原头蚴体外形成棘球蚴的优化培养体系研究
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摘要
目的建立适合细粒棘球绦虫原头蚴在体外形成棘球蚴的培养体系。方法以RPMI1640或DMEM培养基为基质,胎牛血清或绵羊血清为营养成分,人肝细胞株L02为饲养细胞设计配伍2大类、6个组培养体系;培养细粒棘球绦虫原头蚴45 d,根据各组原头蚴的头节外翻率、成囊率和囊泡大小情况,寻找适合原头蚴形成棘球蚴的体外培养体系最佳配伍;在最佳配伍的培养体系继续培养棘球蚴囊泡至180 d。结果在起初的45 d期间,DMEM体系的E组(DMEM+胎牛血清+人肝细胞株L02)培养的棘球蚴生长最快,20 d起原头蚴成囊率高于其他各组(P<0.05),25 d起棘球蚴囊泡大于其他各组(P<0.05),45 d时原头蚴成囊率达到100%;在其后的46～180 d内,棘球蚴囊泡持续增大,至80 d时肉眼可见透明囊泡,至180 d时最大囊泡直径达2.2 mm。结论添加胎牛血清及人肝细胞株L02的DMEM培养基有利于体外培养的原头蚴发育为棘球蚴囊泡。
Objective To search for a suitable culture system for transforming protoscoleces of Echinococcus granulosus into hydatid cysts in vitro. Methods Based on the basic cell culture media RPMI1640 or DMEM, and fetal calf serum or sheep serum as nutrients, and human liver cell line L02 as server cells, culture systems for transformations of protoscoleces into hydatid cysts in vitro were established based on block designs. The culture systems were divided into 2 main groups, and 6 subgroups according the different ingredients of culture media, sera, and sever cells. The protoscoleces were cultured in 6 subgroups for 45 d, and then the most suitable medium for the transformations was screened according to the ratios of evaginated or encysted protoscoleces, and the sizes of protoscoleces. In the selected best culture system, the encysted protoscoleces were kept cultivating up to 180 d for further observation. Results Within the first period of 45 d, it demonstrated that the culture system of DMEM medium+fetal calf serum+L02 cells was the best culture system for transformations of protoscoleces into hydatid cysts in vitro, because of the fastest growth speed of protoscoleces, the maximal ratio of encystation of protoscoleces since 20 d(P<0.05), the biggest size of hydatid cysts since 25 d(P<0.05), and 100% percentage of encystation of protoscoleces at 45 d. In the following culture days(46 to 180 d), the hydatid cysts still grew up, the transparent hydatid cysts were visible to the naked eyes at 80 d, and the biggest cyst size reached to 2.2 mm at 180 d. Conclusion The culture medium of DMEM, added with fetal calf serum and L02 cells, is a suitable culture system for transformations of protoscoleces into hydatid cysts in vitro.
Objective To search for a suitable culture system for transforming protoscoleces of Echinococcus granulosus into hydatid cysts in vitro. Methods Based on the basic cell culture media RPMI1640 or DMEM, and fetal calf serum or sheep serum as nutrients, and human liver cell line L02 as server cells, culture systems for transformations of protoscoleces into hydatid cysts in vitro were established based on block designs. The culture systems were divided into 2 main groups, and 6 subgroups according the different ingredients of culture media, sera, and sever cells. The protoscoleces were cultured in 6 subgroups for 45 d, and then the most suitable medium for the transformations was screened according to the ratios of evaginated or encysted protoscoleces, and the sizes of protoscoleces. In the selected best culture system, the encysted protoscoleces were kept cultivating up to 180 d for further observation. Results Within the first period of 45 d, it demonstrated that the culture system of DMEM medium+fetal calf serum+L02 cells was the best culture system for transformations of protoscoleces into hydatid cysts in vitro, because of the fastest growth speed of protoscoleces, the maximal ratio of encystation of protoscoleces since 20 d(P<0.05), the biggest size of hydatid cysts since 25 d(P<0.05), and 100% percentage of encystation of protoscoleces at 45 d. In the following culture days(46 to 180 d), the hydatid cysts still grew up, the transparent hydatid cysts were visible to the naked eyes at 80 d, and the biggest cyst size reached to 2.2 mm at 180 d. Conclusion The culture medium of DMEM, added with fetal calf serum and L02 cells, is a suitable culture system for transformations of protoscoleces into hydatid cysts in vitro.
