【作者】 方强；

【导师】 沈继龙； 孙新；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2003， 硕士

【摘要】 目的：1) 通过酸性胃蛋白酶消化猪带绦虫孕节建立收集虫卵的新方法； 2) 应用新方法了解猪带绦虫成熟孕节内虫卵数量； 3) 探索次氯酸钠法孵化带绦虫卵的孵化规律和适宜孵化条件； 4) 研究一氧化氮(NO)对猪带绦虫六钩蚴杀伤作用。 方法：1) 以1％酸性胃蛋白酶分别在普通和振荡条件下消化完整和剪碎孕节，观察不同条件下消化孕节收集虫卵时间；分别对以物理法收集的虫卵及以酸性胃蛋白酶法消化物理法剩余的孕节残片收集的虫卵计数，比较酸性胃蛋白酶法与物理法在收集虫卵的数量上的差异；观察酸性胃蛋白酶法和物理法收集的虫卵以次氯酸钠法孵化后虫卵孵化率及六钩蚴存活率；观察1％酸性胃蛋白酶消化孕节40min和120min收集的虫卵以次氯酸钠法孵化后虫卵孵化率及六钩蚴存活率； 2) 对10条猪带绦虫的50片成熟孕节以酸性胃蛋白酶法消化，收集虫卵，以细胞计数板计数： 3) 以7种浓度的次氯酸钠溶液孵化带绦虫卵，观察不同时间的虫卵孵化率和六钩蚴存活率；同时观察1.0％次氯酸钠溶液在3个不同孵化时间的孵化率和六钩蚴存活率；并观察1.0％次氯酸钠溶液孵化带绦虫卵的过程； 4) 以LPS诱导离体的小鼠腹腔巨噬细胞，使其产生NO；加入猪带绦虫六钩蚴，测定48h内六钩蚴的死亡率。同时，为进一步证实NO对六钩蚴的杀伤作用，分别用诱导型一氧化氮合酶(iNOS)的抑制剂地塞米松(Dexamethasone，DEX)和左旋硝基精氨酸(Nω -nitro-L-arginine，L-NNA)抑制NO产生，与未加抑制剂组比较，观察六钩蚴死亡率的变化。 安徽医科大学硕士学位论文结果:1)l%酸性胃蛋白酶振荡条件下消化孕一节收集虫卵时问少于普通条件下听需时间(P<0.01)，消化剪碎孕节时间少于消化完整孕节时间(P<001);酸性胃蛋白酶法收集虫卵的数址较物理法高6%;酸性胃蛋}‘」酶法和物理法收集的虫卵以次氯酸钠法孵化其孵化率及存活率无差异(P>0.05);1%酸性胃蛋白酶消化孕节40min和1 20而n收集的虫卵以次氯酸钠法孵化后孵化率及虫卵存活率无差异(P>0.05); 2)猪带绦虫姆节成熟孕竹内虫卵数ht不等，址少为550个，址多为172500个，平均为26891个;不同虫体间孕节内虫卵数的差异较同一虫体户lJ的差异显著; 3)以不同浓度次氯酸钠溶液孵化带绦虫卵，结果在一定浓度范川内，孵化率随孵化时间而升高，但过长的时间会导致六钩蝴存活率下降。达到最高孵化率的孵化时间随次氯酸钠溶液升高而缩短，但较高浓度下六钩坳存活率下降，高浓度则虫卵迅速完全崩解。在0.4%一1 .0%的浓度，孵化时问为3一6而n时，虫卵孵化率和六钩坳存活率均较高;对孵化过程的镜下观察发现，次认酸钠法通过济解胚膜，致六钩坳逸出; 4)LPS可有效诱导巨噬细胞产生NO，2.0 x 105巨I噬细胞产二生的NO浓度为119.64(X)土5.1154卜mol/L，该浓度的杀虫率为79.83%。)J 11人J印下1川齐11 DXS和L-NNA后，巨噬细胞的NO产量均明显降低，J一冬杀虫率也明显降低。结论:l)成功地建立了较为适川的新的猪带绦虫虫卵收集方法—酸性}‘{蛋白酶法; 2)猪带绦虫成熟孕节内虫卵数量不等，可有口悬殊的差别，较既滚11的文献有新的发现; 3)次氯酸钠溶液孵化带绦虫卵的孵化率与孵化时问呈正相关关系，孵化时问与孵化川次纵酸钠济液浓度’.飞负相关关系;较为适介的条件为04伏一!，O伏的浓度，孵化时间为3一6min;次氯酸钠法孵化带绦虫卵的本质是破坏溶解r!1了:膜，使六钩坳得以逸出; 4)宁舌化j月一噬细胞)卉二‘l几的NO对猪·}}梦绦虫六钩蝴有杀伤作川更多还原

