyzoites were observed under fluorescence microscope after cultured for 24,48,and 72 h.The average fluorescence gray scale values of each group at 48 h were measured and the group having optimal concentration of ARM⁃Lip was selected.After Giemsa staining,the parasitophorous vacuoles in Hela cells and the growth of T.gondii tachyzoites were observed and counted.One‑way ANOVA was used for inter⁃group comparison,and the least significant difference(LSD)test was used for pairwise comparison.Results ARM⁃Lip at a concentration of 25μmol/L or lower had little toxic effect on Hela cells,and its IC50 was 285.1μmol/L.Treated with 100.000,200.000,400.000,and 800.000μmol/L ARM‑Lip,the cell survival rate was 89.03%,76.30%,42.12%and 27.85%,respectively.ARM⁃Lip concentrations of 6.25

关 键 词：刚地弓形虫 速殖子 蒿甲醚脂质体 人宫颈癌细胞 纳虫空泡 

分 类 号：R382.5[医药卫生—医学寄生虫学]