日本血吸虫Mago nashi样蛋白编码基因的功能鉴定
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摘要
血吸虫病是严重危害人民身体健康,阻碍社会经济发展的重大传染病。日本血吸虫致病主要是由于虫卵在肝脏大量沉积形成的虫卵肉芽肿及其纤维化。控制血吸虫性别分化、性成熟及雌虫产卵成为防治血吸虫病的重要策略之一。
Mago nashi基因(Genebank登录号BM735619)所编码的蛋白通过氨基酸序列分析发现在人,果蝇,线虫等绝大多数生物中至少有80%的同源性。已有报道该基因在正常情况下抑制雄性化基因的表达,是决定生殖细胞雄性化作用的基因。由此推测日本血吸虫Mago nashi基因在血吸虫的生殖系统中也起着较为重要的作用。为了解日本血吸虫Mago nashi蛋白的功能,我们首先通过激光共聚焦显微镜观察日本血吸虫在终宿主体内不同发育阶段的形态特征,然后用RNAi技术下调Mago nashi基因的转录水平,再观察干扰作用后虫体的发育(尤其是生殖系统发育)是否与正常虫体之间存在差异。
日本血吸虫于终宿主体内的发育是一个复杂的过程,由适应自然外界生存过渡到适应终宿主体内生存,其生理、形态等方面发生了巨大变化。而这些变化对于我们研究日本血吸虫病的预防以及治疗具有重大的意义。激光共聚焦显微镜(CLSM)技术现已广泛的应用到生物医药和生命科学领域研究中去。曼氏血吸虫的激光共聚焦显微镜形态学研究已有报道,但日本血吸虫的激光共聚焦显微镜形态研究至今未见文献。本文我们设计单性以及双性感染日本血吸虫的方式感染小鼠,在双性感染中我们分别收集感染后15天,20天,25天,30天,35天的虫体;单性感染中我们收集感染后35天的虫体,将这些虫体通过固定,染色,脱色,脱水等一系列的处理后,最后于激光共聚焦显微镜下观察双性感染上述各发育阶段的形态发育特点,观察单性感染35天的形态特点。用激光共聚焦显微镜测量单性及双性感染35天虫体,包括:体长、体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积。激光共聚焦显微镜的观察以及统计学的分析结果发现双性感染中30天成虫各器官发育成熟,雄性成虫睾丸、抱雌沟、贮精囊、雄性生殖孔,雌性成虫卵巢、卵黄腺、卵黄管、卵模、梅氏腺、子宫等结构在激光共聚焦显微镜下结构清晰;各发育阶段虫体形态变化最为显著的是15-25天的虫体。感染后15天,33%雄性虫体已出现明显抱雌沟,8%虫体位于前端的睾丸可见含有少量排列稀疏生殖细胞,13%雌虫出现早期卵巢结构,卵细胞尚未分化;感染后20天,86%雄虫有5-7个含有较15天多的生殖细胞的睾丸,此时期生殖细胞之间界线仍不清楚。雌虫后半部可见卵黄腺,卵黄细胞数量较少且不成熟,排列稀疏。卵巢中可见较前数量多的未成熟卵细胞,但未见细胞分化;感染后25天,所有虫体生殖系统均已发育,卵黄腺中卵黄细胞明显增多,形态不规则,排列较为紧密。卵巢内卵细胞已出现细胞分化。90%以上雌虫子宫内可见少量虫卵。感染30天以及以上虫体均已发育成熟。单性感染35天虫体与双性相比,单性感染虫体体积均明显小于双性感染虫体,且发育均不成熟(雌虫尤为显著)。经SPSS 13.0统计软件分析单性感染35天虫体体长、睾丸长、睾丸面积、卵巢长、卵巢宽、卵巢面积均与双性感染35天虫体有显著性差异。
按照双链RNA(dsRNA)体外合成试剂盒要求,我们从日本血吸虫童虫cDNA文库中扩增出Mago nashi基因,将其片段正、反义链连接上T7启动子,然后把连接有T7启动子的目的基因的正、反义链进行PCR扩增。将PCR产物转录成单链RNA(ssRNA)并将其纯化,最后把一对ssRNA混合合成dsRNA。阴性对照组我们采用试剂盒中提供的基因。在BTX电穿孔仪上,设定参数为125V电压、20ms脉冲时值、1次脉冲次数,将dsRNA电穿孔转染入机械制备的日本血吸虫童虫体内,体外培养于电转后第1,3,5天收集童虫,按TRIzol方法同时提取童虫的总RNA及总蛋白。经实时荧光定量PCR及Western blotting分别检测Mago nashi基因表达水平变化。结果发现Mago nashi基因的转录水平实验组分别于电穿孔后第1, 3, 5天较阴性对照组下降22%,69%,80%。该基因的蛋白水平在第1, 3, 5天较阴性对照组下降12%,39%,56%。实验结果证明dsRNA可以特异性的抑制日本血吸虫靶基因以及蛋白的表达且效果明显。经dsRNA电转的童虫注射入小鼠体内,6周后取出虫体通过一系列处理制成标本,在激光共聚焦显微镜下观察虫体内部各器官形态特征及测量虫体体长、体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积。结果发现SjMago dsRNA组中8条中6条雄虫睾丸内有大量精子出现,而雌虫中卵巢,卵黄腺中却没有明显特征变化;测得各项指标经SPSS 13.0统计软件分析阴性对照组与SjMago dsRNA组的体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积均有显著性差异。由此结果我们得出Mago nashi基因在日本血吸虫中是生殖相关基因,在生殖系统器官的正常发育起一定作用。
Schistosomiasis is still serious infectious diseases which is harmful to people’s health and obstruct the social and economic development. The schistosome eggs are retained in the liver of the final host where they elicit inflammatory immune responses, which lead to formation of the granuloma and fibrosis, the major pathological effects of schistosomiasis. Controlling sexual dimorphism, sexual maturation and labour division may be effective in prevention of schistosomiasis.
Through amino acid sequence analysis, we found the protein which was coded by Mago nashi(Genebank accession No.BM735619) has 80% homology in many animals such as drosophila, nematodes etc and there had been reported that this gene can inhibit the expression of masculinizing genes and has the function of decide the germ cell masculinize in normal condition. So we conclude the Mago nashi gene has played a key role in reproductive system. In order to research the function of the protein coded by gene of SjMago nashi, firstly we observed the morphology of the schistosomiasis japonicum which lived in final host, then leveled down the transcription of the gene of SjMago nashi by RNAi, finally wonder if the test group (RNAi) had any difference from the control group (normal) in the development of the worms (especially in reproductive systems).