【Abstract】 Objective: 1) To establish a new method for collecting eggs of Taenia solium by digesting gravid proglottids by the acidity pepsin.2) To observe the number of eggs in gravid proglottids of Taenia solium by the new acidity pepsin method.3) To study the rule and factors of hatching eggs of Taenia solium by sodium hypochrite method.4) To study the in vitro larvicidal activity of nitric oxide (NO) to the oncospheres of Taenia solium.Methods: 1) The integrity and smashed gravid proglottids of Taenia solium were digested by pepsin solution in common or surge condition , then the time of digesting gravid proglottids was observed; the eggs in gravid proglottids collected by physical method and the remanent eggs in the gravid proglottids collected by pepsin method were counted, then the difference in the number of eggs collected by physical method or pepsin method was observed; the hatching rate of eggs and the viability of oncospheres were observed after the eggs collected by physical method or pepsin method and hatched by sodium hypochrite method; the hatching rate of eggs and the viability of oncospheres were observed after the gravid proglottids were digested 40min or 120min by pepsin solution and hatched by sodium hypochrite method; 2) Gravid proglottids of Taenia solium were digested by pepsin. Then the eggs werecollected and counted;3) The eggs of Taenia solium were hatched by sodium hypochrite solution in different concentration, then the hatching rate of eggs and the viability of oncospheres were observed in different time; the hatching process with 1% sodium hypochrite solution was observed;4) Macrophages were induced by LPS to produce NO, and the oncospheres were added to the medium with activated macrophages, then the larvicidal activity was observed; in order to further comfirm the effect of NO, the inhibitors of iNOS, Dexamethasone(DXS) and N w -nitro-L-arginine (L-NNA), were used to inhibit the production of NO, larvicidal activity was measured by the same methods and the difference of dead larvae ratio was compared between the inhibited and uninhibited groups.Results: l)The time of gravid proglottids digested by pepsin solution in surge condition was less than in common condition(P<0.01), and the time of the integrity gravid proglottids digested by pepsin solution was less than the smashed(P<0.01); the number of eggs collected by pepsin method was more than that by physical method (>6%);the hatching rate of eggs and the viability of oncospheres after the eggs collected by physical method or pepsin method and hatched by sodium hypochrite method were not different(P>0.05); the hatching rate of eggs and the viability of oncospheres after the gravid proglottids were digested 40min or 120min by pepsin solution and then hatched by sodium hypochrite method were not different(P>0.05);2) The number of the eggs in each mature gravid proglottid varied from 550 to 172500, and the average number was 26891. The difference of the eggs in gravid proglottids of different worms of Taenia solium was more prominent than that in gravid proglottids of the same worm of Taenia solium;3) The oncospheres can be obtained in all concentration except for that of 36%. The hatching rate rose with the concentration and the viability of oncospheres descreased 1with increase of the concentration and the eggs showed cytolytic quickly in highconcentration (36%). After the eggs of Taenia solium were hatched 5-6 minutes in 0.4%-1.0% sodium hypoehrite solution, the hatching rate of eggs and the viability was higher than the others; and though the process that the eggs of Taenia solium were hatched by 1% sodium hypoehrite solution was observed, we found the embryo membrane was dissolved and the oncospheres transgressed ;4) LPS can induce macrophages to produce NO effectively. The NO production of 2.0 x 105 macrophages was 119.6400+5.1154 u,mol,L, and the larvicidal effect reached to 79.83%. This activity can be inhibited by DXS and L-NNA. NO production and dead larvae ratio were reduced significantly in the inhibited gr更多还原