共吡喹酮衍生物抗日本血吸虫生物学效应及吡喹酮耐药虫体差异表达蛋白的筛选

发布时间：2018-06-15 01:35

本文选题：日本血吸虫 + PZQ　；参考：《苏州大学》2014年硕士论文

【摘要】：第一部分吡喹酮衍生物DW-3-15、P96及P96异构体体外抗日本血吸虫虫期及性别特异性生物学效应目的：观察两种吡喹酮衍生物DW-3-15、P96体外抗日本血吸虫童虫及雌、雄成虫生物学效应，初步探讨9种P96异构体体外抗日本血吸虫童虫和成虫生物学效应。方法：于小鼠感染日本血吸虫尾蚴后第16d及第5w肝门静脉灌注法分别收集童虫与（雌、雄）成虫，加入不同浓度的DW-3-15、P96及其异构体、青蒿琥酯（Art）、吡喹酮（PZQ）于DMEM培养液中继续培养72h，观察虫体死亡率及活力降低情况。结果：两种PZQ衍生物对日本血吸虫均有较好的杀虫效应。抗童虫效应：DW-3-15和P96作用浓度为50μmol/L时，童虫的存活率分别为33.3%和25.0%，活力降低率为71.1%和91.7%，50μmol/L的Art作用于童虫，存活率和活力降低率分别为42.9%和59.5%。抗成虫效应：DW-3-15作用浓度为50μmol/L时，雄虫与雌虫的存活率分别为20.0%和41.9%，活力降低率为93.3%和82.9%； P96作用浓度为50μmol/L时，雄虫全部死亡，雌虫有33.3%的存活率；PZQ15μmol/L作用雄虫和雌虫时，存活率分别为11.1%和37.8%。9种P96异构体抗成虫作用浓度为100μmol/L时，只有P96-3作用后，虫体存活率和活力降低率分别为60.0%和77.8%，抗童虫作用浓度为50μmol/L，存活率和活力降低率分别为44.1%和66.7%。结论：两种PZQ衍生物体外具有一定的抗日本血吸虫童虫效应，均优于Art，其中以P96抗童虫效果更好。两种化合物及PZQ对日本血吸虫雌、雄成虫作用后，均显示雌虫对药物耐受性较强，而雄虫较敏感，杀雄虫效果优于雌虫。对P96的9种异构体抗虫效果初步筛选结果表明这些化合物抗虫效果不明显，不如P96。第二部分PZQ衍生物DW-3-15与P96对不同动物模型体内抗日本血吸虫生物学效应的观察目的：观察两种PZQ衍生物对感染小鼠和家兔体内日本血吸虫童虫和成虫的杀虫效应。方法：小鼠感染日本血吸虫后，于虫体不同发育时期（3d、14d、35d），以200mg/kg剂量经灌胃给予DW-3-15、P96、Art和PZQ，连续5d，停药后3w解剖小鼠，计算减虫率。家兔感染日本血吸虫后，于第2w和第4w给予不同剂量（150mg/kg、300mg/kg）P96，顿服，于1w后各重复治疗一次，于感染后第10w，剖查虫荷数，计算减虫率和减卵率。结果：小鼠实验:童虫与成虫经DW-3-15、P96和Art作用后平均检获虫数与对照组相比，均显著减少（P0.05），DW-3-15和P96对3d、14d童虫期减虫率分别为64.0%、56.2%和69.1%、70.4%，Art对3d、14d减虫率分别为66.5%、67.4%，两种化合物和Art作用童虫后减虫率均显著高于PZQ组（22.2%、18.0%）(P0.05)；DW-3-15、P96和Art对35d成虫的减虫率分别为81.1%、82.8%和51.1%，低于PZQ组（95.7%），DW-3-15、P96组与PZQ组无显著性差异（P0.05）。9种P96异构体抗14d童虫作用，结果显示，给药剂量为200mg/kg/只时，P96-3的减虫率达到60.0%，与P96相似（76.4%），无显著性差异，具有一定的抗童虫效应。. 家兔实验:家兔于感染后第2w、第4w分别给予150mg/kg P96顿服，于1w后各重复治疗一次，减虫率分别为25.3%和65.2%，减卵率为30.5%和84.0%，在相同感染后时间点（2w、4w）以同样给药方式将P96剂量增加为300mg/kg，减虫率分别为62.2%和91.7%，减卵率为86.2%和97.7%，显著高于150mg/kg P96治疗组(P0.05)，与PZQ组相近（减虫率：95.4%、98.5%；减卵率：94.1%、97.1%）。结论：小鼠实验，DW-3-15、P96对3d、14d的童虫以及35d成虫均具有良好的杀虫作用，抗童虫效果与Art相似，但显著高于PZQ组，抗成虫作用优于Art，与PZQ组相近。9种P96异构体中发现P96-3对14d童虫具有一定的杀灭作用，其构效关系值得进一步深入探讨。家兔实验，高剂量治疗效果优于低剂量治疗效果，增大治疗剂量后减虫率与减卵率均提高并显示良好的抗虫效果；尤其P96对28d成虫的减虫率达到91.7%，接近PZQ的杀虫效应。具有发展为新的抗血吸虫药的潜在应用价值。第三部分Real-time PCR检测日本血吸虫感染家兔血清DNA水平评价P96体内杀虫效应目的：观察家兔疾病模型经P96和PZQ治疗后血清DNA的动态变化，评价其杀虫效应。