There is a complex procession about the development of Schistosoma japonicum in final host. It has great changes in physiology and morphology from living in nature to final host and these changes have important significance to the prevention and treatment of the schistosomiasis.Now CLSM technology is applying to many fields such as biomedicine, life sciences and etc.The morphology research of the Schistosoma Mansoni under CLSM had reported, but not about the Schistosoma japonicum.In our study, we designed mixed and unisexual infection of Schistosoma japonicum in mice. We collected adult worms which are infected 15, 20,25,30,35 days separately in mixed sexual infection and adult worms which infected 35 days in unisexual infection. Then the adult worms are fixed, stained, clarified, dehydrated, and finally observing the morphology development of the worms in different stages in mixed infection and the morphology of the worms which are infected 35 days in unisexual infection. We measure many parameters such as length, width of body; length, width, area of testicular lobes and ovary. The results of the observation and statistic analysis show every organ developed maturely, the structure of the testicular lobes,gynaecophoric canal, seminal vesicle, genital pore and the ovary,vitelline glands,vitelline duct,ootype,mehli' gland, uterus are clear under the CLSM.In each development stages, the morphology changes significantly are worms which were infected after 15-25 days. On the fifeenth day ,33% of male worms had formed a distinct gynaecophoric canal ,8% of them had testicular lobes with free areas around the few germinative cells inside. Of females, 13% had an incipient ovary lacking cellular differentiation. On the twentieth day, most worms had developed reproductive systems .Of male worms, 86% had 5-7 testicular lobes where more germinative cells could be observed, the border of the reproduction cell in this stage is not clear adequately. In female worms, vitelline glands around the digestive canal were composed of several anomalous cells which were sparsely arranged. The ovaries had more immature cells without cellular differentiation. On the twenty-fifth day, all worms had developed complete reproductive systems. In females, vitelline cell numbers were increased while the vitelline glands were irregular arranged closely. Ovaries were differentiated. 90% of them had presented few eggs in the uterus .The worms showed fully developed organs from day 30 onwards. The adult worms which were infected after 35 days in unisexual infection are smaller than in mixed infection, and develop immature especially the female. The adult worms which were infected 35 days in unisexual infection have significant differences from the mixed infection in length of body; length, width, area of testicular lobes and ovary through SPSS13.0 statistics software package.