引文
[1] 温浩,徐明谦.实用包虫病学[M].北京:科学出版社, 2007:1. WEN H, XU M Q. Practical hydatidiology[M]. Beijing:Science Press, 2007:1.
[2] OZDOL C, YILDIRIM A E, DAGLIOGLU E, et al. Alveolar hydatid cyst mimicking cerebellar metastatic tumor[J]. Surg Neurol Int, 2011, 2:13. DOI:10.4103/2152-7806.76281.
[3] ALGROS M P, MAJO F, BRESSON-HADNI S, et al. Intra cerebral alveolar echinococcosis[J]. Infection, 2003, 31(1): 63-65. DOI: 10.1007/s15010-002-2178-y.
[4] MANDAL S, MANDAL M D. Human cystic echinococcosis: epidemiologic, zoonotic, clinical, diagnostic and therapeutic aspects[J]. Asian Pac J Trop Med, 2012, 5(4): 253-260. DOI: 10.1016/S1995-7645(12)60035-2.
[5] 张梦媛, 伍卫平, 官亚宜, 等. 我国棘球蚴病疾病负担分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 15-19. ZHANG M Y, WU W P, GUAN Y Y, et al. Analysis on disease burden of hydatid disease in China[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 15-19.
[6] BUDKE C M, DEPLAZES P, TORGERSON P R. Global socioeconomic impact of cystic echinococcosis[J]. Emerging Infect Dis,2006,12(2):296-303.DOI:10.3201/eid1202.050499.
[7] 尚婧晔, 张光葭, 何伟,等. 细粒棘球蚴病病原分类学与分子流行病学研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(2): 166-173. SHANG J Y, ZHANG G J, HE W, et al. Taxonomy and molecular epidemiology of Echinococcus granulosus complex causing cystic echinococcosis[J]. Chin J Parasitol Parasit Dis, 2018, 36(2): 166-173.
[8] GROSSO G, GRUTTADAURIA S, BIONDI A, et al. Worldwide epidemiology of liver hydatidosis including the Mediterranean area[J].World J Gastroenterol,2012,18(13): 1425-1437. DOI: 10.3748/wjg.v18.i13.1425.
[9] 徐硕,党志胜,张皓冰,等.棘球蚴病药物治疗的研究进展[J].中国寄生虫学与寄生虫病杂志,2018,36(3):297-302. XU S , DANG Z S , ZHANG H B, et al. Research progress on drug treatment for hydatidosis[J]. Chin J Parasitol Parasit Dis, 2018, 36(3): 297-302.
[10] 温浩, 吐尔干艾力·阿吉, 邵英梅,等. 棘球蚴病防治成就及面临的挑战[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(6):466-471. WEN H, TUERGANAILI A J, SHAO Y M, et al. Research achievemets and challenges for echinococcosis control[J]. Chin J Parasitol Prasit Dis, 2015, 33(6):466-471.
[11] 周璇,夏党荣,赵新斐,等.棘球蚴病研究进展[J].中国动物保健,2014,16(11):7-10.DOI:10.3969/j.issn.1008-4754.2014.11.004. ZHOU X, XIA D R, ZHAO X F, et al. Study progress of echinococcosis[J]. China Ani Health, 2014, 16(11):7-10. DOI:10.3969/j.issn.1008-4754.2014.11.004.
[12] 伍卫平,王虎,王谦,等. 2012-2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 1-14. WU W P, WANG H, WANG Q, et al. A nationwide sampling survey on echinococcosis in China during 2012-2016[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 1-14.
[13] 孙艳红,杨亚明.包虫病的治疗研究进展[J].热带病与寄生虫学,2015,13(1):53-58.DOI:10.3969/j.issn.1672-2302.2015.01.017. SUN Y H, YANG Y M. Advances in the treatment of echinococcosis[J]. J Trop Dis Parasit, 2015, 13(1):53-58. DOI:10.3969/j.issn.1672-2302.2015.01.017.
[14] GOTTSTEIN B, FELLEISEN R. Protective immune mechanisms against the metacestode of Echinococcus multilocularis[J]. Parasitol Today(Regul Ed), 1995, 11(9): 320-326. DOI: 10.1016/0169-4758(95)80184-7.
