阿霉素体外抗细粒棘球蚴原头节作用研究

doi:10.12140/j.issn.1000-7423.2019.05.011

中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (5): 571-576.doi: 10.12140/j.issn.1000-7423.2019.05.011

阿霉素体外抗细粒棘球蚴原头节作用研究

田春艳¹(), 陈蓓^2,³, 卢帅^2,³, 文丽梅^2,³, 赵军^2,³, 郑璇¹, 库尔班泥沙·阿马洪¹, 高旖¹, 王建华^2,^3,^*()

1 新疆医科大学 1药学院
2 第一附属医院药学部
3 第一附属医院,省部共建中亚高发病成因与防治国家重点实验室,乌鲁木齐 830054

收稿日期:2019-03-25 出版日期:2019-10-30 发布日期:2019-11-07
通讯作者: 王建华
作者简介:
作者简介：田春艳（1993-）,女,硕士,从事棘球蚴病新药及作用机制研究。E-mail：tcy1416@163.com
基金资助:
国家自然科学基金（No. 81860666,No. 81560607）;省部共建中亚高发病成因与防治国家重点实验室开放课资助项目（No. SKL-HIDCA-2017-11,No. SKL-HIDCA-2017-Y7）;新疆维吾尔自治区药学会项目（No. YXH201704）

Toxicity of doxorubicin on Echinococcus granulosus protoscoleces in vitro

Chun-yan TIAN¹(), Bei CHEN^2,³, Shuai LU^2,³, Li-mei WEN^2,³, Jun ZHAO^2,³, Xuan ZHENG¹, Kuerbannisha·AMAHONG¹, Yi GAO¹, Jian-hua WANG^2,^3,^*()

1 College of Pharmaceutical Sciences, Xinjiang Medical University, Urumqi 830054, China
2 Department of Pharmacy, The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054, China
3 The State Key Laboratory of Pathogenesis, Prevention and Treatment of Diseases Prevalent in Central Asia, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China

Received:2019-03-25 Online:2019-10-30 Published:2019-11-07
Contact: Jian-hua WANG
Supported by:
Supported by the National Natural Science Foundation of China (No. 81860666, No. 81560607), the Ministry-Provincial Cooperation for the Establishment of the State Key Laboratory on the Causes and Control of High Incidence of Disease in Central Asia (No. SKL-HIDCA-2017-11, No. SKL-HIDCA-2017-Y7) and the Project of Xinjiang Uygur Autonomous Region Medical Association (No. YXH201704)

摘要/Abstract

摘要：

目的研究阿霉素（Dox）对细粒棘球蚴原头节的体外抑制作用。方法 4 800个原头节随机分为空白对照组、1% DMSO组、Dox组（25、50、100、200、400、600 μmol/L）,每组200个原头节,设3个平行组,体外干预24 h。伊红拒染实验检测Dox干预后原头节活力,统计存活率。扫描电镜观察Dox干预后原头节表面超微结构变化。单细胞凝胶电泳实验检测Dox干预后原头节DNA损伤程度。实时荧光定量PCR（qRT-PCR）检测Dox干预后DNA损伤相关基因共济失调毛细血管扩张症突变激酶（ATM）、p53、DNA拓扑异构酶2A（Topo2a）的mRNA表达水平。结果伊红拒染实验结果显示,空白对照组、1% DMSO组、25、50、100、200、400、600 μmol/L Dox组细粒棘球蚴原头节的存活率分别为（99.0 ± 0.5）%、（98.6 ± 0.3）%、（96.0 ± 1.4）%、（80.3 ± 4.8）%、（75.6 ± 6.2）%、（53.2 ± 3.0）%、（26.4 ± 8.1）%和0,IC₅₀为（267.9 ± 7.1）μmol/L。与空白对照组相比,1% DMSO组、25 μmol/L Dox组原头节存活率差异无统计学意义（P > 0.05）,50、100、200、400、600 μmol/L Dox组原头节存活率差异有统计学意义（P < 0.01）。扫描电镜结果显示,空白对照组、1% DMSO组原头节表面光滑,头节呈球形或椭圆形,顶突完整,微毛清晰可见;400 μmol/L Dox组原头节吸盘变形,体部塌陷严重,顶突轻微变形,微毛明显脱落,虫体表层发生皱缩。单细胞凝胶电泳实验结果显示,100 μmol/L Dox组原头节出现拖尾现象,彗星尾距（OTM）为16.6 ± 1.7,与空白对照组（0.1 ± 0.0）相比,差异有统计学意义（P < 0.01）。qRT-PCR结果显示,1% DMSO组原头节ATM、p53和Topo2a mRNA相对表达量分别为1.0 ± 0.1、1.1 ± 0.1、1.3 ± 0.9,与空白对照组（1.0 ± 0.1、1.0 ± 0.4、1.0 ± 0.1）相比,差异无统计学意义（P > 0.05）。100 μmol/L Dox组原头节ATM、p53和Topo2a mRNA相对表达量分别为38.6 ± 3.5、10.0 ± 2.5、54.0 ± 0.8,与空白对照组相比,差异有统计学意义（P < 0.05）。结论 Dox具有体外抗细粒棘球蚴原头节作用,可引起虫体DNA损伤,可能与ATM、p53、Topo2a的mRNA相对表达量变化相关。