结果：150mg/kg P96、300mg/kg P96和150mg/kg PZQ于第2w（14d）开始治疗日本血吸虫感染的家兔，家兔血清DNA检测结果总体变化趋势相似，但应用两种治疗剂量的P96治疗后的DNA最高浓度分别为267.8，299.5（拷贝数），均高于PZQ组最高浓度DNA249.5（拷贝数），提示P96的杀童虫效果优于PZQ；而150mg/kg P96、300mg/kg P96和150mg/kgPZQ于第4w（28d）开始治疗日本血吸虫感染的家兔血清DNA变化，结果显示，两种剂量的P96和PZQ均在治疗后第3d血清DNA浓度达到最高值，分别为495.7、1049.5、1222.4（拷贝数），并显示当P96用药量为300mg/kg其杀虫效应与PZQ有效剂量相似。不同剂量P96治疗童虫和成虫效果，结果显示治疗成虫时，大剂量P96组检测到的高浓度DNA（1049.5拷贝数）显著高于小剂量P96组（495.7拷贝数）（P0.05），治疗童虫时，两种剂量组检测结果虽无显著性差异，但大剂量P96组检测到的高浓度DNA（299.5拷贝数）仍高于小剂量P96组（267.8拷贝数），提示大剂量P96杀虫效果优于小剂量P96，但其有效剂量仍需探索。结论：通过Real-time PCR法检测血吸虫感染的家兔动物模型经P96治疗后血清DNA水平的动态变化，进一步证明了P96体内杀虫效果，结果表明P96体内杀童虫效果优于PZQ，且剂量越高，杀虫效果越好，当用药剂量达300mg/kg时其杀成虫效果与PZQ有效剂量相似，显示出作为抗血吸虫候选新药的潜在价值。第四部分PZQ压力下日本血吸虫耐受性诱导及其差异表达蛋白的筛选目的：分析经PZQ ED50体内诱导日本血吸虫感染小鼠的耐药性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：应用半数有效量（ED50）的PZQ，经灌胃连续诱导30天，停药21d后，给予治疗剂量（200mg/kg）连续灌胃5d，观察虫体对PZQ及其衍生物的敏感性。收集诱导虫体和未诱导虫体，应用2D-DIGE技术分析筛选差异表达蛋白，再对候选蛋白点进行质谱鉴定，结果通过NCBI数据库检索，在线uniprot检索差异蛋白的功能注释。结果：经PZQ ED50压力诱导的虫体体外分别暴露于14μmol/L、28μmol/L、56μmol/L和112μmol/L的PZQ作用后72h，虫体存活率分别为87.5%、82.0%、77.3%和75.6%，与对照组（存活率100%）相比无显著性差异（P0.05）。随着PZQ临界致死浓度的倍数增加，诱导后虫体的存活率与活力分值虽然有所下降，但变化不明显，特别是PZQ浓度增至112μmol/L，即8倍的正常血吸虫临界致死浓度，仍有75.6%虫体存活，可见诱导后虫体对PZQ的敏感性显著下降，显示耐药趋势。诱导虫体对不同浓度PZQ衍生物DW-3-15和P96交叉抗性观察，结果显示，DW-3-15和P96作用浓度为正常虫体临界致死浓度时（分别为15μmol/L和25μmol/L），存活率分别为95.8%和86.7%，活力降低率分别为26.1%和42.2%。但当两种衍生物DW-3-15和P96的浓度增至临界致死浓度的4倍时(分别为60μmol/L和100μmol/L)，虫体存活率仅为10.0%和20.0%，活力降低率达到96.7%和93.3%，两组间差异不明显，与PZQ组相比有显著性差异（112μmol/L时存活率为75.6%）（P0.05）。提示2种PZQ衍生物的作用靶点与PZQ可能存在差异。收集诱导虫体和未诱导虫体，应用2D-DIGE和质谱技术共筛选鉴定出34个差异蛋白点，质谱结果通过MASCOT软件搜索并通过NCBI数据库Blast比对氨基酸序列与去重复分析后共确认有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：经诱导的虫体对PZQ敏感性下降显著，显示出明显的耐受性，诱导虫体对DW-3-15和P96的耐受性比PZQ低，提示两种PZQ衍生物的作用靶点与PZQ可能存在差异。对诱导虫体和未诱导虫体差异蛋白分析，部分蛋白分子呈现差异表达。这些差异蛋白的表达上调或下调，提示PZQ诱导促进或抑制了某些特定基因的表达。对诱导虫体和未诱导虫体差异表达蛋白的筛选与分析，有助于深入阐明PZQ作用机制，，并为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。
[Abstract]:Part one: in vitro anti Schistosoma japonicum worm stage and sex specific biological effects of praziquantel derivatives DW-3-15, P96 and P96 isoforms.
Objective: To observe the biological effects of two kinds of praziquantel derivatives DW-3-15, P96 in vitro against Schistosoma japonicum and female and male adults, and preliminarily explore the biological effects of 9 P96 isomers against Schistosoma japonicum and adult in vitro.