According to in vitro dsRNA synthesis kit demand, we amplified Mago nashi gene from schistosomulum cDNA library. Then we amplified the positive and anti-sense strand with promoter T7 by PCR then transcript into ssRNA and purification, finally syntheses dsRNA with a couple of ssRNA.We use the gene provided by the kit in control group. We electroporated with dsRNA to schistosomulum at 125V for 20ms,1 pulse using an Electro Square PoratorTM ECM830(BTX).Aliquots of parasites were harvested respectively in day 1,3,5 after electroporation.Total RNA,DNA and proteins were isolated using TRIZOL Reagent according to the manufacturer's guidelines. Levels of Mago nashi mRNA and proteins were determined by RT-PCR and Western blotting analysis. The results showed that SjMago mRNA levels were decreased by22%,69%,80% in test group,respectively,at day 1,3,5 after electroporation.The SjMago protein expression levels were decreased by12%,39%,56% in test group,respectively,at day 1,3,5 after electroporation.The results suggested the dsRNA could inhibit the expression of the target gene and the protein specifically and efficiently. The schistosomula which were electroporated with dsRNA were injected into the mice,and we made them into specimen through a procession of methods after 6 weeks,then oberserved the morphology characteristics of each organ and measured the length and width of the worms,the length,width and area of the testicular lobes,the length,width and area of the ovaries under the confocal laser scanning microscopy.The results show there are many spermatozoa in testicular lobes of six worms of eight male in SjMago group and no changes in ovary and vitelline gland of female. The adult worms which were infected 6 weeks in SjMago dsRNA group have significant differences from control group in width of the worms, the length, width and area of the testicular lobes, the length, width and area of the ovaries through SPSS13.0 statistics software package. So we concluded that Mago nashi is the gene associated with reproduction in schistosomiasis japonicum.
Mago nashi基因(Genebank登录号BM735619)所编码的蛋白通过氨基酸序列分析发现在人,果蝇,线虫等绝大多数生物中至少有80%的同源性。已有报道该基因在正常情况下抑制雄性化基因的表达,是决定生殖细胞雄性化作用的基因。由此推测日本血吸虫Mago nashi基因在血吸虫的生殖系统中也起着较为重要的作用。为了解日本血吸虫Mago nashi蛋白的功能,我们首先通过激光共聚焦显微镜观察日本血吸虫在终宿主体内不同发育阶段的形态特征,然后用RNAi技术下调Mago nashi基因的转录水平,再观察干扰作用后虫体的发育(尤其是生殖系统发育)是否与正常虫体之间存在差异。
日本血吸虫于终宿主体内的发育是一个复杂的过程,由适应自然外界生存过渡到适应终宿主体内生存,其生理、形态等方面发生了巨大变化。而这些变化对于我们研究日本血吸虫病的预防以及治疗具有重大的意义。激光共聚焦显微镜(CLSM)技术现已广泛的应用到生物医药和生命科学领域研究中去。曼氏血吸虫的激光共聚焦显微镜形态学研究已有报道,但日本血吸虫的激光共聚焦显微镜形态研究至今未见文献。本文我们设计单性以及双性感染日本血吸虫的方式感染小鼠,在双性感染中我们分别收集感染后15天,20天,25天,30天,35天的虫体;单性感染中我们收集感染后35天的虫体,将这些虫体通过固定,染色,脱色,脱水等一系列的处理后,最后于激光共聚焦显微镜下观察双性感染上述各发育阶段的形态发育特点,观察单性感染35天的形态特点。用激光共聚焦显微镜测量单性及双性感染35天虫体,包括:体长、体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积。激光共聚焦显微镜的观察以及统计学的分析结果发现双性感染中30天成虫各器官发育成熟,雄性成虫睾丸、抱雌沟、贮精囊、雄性生殖孔,雌性成虫卵巢、卵黄腺、卵黄管、卵模、梅氏腺、子宫等结构在激光共聚焦显微镜下结构清晰;各发育阶段虫体形态变化最为显著的是15-25天的虫体。感染后15天,33%雄性虫体已出现明显抱雌沟,8%虫体位于前端的睾丸可见含有少量排列稀疏生殖细胞,13%雌虫出现早期卵巢结构,卵细胞尚未分化;感染后20天,86%雄虫有5-7个含有较15天多的生殖细胞的睾丸,此时期生殖细胞之间界线仍不清楚。雌虫后半部可见卵黄腺,卵黄细胞数量较少且不成熟,排列稀疏。卵巢中可见较前数量多的未成熟卵细胞,但未见细胞分化;感染后25天,所有虫体生殖系统均已发育,卵黄腺中卵黄细胞明显增多,形态不规则,排列较为紧密。卵巢内卵细胞已出现细胞分化。90%以上雌虫子宫内可见少量虫卵。感染30天以及以上虫体均已发育成熟。单性感染35天虫体与双性相比,单性感染虫体体积均明显小于双性感染虫体,且发育均不成熟(雌虫尤为显著)。经SPSS 13.0统计软件分析单性感染35天虫体体长、睾丸长、睾丸面积、卵巢长、卵巢宽、卵巢面积均与双性感染35天虫体有显著性差异。