[15] ELISSONDO M C, DOPCHIZ M C, BRASESCO M, et al. Echinococcus granulosus: first report of microcysts formation from protoscoleces of cattle origin using the in vitro vesicular culture technique[J]. Parasite, 2004, 11(4): 415-418. DOI: 10.1051/parasite/2004114415.
[16] 王慧,李军,郭宝平,等. 微囊法棘球蚴继发感染小鼠动物模型的建立[J]. 中国人兽共患病学报, 2016, 32(9): 784-788. DOI:10.3969/j.issn.1002-2694.2016.09.004. WANG H, LI J, GUO B P, et al. Establishment of secondary hydatid disease infection in mice with cystic and alveolar Echinococcus cysts cultured in vitro[J]. Chin J Zoonoses, 2016,32(9):784-788.DOI:10.3969/j.issn.1002-2694.2016.09.004.
[17] ZHANG W B, JONES M K, LI J, et al. Echinococcus granulosus: pre-culture of protoscoleces in vitro significantly increases development and viability of secondary hydatid cysts in mice[J]. Exp Parasitol, 2005, 110(1): 88-90. DOI: 10.1016/j.exppara.2005.02.003.
[18] LIU C S, ZHANG H B, YIN J H, et al. Echinococcus granulosus: suitable in vitro protoscolices culture density[J]. Biomed Environ Sci, 2013, 26(11): 912-915. DOI:10.3967/bes2013.020.
[19] 袁丽英,张壮志,石保新,等. 细粒棘球绦虫-原头蚴在两种细胞培养液中体外培养的初步观察[J]. 畜牧兽医杂志, 2008, 27(5): 16-18. YUAN L Y, ZHANG Z Z, SHI B X, et al. In vitro cultivation protoscoleces of the protoslices of Echinococcus granulosus in medium RPMI-1640 and MEM[J]. J Ani Sci Vet Med, 2008, 27(5): 16-18.
[20] 袁丽英,张壮志,石保新,等. 细粒棘球蚴-原头蚴体外培养成囊模型的建立[J]. 畜牧与兽医, 2009, 41(7): 29-31. YUAN L Y, Zhang Z Z, Shi B X, et al. Establishment of the in vitro cultivation model of protoscolex of Echinococcus cyst[J]. Ani Husbandry Veter Med,2009, 41(7): 29-31.
[21] 马海龙,李兆勇,孙世安,等. 肝癌细胞及其上清液体外培养多房棘球蚴的实验研究[J]. 武警后勤学院学报(医学版), 2012,21(11):864-867.DOI:10.3969/jissn.2095-3720.2012.11.008. MA H L, LI Z Y, SUN S A, et al. Experimental studies on the in vitro culture of Echinococcus multilocularis metacestodes with Bel-7404 cells and its supematant fluid[J]. J Logist Univ CAPF(Med Sci), 2012, 21(11): 864-867. DOI:10.3969/jissn.2095-3720.2012.11.008.
[22] 孙萃萍. 多房棘球绦虫HSP20蛋白的生物信息学分析、重组表达及泡球蚴的体外培养[D]. 兰州:兰州大学, 2015. SUN C P. Bioinformatic analysis, recombination and expression of HSP20 from Echinococcus multilocularis as well as the culture of alveolar hydatid in vitro[D]. Lanzhou: Lanzhou Univ, 2015.
[23] SPILIOTIS M, TAPPE D, SESTERHENN L, et al. Long-term in vitro cultivation of Echinococcus multilocularis metacestodes under axenic conditions[J]. Parasitol Res, 2004, 92(5):430-432. DOI: 10.1007/s00436-003-1046-8.
[24] HEMPHILL A, GOTTSTEIN B. Immunology and morphology studies on the proliferation of in vitro cultivated Echinococcus multilocularis metacestodes[J]. Parasitol Res, 1995, 81(7): 605-614. DOI: 10.1007/BF00932028.
[25] JURA H, BADER A, HARTMANN M, et al. Hepatic tissue culture model for study of host-parasite interactions in alveolar echinococcosis[J]. Infect Immun, 1996, 64(9): 3484-3490.
[26] SMYTH J D, DAVIES Z. In vitro culture of the strobilar state of Echinococcus granulosus (sheep strain): a review of basic problems and results[J]. Int J Parasitol, 1974, 4(6): 631-644. DOI:10.1016/0020-7519(74)90028-9.