关键词: 阿霉素, 细粒棘球蚴原头节, DNA损伤, 共济失调毛细血管扩张症突变激酶, p53, Topo2a

Abstract:

Objective To evaluate the toxicity of doxorubicin (Dox) on Echinococcus granulosus protoscoleces in vitro. Methods Total 200 E. granulosus protoscoleces in each group were incubated with different concentration of doxorubicin (25, 50, 100, 200, 400, 600 μmol/L) in triplicate for 24 hours in vitro. Other two groups were incubated with medium(blank) or 1% DMSO controls. The viability of protoscoleces was determined by eosin staining and survival rate was calculated. The structural damage of treated protoscoleces was observed under scanning electron microscope (SEM). Single cell gel electrophoresis was used to detect DNA damage in treated protoscoleces. Real-time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of ataxia telangiectasia mutated gene (ATM), p53 and DNA topoisomerase 2-alpha (Topo2a) in treated protoscoleces. Results Based on the eosin dye staining, the survival rates of treated protoscoleces were (96.0 ± 1.4)%, (80.3 ± 4.8)%, (75.6 ± 6.2)%, (53.2 ± 3.0)%, (26.4 ± 8.1)% and 0 when the concentrations of Dox were 25, 50, 100, 200, 400, 600 μmol/L, respectively, compared to that of protoscoleces incubated with blank control [(99.0 ± 0.5)%] and 1% DMSO (98.6 ± 0.3)%], with significant difference when incubated concentration is over 25 μmol/L (P < 0.01). The Dox IC₅₀ on protoscoleces was (267.92 ± 7.14) μmol/L. SEM results showed that E. granulosus protoscoleces in the blank control group and 1% DMSO group had smooth surface with a spherical or elliptic shaped scolex and complete apical structure. There were clearly visible microtricles on the surface. When incubated with 400 μmol/L Dox, the protoscoleces scolex was distorted and damaged and whole body collapsed. The apical structure slightly distorted. The microtriches on the surface were lost obviously. The body surface was shrinked. The single cell gel electrophoresis showed trailed cell when the concentration of Dox reached to 100 μmol/L, indicating the cell damage. The olive tail moment (OTM) was 16.6 ± 1.7 for cells treated with 100 μmol/L of Dox, which was significantly longer compared with the blank control group(0.1 ± 0.0) (P < 0.01). The qRT-PCR results showed that the relative expressions of ATM, p53 and Topo2a mRNA in 1% DMSO group were 1.0 ± 0.1, 1.1 ± 0.1 and 1.3 ± 0.9, respectively, with no statistical significance compared with the blank control group (1.0 ± 0.1, 1.0 ± 0.4, 1.0 ± 0.1, respectively) (P > 0.05). However, the relative expression levels of ATM, p53 and Topo2a mRNA in Dox group were 38.6 ± 3.5, 10.0 ± 2.5 and 54.0 ± 0.8, respectively, which were significantly increased compared with the blank control group (P < 0.05). Conclusion Dox has toxicity on the protoscoleces of E. granuloides in vitro, leading to cell and DNA damage and rpossibly related to the mRNA expression of of ATM, p53 and Topo2a.

