全反式维甲酸对多房棘球蚴原头节体外活性及生长的影响  

Characteristics of genetic differentiation of Echinococcus multilocularis and E. shiquicus in Qinghai region

作　　者：葛宇飞 徐刚 张宏伟[1] 李江[1] 张永国[1] 孙红[1] 杨婧[1] 张示杰[2] GE Yufei;XU Gang;ZHANG Hongwei;LI Jiang;ZHANG Yongguo;SUN Hong;YANG Jing;ZHANG Shijie(First Affiliated Hospital of School of Medicine,Shihezi University,Shihezi 832000,Xinjiang,,China;Department of Hepatobiliary Surgery,First Affliated Hospital of School of Medicine,Shihezi University,Shihezi 832000,Xinjiang,China)

机构地区：[1]石河子大学第一附属医院,新疆石河子832000 [2]石河子大学第一附属医院肝胆外科,新疆石河子832000

出　　处：《中国寄生虫学与寄生虫病杂志》2024年第3期303-308,315,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(8176120052,81860363)。

摘　　要：目的 探究全反式维甲酸(ATRA)对多房棘球蚴原头节体外活性及生长的影响。方法 从保种沙鼠体内无菌剖取包囊,经过研磨、过滤、清洗获取活性良好的多房棘球蚴原头节。将原头节分为不同浓度ATRA组(10、25、50、100μmol/L组,添加对应终浓度的ATRA)、二甲基亚砜(DMSO)组(DMSO终浓度为0.1%)和空白组(加入等量完全培养基),共干预9 d,伊红染色后于显微镜下观察其活力和形态变化,计算原头节的存活率并绘制存活率曲线。将原头节与大鼠肝癌RH35细胞共培养5~6周,获得多房棘球蚴微囊泡后,使用不同终浓度ATRA (10、100μmol/L)干预微囊泡9 d,显微镜下观察其活力和形态变化,同时设置DMSO组和空白组。干预原头节48 h后,分别使用EdU细胞成像检测原头节EdU阳性率,使用半胱天冬氨酸蛋白酶-3 (Caspase-3)试剂盒检测原头节中Caspase-3相对表达量;干预原头节24 h后,活性氧(ROS)检测试剂盒测定各组原头节ROS水平。两组样品的比较采用独立样本t检验,不同浓度ATRA组与DMSO组差异比较采用单因素方差分析(ANOVA)。结果 10、25、50、100μmol/L组中的死亡原头节体积明显缩小,可被伊红染液染成红色,随着时间延长和药物浓度升高出现轮廓模糊、透光性减弱、小钩脱落增多等。第4天时,10、25、50、100μmol/L组原头节存活率分别为(87.33±4.90)%、(74.00±2.08)%、(64.33±2.03)%、(53.33±1.86)%,均低于DMSO组[(95.67±1.20)%](F=98.41,P <0.05);第9天时,10、25、50、100μmol/L组原头节存活率仅为(62.00±2.64)%、(36.33±2.52)%、(7.67±1.53)%、0,均低于DMSO组[(85.67±2.08)%](F=1 154.34,P <0.05)。不同浓度的ATRA与多房棘球蚴微囊泡共培养9 d时,随ATRA浓度的升高,囊泡生长缓慢,囊内结构逐渐浑浊;空白组和DMSO组的微囊泡生发层和角质层结构完整。EdU细胞成像可见不同浓度ATRA组均呈现红色及蓝色荧光,10、 25、 50、 100μmol/L组原头节的EdU阳性率分别为(51.63�Objective To analyze the genetic differentiation characteristics of Echinococcus multilocularis and E. shiquicus in Qinghai region to provide theoretical support for the prevention and control of echinococcosis in Qinghai Province.Methods Small mammals were captured in the main natural endemic areas of Echinococcus spp. in Yushu, Guoluo, and Huangnan Tibetan Autonomous Prefecture and were dissected to collect cysts. The genomic DNA from cysts tissue was extracted and the cytochrome oxidase 1(cox1) gene was amplified using PCR and sequenced.DnaSP v6, Iqtree, BEAST v2.7.4 and other software were used for haplotype analysis, nucleotide polymorphism analysis, construction of a phylogenetic tree, and estimation of the divergence time of the Echinococcus genus.Results A total of 55 hydatid cysts were obtained from 2 864 small mammals. All 55 cyst samples were amplified for cox1bands with a length of approximately 800 bp, of which 37 were E. multilocularis, and 18 were E. shiquicus, the prevalence of E. multilocularis in Neodon fuscus was 1.96%(37/1884). The prevalence of E. shiquicus in Ochotona curzoniae was 1.84%(18/980). In the 37 cox1 sequences of E. multilocularis, there were 5 haplotypes in the 37 cox1 sequences of E. multilocularis with EmH3 being the predominant one(33/37), the haplotype diversity index was 0.207, the nucleotide diversity index was 0.033 55, and there were 156 variable sites. In the 18 cox1 sequences of E. shiquicus, There were 8 haplotypes, with the EsH2 haplotype being the predominant one(8/18), the haplotype diversity index was 0.778,the nucleotide diversity index was 0.060 52, and there were 14 variable sites. Thirteen haplotypes of E. multilocularis and E. shiquensis were uploaded to GenBank. The accession numbers of haplotypes EmH1-EmH5 are OR821706,OR821707, OR830343, OR830344, OR826123, respectively. The accession numbers of haplotypes ESH1-ESH8 are OR835156, OR835157, OR830376, OR830378, OR831110, OR875250, OR835161, OR841080. The phylogenetic tree shows that the 5 haplotypes of E. mul

关 键 词：全反式维甲酸 多房棘球蚴原头节 增殖活性 凋亡 活性氧 

分 类 号：R383.33[医药卫生—医学寄生虫学]