Methods: in mice infected with Schistosoma japonicum cercariae, 16d and 5W hepatic portal vein perfusion method were used to collect the adult worms and (female and male) adults respectively, adding different concentrations of DW-3-15, P96 and their isomers, artesunate (Art), and praziquantel (PZQ) in DMEM culture, to continue to cultivate 72h, and to observe the mortality and vitality of the insect body.
Results: two PZQ derivatives have good insecticidal effect on Schistosoma japonicum. When the action concentration of DW-3-15 and P96 is 50 mol/L, the survival rate of children is 33.3% and 25%, the decrease of vitality is 71.1% and 91.7%, and 50 mu mol/L Art acts on the worm, and the survival rate and vitality decrease rate are 42.9% and 59.5%. respectively. The survival rate of male and female worms was 20% and 41.9% when the concentration of DW-3-15 was 50 mu mol/L, and the survival rate was 93.3% and 82.9% respectively. When the concentration of P96 was 50 mol/L, the males died and the females had 33.3% survival rate; the survival rates of the males and females with PZQ15 mu mol/L were 11.1% and 37.8%.9 P96 isomers respectively. When the action concentration was 100 mu mol/L, the survival rate and activity reduction rate of the insect body were 60% and 77.8% respectively after the action of P96-3, and the concentration of anti child insect was 50 mu mol/L, and the survival rate and the reduction rate of vitality were 44.1% and 66.7%., respectively.
Conclusion: two kinds of PZQ derivatives have a certain effect on the resistance to Schistosoma japonicum, which are better than Art, and the effect of P96 on children is better. The two compounds and PZQ are more tolerant to the drug after the action of the female and male adults of Schistosoma japonicum, and the male is better than the female. The 9 isomers of P96 are better than those of the female. The preliminary screening results of insect resistance showed that these compounds had no obvious effect on insect resistance than P96..
