吡喹酮压力下日本血吸虫耐药性的诱导和虫体差异表达蛋白的分析  

Drug resistance induction and analysis of differential expression protein on adult Schistosoma japonicuminduced by ED_(50) PZQ

作　　者：董兰兰[1] 许静[1] 赵波[1] 梁松[1] 王言言[1] 关志勋 曹蕴[1] 夏超明[1] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院病原生物学系,苏州215123

出　　处：《中国人兽共患病学报》2014年第12期1171-1180,共10页Chinese Journal of Zoonoses

基　　金：国家863项目(No.2012AA0203006)~~

摘　　要：目的分析经吡喹酮(PZQ)半数有效量(ED50)诱导虫体与未诱导虫体之间的差异表达蛋白,为阐明PZQ的作用机制,探索候选疫苗靶抗原及药物治疗靶点,提供实验依据。方法应用PZQ ED50(25.98mg/kg),对单性感染4周后ICR鼠连续灌胃给药30d,停药21d后,再给予治疗剂量(200mg/kg)连续灌胃5d,肝门静脉灌注法收集成虫体外培养,观察虫体对PZQ的敏感性。收集诱导虫体和未诱导虫体,利用2D-DIGE分离差异蛋白,并予MALDI-TOF-MS鉴定,进而在线Uniprot软件检索差异蛋白功能。结果诱导成虫在8倍未诱导成虫临界致死浓度(112μmol/L)PZQ作用下,仍有75.6%存活率,与未诱导成虫临界致死浓度组相比差异有统计学意义(P<0.05)。诱导虫体和未诱导虫体共确认有30个差异表达蛋白,其中12个蛋白表达上调,18个蛋白表达下调,主要是细胞骨架相关蛋白、细胞中参与糖代谢和能量代谢的酶类、氧化还原酶类以及应激蛋白和参与解毒代谢的蛋白酶等。结论诱导虫体对PZQ敏感性下降显著,显示出明显的耐受性。诱导后,蛋白表达上调或下调提示PZQ诱导促进或抑制了某些特定基因的表达。为深入阐明PZQ作用机制,探索新的疫苗候选抗原及药物治疗的靶点,提供科学依据,同时为研发抗血吸虫新药开拓新途径和新思路。In the present study, we aimed to identify differentially expressed proteins between induced worms （the infec- ted mice were treated intragastrically with EDs0 PZQ） and uninduced worms （control group） for clarifying the mechanism of PZQ. EDs0 PZQ was used to administrate mice that were infected with S. japonicum via intragastric incubation for consecutive- ly 30 days. Twenty-one days later, mice were sacrificed after treatment with 200 mg/kg PZQ for continuously five days, and the male worms were obtained and some of them were subjected in DMEM medium with different concentrations of PZQ in vitro for 16 hours. Then the worms were washed twice and incubated in PZQ-free medium for 72 hours. Compared with control group, the induced worms had lesser sensitivity to PZQ. The survival rate of induced worms was 75.6% in vitro when the con- centration of PZQ was 112 mol/L （the concentration was 8 times of uninduced worms Lethal Concentration）, significantly higher than that in the uninduced worms （11.1%, P〈0.05）, showing obviously tolerance. The other induced and uninduced worms were acquired and collected for 2D-DIGE and MALDI-TOF-MS, and combined with bioinformatics to analyse the func- tion of the identified protein. Thirty differential expression proteins were confirmed between induced and uninduced worms, in- cluding 12 proteins up-regulated and 18 proteins down-regulated. These proteins respectively ascribed to cytoskeleton-associat- ed protein, glucose and energy metabolism enzymes, stress proteins, thioredoxin peroxidase enzymes, and other protease. Up- or down-regulation of these differential proteins indicated that PZQ promote or inhibit the expression of some specific genes. These findings may help to clarify the mechanism of PZQ, simultaneously, providing a scientific basis for exploring new vaccine candidate antigens and targets for drug therapy.

关 键 词：日本血吸虫 吡喹酮 ED50 耐药性 蛋白质组学 差异表达 

分 类 号：R383.2[医药卫生—医学寄生虫学]