[27] 周启锋, 任利,张灵强,等. 肝泡型包虫病动物模型建立的研究进展[J]. 临床肝胆病杂志, 2017,33(11):1-5. ZHOU Q F, REN L, ZHANG L Q, et al. Research advance in animal models of hepatic alveolar echinococcosis[J].J Clin Hepatol, 2017,33(11):1-5.
[28] 倪兴维, 贾万忠, 者永辉,等. 棘球蚴体外培养技术的应用[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(4):314-318. NI X Y, JIA W Z, ZHE Y H, et al. Application of in vitro cultivation technique for metacestodes in study of Echinococcus spp.[J]. Chin J Parasitol Parasit Dis, 2011, 29(4):314-318.
[29] 杨梅, 梁小弟, 李军,等. 细粒棘球绦虫新疆株胆汁酸钠协同转运蛋白基因的克隆及序列分析[J]. 科技导报, 2016, 34(2):215-220.DOI:10.3981/j.issn.1000-7857.2016.2.036. YANG M, LIANG X D, LI J, et al. Molecular cloning and sequence analysis of sodium-bile acid cotransporter from Echinococcus granulosus in Xinjiang, China[J]. Sci & Technol Rev, 2016, 34(2):215-220. DOI:10.3981/j.issn.1000-7857.2016.2.036.
[30] SMYTH J D. In vitro cultivation of parasitic helminths[M]. London: Macmillan Education UK, 1990:77-154.
[31] ZHENG H, ZHANG W, ZHANG L, et al. The genome of the hydatid tapeworm Echinococcus granulosus[J]. Nat Genet, 2013, 45(10):1168-1175. DOI: 10.1038/ng.2757.
[32] LI S, KIM T I, YOO W G, et al. Bile components and amino acids affect survival of the newly excysted juvenile Clonorchis sinensis, in maintaining media[J]. Parasitol Res, 2008, 103(5):1019-1024. DOI: 10.1007/s00436-008-1084-3.
[33] 王慧. 棘球蚴囊液蛋白质组学及主要分泌蛋白对宿主免疫调节的影响[D]. 乌鲁木齐:新疆医科大学, 2015. WANG H. Proteomic analysis of hydatid cyst fluid and effect of mainly secreted protein on host immune regulation[D]. Urumchi:Xinjiang Med Univ, 2015.
[2] OZDOL C, YILDIRIM A E, DAGLIOGLU E, et al. Alveolar hydatid cyst mimicking cerebellar metastatic tumor[J]. Surg Neurol Int, 2011, 2:13. DOI:10.4103/2152-7806.76281.
[3] ALGROS M P, MAJO F, BRESSON-HADNI S, et al. Intra cerebral alveolar echinococcosis[J]. Infection, 2003, 31(1): 63-65. DOI: 10.1007/s15010-002-2178-y.
[4] MANDAL S, MANDAL M D. Human cystic echinococcosis: epidemiologic, zoonotic, clinical, diagnostic and therapeutic aspects[J]. Asian Pac J Trop Med, 2012, 5(4): 253-260. DOI: 10.1016/S1995-7645(12)60035-2.
[5] 张梦媛, 伍卫平, 官亚宜, 等. 我国棘球蚴病疾病负担分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 15-19. ZHANG M Y, WU W P, GUAN Y Y, et al. Analysis on disease burden of hydatid disease in China[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 15-19.
[6] BUDKE C M, DEPLAZES P, TORGERSON P R. Global socioeconomic impact of cystic echinococcosis[J]. Emerging Infect Dis,2006,12(2):296-303.DOI:10.3201/eid1202.050499.
[7] 尚婧晔, 张光葭, 何伟,等. 细粒棘球蚴病病原分类学与分子流行病学研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(2): 166-173. SHANG J Y, ZHANG G J, HE W, et al. Taxonomy and molecular epidemiology of Echinococcus granulosus complex causing cystic echinococcosis[J]. Chin J Parasitol Parasit Dis, 2018, 36(2): 166-173.
[8] GROSSO G, GRUTTADAURIA S, BIONDI A, et al. Worldwide epidemiology of liver hydatidosis including the Mediterranean area[J].World J Gastroenterol,2012,18(13): 1425-1437. DOI: 10.3748/wjg.v18.i13.1425.
