蒿甲醚脂质体体外抑制刚地弓形虫增殖作用的研究

doi:10.12140/j.issn.1000-7423.2023.04.008

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (4): 446-451.doi: 10.12140/j.issn.1000-7423.2023.04.008

蒿甲醚脂质体体外抑制刚地弓形虫增殖作用的研究

赵紫琪¹(), 吕芳丽²^,³^,⁴^,^*()

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

收稿日期:2023-01-29 修回日期:2023-04-18 出版日期:2023-08-30 发布日期:2023-09-06
通讯作者: *吕芳丽，女，博士，教授，从事弓形虫感染免疫研究。E-mail：lvfangli@mail.sysu.edu.cn
作者简介:赵紫琪（1996-），女，硕士研究生，从事弓形虫病防治研究。E-mail：843377801@qq.com
基金资助:
国家自然科学基金面上项目(82272366);国家自然科学基金面上项目(81971955);广东省自然科学基金面上项目(2021A1515012115);广东省自然科学基金面上项目(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(SYSU Undergraduate Education [2022]91);中山大学校级本科教学质量工程项目(SYSU Undergraduate Education [2022]93)

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

ZHAO Ziqi¹(), LV Fangli²^,³^,⁴^,^*()

1 Artemisinin Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong, China
2 Department of Clinical Laboratory, The Seventh Affiliated Hospital, Sun Yat-sen University; Department of Parasitology, School of Medicine,Sun Yat-sen University, Shenzhen 518107, Guangdong, China
3 Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, Guangdong, China
4 Key Laboratory of Tropical Disease of Ministry of Education, Sun Yat-sen University, Guangzhou 510080, Guangdong, China

Received:2023-01-29 Revised:2023-04-18 Online:2023-08-30 Published:2023-09-06
Contact: *E-mail: lvfangli@mail.sysu.edu.cn
Supported by:
National Natural Science Foundation of China(82272366);National Natural Science Foundation of China(81971955);Natural Science Foundation of Guangdong Province(2021A1515012115);Natural Science Foundation of Guangdong Province(2019A1515011667);Shenzhen Municipal Natural Science Foundation(JCYJ20220530145002006);Graduate Education Innovation Plan Project of Guangdong Province(2021SFKC003);Undergraduate Teaching Quality Engineering Project of Sun Yat-sen University, China(SYSU Undergraduate Education [2022]91);Undergraduate Teaching Quality Engineering Project of Sun Yat-sen University, China(SYSU Undergraduate Education [2022]93)

摘要/Abstract

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100 μl，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800.000 μmol/L），对照组（仅含1 × 10⁴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100 μl，48 h后各孔均加入10 μl 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ARM-Lip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400 μmol/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25 μmol/L或以下浓度对人宫颈癌细胞几乎无毒性，IC₅₀为285.1 μmol/L。经100.000、200.000、400.000和800.000 μmol/L的ARM-Lip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100.000 μmol/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100.000 μmol/L，最佳作用时间为48 h。未加药组、ARM-Lip组（100.000 μmol/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 = 7 916.301，P < 0.01），ARM-Lip组和SDZ组的平均灰度值相比未加药组均下降（t = 81.247、123.831，均P < 0.01），SDZ组低于ARM-Lip组（t = -42.584，P < 0.01）。经吉氏染色，未加药组、ARM-Lip组和SDZ组的纳虫空泡数分别为（28.667 ± 2.658）、（16.667 ± 0.817）和（12.667 ± 1.211）个（F = 135.652，P < 0.01），弓形虫RH-GFP株速殖子数分别为（176.167 ± 13.273）、（105.333 ± 7.789）和（70.167 ± 5.947）个（F = 192.763，P < 0.01），ARM-Lip组和SDZ组纳虫空泡数量相比未加药组均减少（t = 0.083、0.063，均P < 0.01），弓形虫RH-GFP株速殖子数量也减少（t = 0.014、0.009，均P < 0.01），SDZ组的纳虫空泡和速殖子数量均少于ARM-Lip组（t = -0.500、-0.057，均P < 0.01）。结论 ARM-Lip具有较好的安全性，在体外可抑制弓形虫速殖子的增殖，但效果不及SDZ。

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

Abstract:

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 100 μl cell culture medium in each well containing 1 × 10⁴ Hela cells. Setting ARM-Lip groups with different concentrations (3.125, 6.250, 12.500, 25.000, 50.000, 100.000, 200.000, 400.000 and 800.000 μmol/L), a control group (only containing 1 × 10⁴ human cervical cancer cells), and a blank group (only containing culture medium). A total of 100 μl corresponding drugs were added to each well in the ARM-Lip groups after 24 h, and 10 μl 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 400 μmol/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 25 μmol/L or lower had little toxic effect on Hela cells, and its IC₅₀ was 285.1 μmol/L. Treated with 100.000, 200.000, 400.000, and 800.000 μmol/L ARM-Lip, the cell survival rate was 89.03%, 76.30%, 42.12% and 27.85%, respectively. ARM-Lip concentrations of 6.250, 12.500, 25.000, 50.000, and 100.000 μmol/L were selected to conduct subsequent experiments. Under a fluorescence microscope, a large amount of T. gondii RH-GFP strain tachyzoites were observed in the untreated group, with normal morphology and high intensity of green fluorescence. As the concentration of ARM-Lip was increased, the intensity of green fluorescence was gradually decreased. The optimal concentration for ARM-Lip to inhibit T. gondii growth in vitro was 100.000 μmol/L and the optimal action time was 48 h. After cultured for 48 h, the average gray scale values of the untreated group, ARM-Lip group (100.000 μmol/L) and SDZ group were 89.92 ± 1.12, 35.74 ± 1.55 and 7.34 ± 0.60 (F = 7 916.301, P < 0.01). Compared with the untreated control group, the average grey scale values of the ARM-Lip group and SDZ group were significantly decreased (t = 81.247, 123.831, both P < 0.01); compared with the ARM-Lip group, the average gray scale value of the SDZ group was significantly decreased (t = -42.584, P < 0.01). After Giemsa staining, the numbers of parasitophorous vacuoles of untreated group, ARM-Lip group, and SDZ group were 28.667 ± 2.658, 16.667 ± 0.817 and 12.667 ± 1.211 (F = 135.652, P < 0.01); the numbers T. gondii tachyzoites in each group were 176.167 ± 13.273, 105.333 ± 7.789 and 70.167 ± 5.947 (F = 192.763, P < 0.01). Compared with the untreated control group, the numbers of parasitophorous vacuoles of ARM-Lip group and SDZ group were significantly decreased (t = 0.083, 0.063, both P < 0.01), and the numbers of T. gondii tachyzoites of ARM-Lip group and SDZ group were significantly decreased (t = 0.014, 0.009, both P < 0.01). The numbers of parasitophorous vacuoles and T. gondii tachyzoites in the SDZ group were significantly fewer than in the ARM-Lip group (t = -0.500, -0.057, both P < 0.01). Conclusion ARM-Lip have relatively good safety and can inhibit the proliferation of T. gondii tachyzoites in vitro, whereas its effectiveness remains insufficient when compared with SDZ.

Key words: Toxoplasma gondii, Tachyzoite, Artemether liposome, Hela cell, Parasitophorous vacuole

中图分类号:

R382.5

赵紫琪, 吕芳丽. 蒿甲醚脂质体体外抑制刚地弓形虫增殖作用的研究[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 446-451.

ZHAO Ziqi, LV Fangli. Study on the inhibitory effect of artemether liposome on Toxoplasma gondii proliferation in vitro[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(4): 446-451.

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参考文献 19

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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

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