Key words: Doxorubicin, Echinococcus granulosus protoscoleces, DNA damage, Ataxia telangiectasia mutated, p53, Topo2a

中图分类号:

R383.33

田春艳, 陈蓓, 卢帅, 文丽梅, 赵军, 郑璇, 库尔班泥沙·阿马洪, 高旖, 王建华. 阿霉素体外抗细粒棘球蚴原头节作用研究[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(5): 571-576.

Chun-yan TIAN, Bei CHEN, Shuai LU, Li-mei WEN, Jun ZHAO, Xuan ZHENG, Kuerbannisha·AMAHONG, Yi GAO, Jian-hua WANG. Toxicity of doxorubicin on Echinococcus granulosus protoscoleces in vitro[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(5): 571-576.

图/表 5

表1

图1

图2

图3

图4

参考文献 25

[1]	Tanki H, Singh H, Raswan US, et al. Pediatric intracranial hydatid cyst: a series with literature review[J]. Pediatr Neurosurg, 2018, 53(5): 299-304.
[2]	庞楠楠, 安梦婷, 张峰波, 等. 细粒棘球蚴感染中TGF-β/Smad信号通路对IL-9调控作用的研究[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(5): 460-463, 468.
[3]	刘平, 李金花, 李印, 等. 包虫病病原在我国的流行现状及成因分析[J]. 中国动物检疫, 2016, 33(1): 48-51.
[4]	Fadel SA, Asmar K, Faraj W, et al. Clinical review of liver hydatid disease and its unusual presentations in developing countries[J]. Abdom Radiol (NY), 2019, 44(4): 1331-1339.
[5]	张梦媛, 伍卫平, 官亚宜, 等. 我国棘球蚴病疾病负担分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 15-19, 25.
[6]	伍卫平, 王虎, 王谦, 等. 2012-2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 1-14.
[7]	王永珍, 韩秀敏, 郭亚民. 肝包虫病的诊断与治疗研究进展[J]. 寄生虫病与感染性疾病, 2018, 16(1): 47-51.
[8]	Goja S, Saha SK, Yadav SK, et al. Surgical approaches to hepatic hydatidosis ranging from partial cystectomy to liver transplantation[J]. Ann Hepatobiliary Pancreat Surg, 2018, 22(3): 208-215.
[9]	Das AK.Anticancer effect of antimalarial artemisinin compounds[J]. Ann Med Health Sci Res, 2015, 5(2): 93-102.
[10]	Ashley EA, Dhorda M, Fairhurst RM, et al. Spread of artemisinin resistance in Plasmodium falciparum malaria[J]. N Engl J Med, 2014, 371(5): 411-423.
[11]	魏晗. 新剂型阿苯达唑乳剂对肝囊型包虫病患者的疗效观察[J]. 临床医药文献电子杂志, 2016, 3(10): 1977-1977, 1980.
[12]	高惠静, 陈蓓, 张海波, 等. 阿苯达唑纳米脂质体冻干粉在大鼠体内药动学及肝靶向研究[J]. 中国医院药学杂志, 2017, 37(8): 702-706.
[13]	阿卜来海提·麦提色依提. 阿苯达唑壳聚糖微球抗小鼠泡球蚴药效实验研究[D]. 石河子: 石河子大学, 2015.
[14]	李亚芬, 赵军, 吕国栋, 等. Veliparib联合青蒿琥酯体外抗细粒棘球蚴作用机制的研究[J]. 中国病原生物学杂志, 2018, 13(7): 733-739.
[15]	史红娟, 吕海龙, 雷颖, 等. 布洛芬抑制体外细粒棘球蚴原头节生长的实验研究[J]. 中国病原生物学杂志, 2016, 11(3): 220-224.
[16]	郑璇, 卢帅, 赵军, 等. siRNA特异性干扰EgRad9基因表达对细粒棘球蚴原头节DNA氧化损伤机制的影响[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 343-349.
[17]	郑海亚, 文丽梅, 吕国栋, 等. 青蒿琥酯体外抗细粒棘球蚴活性氧对DNA作用机制影响的研究[J]. 中国病原生物学杂志, 2017, 12(8): 751-761.
[18]	刘晓, 邵方元, 陈宏远. 阿霉素抗肿瘤分子机制的研究进展[J]. 中国医药生物技术, 2012, 7(5): 373-375.
[19]	Deterding A, Dungey FA, Thompson KA, et al. Anti-trypanosomal activities of DNA topoisomerase inhibitors[J]. Acta Tropica, 2005, 93(3): 311-316.
[20]	相琳琳. EZH2在电离辐射诱导Hep3B细胞DNA损伤修复中对ATM/p53通路的调控作用研究[D]. 长春: 吉林大学, 2018.
[21]	Harper JW, Elledge SJ.The DNA damage response: ten years after[J]. Mol Cell, 2007, 28(5): 739-745.
[22]	Yeo CQX, Alexander I, Lin ZR, et al. P53 maintains genomic stability by preventing interference between transcription and replication[J]. Cell Rep, 2016, 15(1): 132-146.
[23]	赵芳, 徐斌, 蒋敬庭, 等. P53在肿瘤微环境、细胞代谢中的调控机制及检测技术的研究进展[J]. 临床检验杂志, 2017, 35(8): 621-623.
[24]	江阔. 辐射诱导DNA损伤修复中EZH2与ATM调控作用的研究[D]. 长春: 吉林大学, 2018.
[25]	李晶, 汪竹, 袁昕, 等. Ki67和TOPOⅡα在胃癌中的表达及临床意义[J]. 现代肿瘤医学, 2015, 23(5): 666-668.