The second part is the observation of the biological effects of PZQ derivative DW-3-15 and P96 on Schistosoma japonicum in different animal models.
Objective: To observe the insecticidal effects of two PZQ derivatives on Schistosoma japonicum larvae and adults in infected mice and rabbits.
Methods: mice infected with Schistosoma japonicum (3D, 14d, 35d) were given DW-3-15, P96, Art and PZQ at a dose of 200mg/kg at a dose of 200mg/kg, and the mice were dissected to calculate the rate of worm reduction. After infected with Schistosoma japonicum, the rabbits were given different doses in 2W and first 4W. After treatment, the number of worm load was counted at 10W after infection, and worm reduction rate and egg reduction rate were calculated.
Results: the average number of insects detected by DW-3-15, P96 and Art decreased significantly compared with the control group (P0.05). DW-3-15 and P96 were 64%, 56.2% and 69.1%, 70.4%, Art to 3D, and Art were 66.5%, 67.4%, respectively, and two compound and Art action worm reduction rates were both significant. Higher than group PZQ (22.2%, 18%) (P0.05), DW-3-15, P96 and Art were respectively 81.1%, 82.8% and 51.1% of 35d adults, lower than PZQ group (95.7%), DW-3-15, P96 group and PZQ group had no significant difference (P0.05). There is no significant difference, it has certain anti insect effect.
Rabbit experiment: the rabbits were treated with 150mg/kg P96 after infection at 2W and 4W respectively. After 1W, the rate of worm reduction was 25.3% and 65.2% respectively. The rate of egg reduction was 30.5% and 84% respectively. The dosage of P96 increased to 300mg/kg at the same time point (2W, 4W), the rate of worm reduction was 62.2% and 91.7%, the rate of egg reduction was 86.2% and 9, respectively. 7.7%, significantly higher than 150mg/kg P96 treatment group (P0.05), similar to group PZQ (worm reduction rate: 95.4%, 98.5%; egg reduction rate: 94.1%, 97.1%).
Conclusion: the mice experiment, DW-3-15 and P96 have good insecticidal effect on 3D, 14d and 35d adults, and the effect of anti child insect is similar to that of Art, but it is significantly higher than that of the PZQ group. The anti adult effect is superior to Art, and the killing effect of P96-3 to the child is found in the.9 P96 isomers of the PZQ group, and its structure-activity relationship should be further explored. Please.
In the rabbit experiment, the effect of high dose treatment was better than that of low dose treatment. After increasing the treatment dose, the rate of worm reduction and egg reduction increased and the effect of insect resistance showed good resistance. Especially, the reduction rate of P96 to 28d adult was 91.7%, close to the insecticidal effect of PZQ, and it was of potential application value for the development of new anti schistosomiasis medicine.
In the third part, Real-time PCR was used to detect serum DNA level in rabbits infected with Schistosoma japonicum, and to evaluate the killing effect of P96 in vivo.
Objective: To observe the dynamic changes of serum DNA in rabbits with disease models after P96 and PZQ treatment, and to evaluate their insecticidal effects.
Results: 150mg/kg P96300mg/kg P96 and 150mg/kg PZQ began to treat the rabbits infected with Schistosoma japonicum in 2W (14d). The overall change trend of serum DNA detection results of rabbit serum was similar, but the highest concentration of DNA after P96 treatment with two kinds of therapeutic doses was 267.8299.5 (copy number), which was higher than that of the highest concentration of PZQ group (copy number). The effect of P96 was better than that of PZQ, while 150mg/kg P96300mg/kg P96 and 150mg/kgPZQ began to treat the serum DNA changes in rabbits infected with Schistosoma japonicum by 4W (28d). The results showed that the two doses of P96 and PZQ were all at the highest value after the treatment. The insecticidal effect of 300mg/kg was similar to that of PZQ.
The effect of different doses of P96 on children and adults showed that the high concentration of DNA (1049.5 copy number) detected in the large dose P96 group was significantly higher than that of the small dose P96 group (495.7 copies) (P0.05). Although there was no significant difference in the test results of the two dose groups, the high concentration of DNA (299.5 copies) detected by the large dose P96 group (299.5 copies) The number is still higher than the low dose P96 group (267.8 copy number), suggesting that the high dose P96 insecticidal effect is superior to the small dose P96, but its effective dose still needs to be explored.
