胰蛋白酶和胆汁对细粒棘球绦虫原头蚴外翻和成虫发育的影响  

Effects of trypsin and canine bile on evagination and development of Echinococcus granulosus protoscoleces in vitro

作　　者：刘文英 李军 王甜 郑雪婷[3] 郭刚 张文宝 LIU Wenying;LI Jun;WANG Tian;ZHENG Xueting;GUO Gang;ZHANG Wenbao(School of Basic Medicine, Xinjiang Medical University, Urumqi 830011, China;Instiute of Clinical Medicine, The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054, China;State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi 830054, China)

机构地区：[1]新疆医科大学基础医学院,乌鲁木齐830011 [2]新疆医科大学第一附属医院临床医学研究院,乌鲁木齐830054 [3]新疆医科大学第一附属医院临床医学研究,乌鲁木齐830054 [4]新疆医科大学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

出　　处：《新疆医科大学学报》2019年第5期599-603,共5页Journal of Xinjiang Medical University

基　　金：科技部国家重点研发计划(2017YFD0501301);省部共建中亚高发病成因与防治国家重点实验室开放课题(SKL-HIDCA-2017-5)

摘　　要：目的探讨胰蛋白酶和犬胆汁对细粒棘球绦虫原头蚴外翻和体外成虫培养发育的影响。方法无菌条件下采集羊源细粒棘球绦虫原头蚴,用磷酸盐缓冲液(PBS)漂洗,经胃蛋白酶处理后,用0.1%亚甲基蓝染色,检测虫体活率>98%的原头蚴用不同浓度的犬胆汁(0.005%～5%)和胰蛋白酶(0.01%～5%)及纯水刺激处理,在不同的培养条件及时间点采集样品,观察原头蚴的死亡率及头节外翻情况。结果当胆汁终浓度为0.02%时,培养48 h头节外翻率最高,可达62.7%;原头蚴可以耐受0.25%的胰蛋白酶,但其浓度为0.5%,原头蚴培养48 h时,死亡率为13.7%。研究发现,原头蚴培养的前3 d,其培养基中添加0.25%的胰蛋白酶和0.02%的犬胆汁,培养3 d后去除胰蛋白酶,仅在培养基中加入0.02%的犬胆汁原头蚴可以保持高的成虫发育率,达60%以上。原头蚴经两次在室温下30 s的刺激,原头蚴外翻率达到100%,但培养24 h后外翻的原头蚴回翻,外翻率与对照无差别(t=0.27,P=0.79)。结论犬胆汁对细粒棘球绦虫原头蚴的外翻和成虫的维持和发育是必须的。适量(0.25%)胰蛋白酶有利于已外翻原头蚴成虫性状发育。仅纯水刺激原头蚴,可以使原头蚴外翻,但不能提高后成虫的发育率。Objective To study the effects and role of trypsin and dog bile on the evagination and development of Echinococcus granulosus protoscoleces in vitro. Methods E. granulosus protoscoleces were aspirated and pooled from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi,then rinsed with phosphate buffered saline(PBS) under sterile conditions. Trypsin digested the protoscoleces of E. granulosus, then stained with 0.1% methylene blue and the worm survival rate were calculated. The protoscoleces of E. granulosus with the survival rate higher than 98% were treated with different concentrations of canine bile(0.005%-5%) and trypsin(0.01%-5%). Protoscolex evagination and adult worm development were observed at different time points. Results Canine bile at concentration of 0.02% showed a high rate of evagination up to 62.7%. Protoscoleces tolerated trypsin at concentration of 0.5% with 13.7% dead after 48 h of cultivation. The optimal conditions for the development of the E. granulosus protoscoleces were 0.25% trypsin and 0.02% dog bile in the medium for the first 3 days of culture. After 3 days of culture, trypsin was removed, and only 0.02% of dog bile was added to the medium. This allowed more than 60% of protoscoleces developed to adult worms. Pure water showed a good stimulator for up to 100% of protoscoleces evaginated with 2 time of 30 s stimulation at room temperature. However, this did not increase adult development after 24 hours of culture compared with non-stimulation(t=0.27, P=0.79). Conclusion Canine bile is essential for the evagination of E. granulosus and the maintenance and development of adult worm development. Pure water stimulates protoscolex evagination, but do not increase the development of adult development in vitro.

关 键 词：细粒棘球绦虫 原头蚴 外翻 成虫发育 胰蛋白酶 犬胆汁 

分 类 号：R383.3[医药卫生—医学寄生虫学]