基因 Gene	引物序列（5′→3′） Primer sequences（5′→3′）	退火温度/℃ Tm/℃	长度/bp Length/bp
ATM	F：GTTCCTACAGTCCATCCTAAT	73.3	146
	R：CTCCATCAAGCCAGCATT
p53	F：AACCACCGAACTCACAAC	78.2	216
	R：AACCGACACAACTCATCAA
Topo2a	F：ACCTTCCTCGCCAACTAT	82.4	256
	R：GTCTGTCTCCTCCATCAAC
β-actin	F：GCGATGTATGTAGCTATCCAGGC- AGTGCTCTCGCT	81.5	431
	R：CAATCCAGACAGAGTATTTGCGT- TCCGGAGGA

阿霉素体外抗细粒棘球蚴原头节作用研究

Toxicity of doxorubicin on Echinococcus granulosus protoscoleces in vitro

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 25

相关文章 3

编辑推荐

Metrics

本文评价

[1]	郑璇, 卢帅, 赵军, 吕国栋, 文丽梅, 李亚芬, 田春艳, 王建华. siRNA特异性干扰EgRad9基因表达对细粒棘球蚴原头节DNA氧化损伤机制的影响[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 343-349.
[2]	辛奇, 高海军, 宋晓霞, 孙旭东, 吕薇, NabilPERVAIZ, 鲁俊, 景涛. 氯舒隆和奥硝唑体外抗细粒棘球蚴原头节和多房棘球蚴的效果评价[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 352-356.
[3]	罗强1，孙黎1 *，田青青1，任鸿2，刘华1，杨翠军1，李鑫1. 刚地弓形虫速殖子培养上清液对人胃癌细胞BGC-823增殖和凋亡的影响[J]. 中国寄生虫学与寄生虫病杂志, 2014, 32(2): 8-123-127.