Conclusion: the dynamic changes of serum DNA level after P96 treatment in rabbit model of Schistosoma infection by Real-time PCR method further proved the killing effect of P96 in the body. The results showed that the effect of P96 in the body was better than that of PZQ, and the higher the dose, the better the effect of insecticidal effect. When the dosage reached 300mg/kg, the effect of the insecticide on the adult was effective and the PZQ was effective. Similar doses showed a potential value as a new candidate for schistosomiasis control.
The fourth part is the induction of tolerance and differentially expressed proteins of Schistosoma japonicum under PZQ stress.
Objective: to analyze the differentially expressed proteins between drug resistant and uninduced bodies induced by PZQ ED50 in mice infected with Schistosoma japonicum, in order to elucidate the mechanism of PZQ, and to explore the candidate vaccine target antigen and the target of drug treatment.
Methods: using PZQ of half effective dose (ED50), after continuous induction of gastric perfusion for 30 days, after stopping drug 21d, the treatment dose (200mg/kg) was given to 5D continuously. The sensitivity of the insect body to PZQ and its derivatives was observed. The insect body and the uninduced body were collected. The differential expression protein was screened by 2D-DIGE technique, and then the candidate protein points were identified by mass spectrometry. Results through NCBI database retrieval, online UniProt retrieved functional annotation of differential proteins.
Results: the strain induced by PZQ ED50 was exposed to 14 mu mol/L, 28 mol/L, 56 mu mol/L and 112 mol/L PZQ, and the survival rate of the insect body was 87.5%, 82%, 77.3% and 75.6%, respectively, compared with the control group (survival rate 100%). Although the ratio and vitality score decreased, the change was not obvious, especially the PZQ concentration increased to 112 mu mol/L, or 8 times the critical lethal concentration of normal Schistosoma, and there were still 75.6% insect bodies, which showed that the sensitivity of the insect body to PZQ decreased significantly and showed the resistance trend. The cross resistance of the insect body to the different concentration of PZQ derivatives DW-3-15 and P96 was induced. The results showed that the concentration of DW-3-15 and P96 was at the critical lethal concentration of the normal insect body (15 mu mol/L and 25 mol/L respectively), the survival rate was 95.8% and 86.7% respectively, and the decrease of vitality was 26.1% and 42.2%., respectively, but when the concentration of two derivatives DW-3-15 and P96 increased to 4 times the critical lethal concentration (60 mu mol/L and 100 u mol/L respectively), the insect body The survival rate was only 10% and 20%, the rate of vitality decreased to 96.7% and 93.3%, the difference between the two groups was not obvious, and there was a significant difference compared with the PZQ group (112 mu mol/L survival rate 75.6%) (P0.05). It suggested that the target of 2 PZQ derivatives may be different from PZQ.
34 differential proteins were screened and identified by 2D-DIGE and mass spectrometry. The results of mass spectrometry were searched by MASCOT software and 30 differentially expressed proteins were confirmed by NCBI database Blast comparison and repeated analysis of amino acid sequences. The expression of 12 proteins was up and 18 proteins were downregulated. Mainly cytoskeleton related proteins, enzymes involved in glycometabolism and energy metabolism, oxidoreductases, stress proteins and proteases involved in detoxification.
Conclusion: the induced susceptibility to PZQ decreased significantly, showing obvious tolerance and the tolerance to DW-3-15 and P96 was lower than that of PZQ, suggesting that the target points of the two PZQ derivatives may be different from PZQ. The expression of protein is up or down, suggesting that PZQ induces or inhibits the expression of certain specific genes. Screening and analysis of differentially expressed proteins in the induced and uninduced bodies can help to elucidate the mechanism of PZQ action, and provide a scientific basis for exploring new vaccine candidate antigens and targets for the treatment of drugs. New ways and new ideas for new drug development of Schistosoma japonicum.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R532.21

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