CAJ | 학술논문

目的探究一种体外培养日本血吸虫胞蚴的新方法,并通过体外注射,感染阴性钉螺,培养阳性钉螺.方法将日本血吸虫虫卵孵化的毛蚴与作为滋养层的草地夜蛾(Spodoptera frugiperda)卵巢细胞(sf9细胞)共培养,观察毛蚴的生长情况,并拍摄生长照片.用显微注射针将3～5只母胞蚴注射到钉螺体内,计算钉螺的存活率及尾蚴的阳性率,用钉螺逸出的尾蚴人工感染昆明鼠,检测尾蚴的活力,用组织化学染色法和PCR方法鉴定尾蚴的相关基因特性.结果毛蚴培养至第12小时观察到纤毛板已脱落,培养2～3 d的虫体呈不断的伸缩运动,此后母胞蚴维持该状态并不转化成子胞蚴.3次实验的钉螺存活率依次为24.72%、28.23%和57.89%,活螺阳性率依次为4.55%、8.57%和14.29%.阳性钉螺释放的尾蚴具有感染性,且具有与日本血吸虫相同的基因特性.结论体外培养毛蚴通过显微注射可以人工感染钉螺并逸出有感染活力的尾蚴,为日本血吸虫幼虫的体外操作提供一种新的方法.
목적 탐구일충체외배양일본혈흡충포유적신방법,병통과체외주사,감염음성정라,배양양성정라.방법 장일본혈흡충충란부화적모유여작위자양층적초지야아(Spodoptera frugiperda)란소세포(sf9세포)공배양,관찰모유적생장정황,병박섭생장조편.용현미주사침장3～5지모포유주사도정라체내,계산정라적존활솔급미유적양성솔,용정라일출적미유인공감염곤명서,검측미유적활력,용조직화학염색법화PCR방법감정미유적상관기인특성.결과 모유배양지제12소시관찰도섬모판이탈락,배양2～3 d적충체정불단적신축운동,차후모포유유지해상태병불전화성자포유.3차실험적정라존활솔의차위24.72%、28.23%화57.89%,활라양성솔의차위4.55%、8.57%화14.29%.양성정라석방적미유구유감염성,차구유여일본혈흡충상동적기인특성.결론 체외배양모유통과현미주사가이인공감염정라병일출유감염활력적미유,위일본혈흡충유충적체외조작제공일충신적방법.
Objective To explore a new method of in vitro cultivation of Schistosoma japonicum sporocysts,infection of negative snails through in vitro-injection,and cultivation of positive snails.Methods Isolated miracidia were directly cultivated in sf9 cells-conditioned medium,the growth of miracidia was observed and the growth picture was taken.3-5 mother sporocysts were inoculated into the snails body using a microscopic needle.The survival rate of the snails and the positive rate of cercariae were calculated.Kunming mice were infected with the cercariac and the activity of cercariae Was detected.The related gene characteristics of cercariae was identified by histochemical examination and PCR method.Results The pictures of miracidia were recorded after co-cultivation for 1 h,12 h,24 h and 3 d.The shedding of the cilia of miracidia occurred within 12 h,parasites exhibited continuous stretching movements after co-cultivated for 2-3 d.Since then the mother sporocysts kept the status,did not develop to daughter sporocysts.The survival rates of snails in 3 experiments were 24.72%,28.23%and 57.89%respectively,while the positive rates of cercariae were 4.55%,8.57% and 14.29%respectively.Animal experiments showed that the cercariae released from artificial cultivation of positive snails were infectious and had same gene characteristics of Schistosoma japonicum.Conclusion Sf9 cells could be feeder cells for in vitro-cultivation of miracidia and positive snails could be obtained by in vitro micro-injection of cercariae thus to provide a new approach for in vitro operation of Schistosoma japonicum lar