[9] 徐硕,党志胜,张皓冰,等.棘球蚴病药物治疗的研究进展[J].中国寄生虫学与寄生虫病杂志,2018,36(3):297-302. XU S , DANG Z S , ZHANG H B, et al. Research progress on drug treatment for hydatidosis[J]. Chin J Parasitol Parasit Dis, 2018, 36(3): 297-302.
[10] 温浩, 吐尔干艾力·阿吉, 邵英梅,等. 棘球蚴病防治成就及面临的挑战[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(6):466-471. WEN H, TUERGANAILI A J, SHAO Y M, et al. Research achievemets and challenges for echinococcosis control[J]. Chin J Parasitol Prasit Dis, 2015, 33(6):466-471.
[11] 周璇,夏党荣,赵新斐,等.棘球蚴病研究进展[J].中国动物保健,2014,16(11):7-10.DOI:10.3969/j.issn.1008-4754.2014.11.004. ZHOU X, XIA D R, ZHAO X F, et al. Study progress of echinococcosis[J]. China Ani Health, 2014, 16(11):7-10. DOI:10.3969/j.issn.1008-4754.2014.11.004.
[12] 伍卫平,王虎,王谦,等. 2012-2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 1-14. WU W P, WANG H, WANG Q, et al. A nationwide sampling survey on echinococcosis in China during 2012-2016[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 1-14.
[13] 孙艳红,杨亚明.包虫病的治疗研究进展[J].热带病与寄生虫学,2015,13(1):53-58.DOI:10.3969/j.issn.1672-2302.2015.01.017. SUN Y H, YANG Y M. Advances in the treatment of echinococcosis[J]. J Trop Dis Parasit, 2015, 13(1):53-58. DOI:10.3969/j.issn.1672-2302.2015.01.017.
[14] GOTTSTEIN B, FELLEISEN R. Protective immune mechanisms against the metacestode of Echinococcus multilocularis[J]. Parasitol Today(Regul Ed), 1995, 11(9): 320-326. DOI: 10.1016/0169-4758(95)80184-7.
[15] ELISSONDO M C, DOPCHIZ M C, BRASESCO M, et al. Echinococcus granulosus: first report of microcysts formation from protoscoleces of cattle origin using the in vitro vesicular culture technique[J]. Parasite, 2004, 11(4): 415-418. DOI: 10.1051/parasite/2004114415.
[16] 王慧,李军,郭宝平,等. 微囊法棘球蚴继发感染小鼠动物模型的建立[J]. 中国人兽共患病学报, 2016, 32(9): 784-788. DOI:10.3969/j.issn.1002-2694.2016.09.004. WANG H, LI J, GUO B P, et al. Establishment of secondary hydatid disease infection in mice with cystic and alveolar Echinococcus cysts cultured in vitro[J]. Chin J Zoonoses, 2016,32(9):784-788.DOI:10.3969/j.issn.1002-2694.2016.09.004.
[17] ZHANG W B, JONES M K, LI J, et al. Echinococcus granulosus: pre-culture of protoscoleces in vitro significantly increases development and viability of secondary hydatid cysts in mice[J]. Exp Parasitol, 2005, 110(1): 88-90. DOI: 10.1016/j.exppara.2005.02.003.
[18] LIU C S, ZHANG H B, YIN J H, et al. Echinococcus granulosus: suitable in vitro protoscolices culture density[J]. Biomed Environ Sci, 2013, 26(11): 912-915. DOI:10.3967/bes2013.020.
[19] 袁丽英,张壮志,石保新,等. 细粒棘球绦虫-原头蚴在两种细胞培养液中体外培养的初步观察[J]. 畜牧兽医杂志, 2008, 27(5): 16-18. YUAN L Y, ZHANG Z Z, SHI B X, et al. In vitro cultivation protoscoleces of the protoslices of Echinococcus granulosus in medium RPMI-1640 and MEM[J]. J Ani Sci Vet Med, 2008, 27(5): 16-18.
[20] 袁丽英,张壮志,石保新,等. 细粒棘球蚴-原头蚴体外培养成囊模型的建立[J]. 畜牧与兽医, 2009, 41(7): 29-31. YUAN L Y, Zhang Z Z, Shi B X, et al. Establishment of the in vitro cultivation model of protoscolex of Echinococcus cyst[J]. Ani Husbandry Veter Med,2009, 41(7): 29-31.
[21] 马海龙,李兆勇,孙世安,等. 肝癌细胞及其上清液体外培养多房棘球蚴的实验研究[J]. 武警后勤学院学报(医学版), 2012,21(11):864-867.DOI:10.3969/jissn.2095-3720.2012.11.008. MA H L, LI Z Y, SUN S A, et al. Experimental studies on the in vitro culture of Echinococcus multilocularis metacestodes with Bel-7404 cells and its supematant fluid[J]. J Logist Univ CAPF(Med Sci), 2012, 21(11): 864-867. DOI:10.3969/jissn.2095-3720.2012.11.008.
[22] 孙萃萍. 多房棘球绦虫HSP20蛋白的生物信息学分析、重组表达及泡球蚴的体外培养[D]. 兰州:兰州大学, 2015. SUN C P. Bioinformatic analysis, recombination and expression of HSP20 from Echinococcus multilocularis as well as the culture of alveolar hydatid in vitro[D]. Lanzhou: Lanzhou Univ, 2015.
[23] SPILIOTIS M, TAPPE D, SESTERHENN L, et al. Long-term in vitro cultivation of Echinococcus multilocularis metacestodes under axenic conditions[J]. Parasitol Res, 2004, 92(5):430-432. DOI: 10.1007/s00436-003-1046-8.
[24] HEMPHILL A, GOTTSTEIN B. Immunology and morphology studies on the proliferation of in vitro cultivated Echinococcus multilocularis metacestodes[J]. Parasitol Res, 1995, 81(7): 605-614. DOI: 10.1007/BF00932028.
[25] JURA H, BADER A, HARTMANN M, et al. Hepatic tissue culture model for study of host-parasite interactions in alveolar echinococcosis[J]. Infect Immun, 1996, 64(9): 3484-3490.
[26] SMYTH J D, DAVIES Z. In vitro culture of the strobilar state of Echinococcus granulosus (sheep strain): a review of basic problems and results[J]. Int J Parasitol, 1974, 4(6): 631-644. DOI:10.1016/0020-7519(74)90028-9.
[27] 周启锋, 任利,张灵强,等. 肝泡型包虫病动物模型建立的研究进展[J]. 临床肝胆病杂志, 2017,33(11):1-5. ZHOU Q F, REN L, ZHANG L Q, et al. Research advance in animal models of hepatic alveolar echinococcosis[J].J Clin Hepatol, 2017,33(11):1-5.
[28] 倪兴维, 贾万忠, 者永辉,等. 棘球蚴体外培养技术的应用[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(4):314-318. NI X Y, JIA W Z, ZHE Y H, et al. Application of in vitro cultivation technique for metacestodes in study of Echinococcus spp.[J]. Chin J Parasitol Parasit Dis, 2011, 29(4):314-318.
[29] 杨梅, 梁小弟, 李军,等. 细粒棘球绦虫新疆株胆汁酸钠协同转运蛋白基因的克隆及序列分析[J]. 科技导报, 2016, 34(2):215-220.DOI:10.3981/j.issn.1000-7857.2016.2.036. YANG M, LIANG X D, LI J, et al. Molecular cloning and sequence analysis of sodium-bile acid cotransporter from Echinococcus granulosus in Xinjiang, China[J]. Sci & Technol Rev, 2016, 34(2):215-220. DOI:10.3981/j.issn.1000-7857.2016.2.036.
[30] SMYTH J D. In vitro cultivation of parasitic helminths[M]. London: Macmillan Education UK, 1990:77-154.
[31] ZHENG H, ZHANG W, ZHANG L, et al. The genome of the hydatid tapeworm Echinococcus granulosus[J]. Nat Genet, 2013, 45(10):1168-1175. DOI: 10.1038/ng.2757.
[32] LI S, KIM T I, YOO W G, et al. Bile components and amino acids affect survival of the newly excysted juvenile Clonorchis sinensis, in maintaining media[J]. Parasitol Res, 2008, 103(5):1019-1024. DOI: 10.1007/s00436-008-1084-3.
[33] 王慧. 棘球蚴囊液蛋白质组学及主要分泌蛋白对宿主免疫调节的影响[D]. 乌鲁木齐:新疆医科大学, 2015. WANG H. Proteomic analysis of hydatid cyst fluid and effect of mainly secreted protein on host immune regulation[D]. Urumchi:Xinjiang Med Univ, 2015.