按照双链RNA(dsRNA)体外合成试剂盒要求,我们从日本血吸虫童虫cDNA文库中扩增出Mago nashi基因,将其片段正、反义链连接上T7启动子,然后把连接有T7启动子的目的基因的正、反义链进行PCR扩增。将PCR产物转录成单链RNA(ssRNA)并将其纯化,最后把一对ssRNA混合合成dsRNA。阴性对照组我们采用试剂盒中提供的基因。在BTX电穿孔仪上,设定参数为125V电压、20ms脉冲时值、1次脉冲次数,将dsRNA电穿孔转染入机械制备的日本血吸虫童虫体内,体外培养于电转后第1,3,5天收集童虫,按TRIzol方法同时提取童虫的总RNA及总蛋白。经实时荧光定量PCR及Western blotting分别检测Mago nashi基因表达水平变化。结果发现Mago nashi基因的转录水平实验组分别于电穿孔后第1, 3, 5天较阴性对照组下降22%,69%,80%。该基因的蛋白水平在第1, 3, 5天较阴性对照组下降12%,39%,56%。实验结果证明dsRNA可以特异性的抑制日本血吸虫靶基因以及蛋白的表达且效果明显。经dsRNA电转的童虫注射入小鼠体内,6周后取出虫体通过一系列处理制成标本,在激光共聚焦显微镜下观察虫体内部各器官形态特征及测量虫体体长、体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积。结果发现SjMago dsRNA组中8条中6条雄虫睾丸内有大量精子出现,而雌虫中卵巢,卵黄腺中却没有明显特征变化;测得各项指标经SPSS 13.0统计软件分析阴性对照组与SjMago dsRNA组的体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积均有显著性差异。由此结果我们得出Mago nashi基因在日本血吸虫中是生殖相关基因,在生殖系统器官的正常发育起一定作用。
Schistosomiasis is still serious infectious diseases which is harmful to people’s health and obstruct the social and economic development. The schistosome eggs are retained in the liver of the final host where they elicit inflammatory immune responses, which lead to formation of the granuloma and fibrosis, the major pathological effects of schistosomiasis. Controlling sexual dimorphism, sexual maturation and labour division may be effective in prevention of schistosomiasis.
Through amino acid sequence analysis, we found the protein which was coded by Mago nashi(Genebank accession No.BM735619) has 80% homology in many animals such as drosophila, nematodes etc and there had been reported that this gene can inhibit the expression of masculinizing genes and has the function of decide the germ cell masculinize in normal condition. So we conclude the Mago nashi gene has played a key role in reproductive system. In order to research the function of the protein coded by gene of SjMago nashi, firstly we observed the morphology of the schistosomiasis japonicum which lived in final host, then leveled down the transcription of the gene of SjMago nashi by RNAi, finally wonder if the test group (RNAi) had any difference from the control group (normal) in the development of the worms (especially in reproductive systems).
There is a complex procession about the development of Schistosoma japonicum in final host. It has great changes in physiology and morphology from living in nature to final host and these changes have important significance to the prevention and treatment of the schistosomiasis.Now CLSM technology is applying to many fields such as biomedicine, life sciences and etc.The morphology research of the Schistosoma Mansoni under CLSM had reported, but not about the Schistosoma japonicum.In our study, we designed mixed and unisexual infection of Schistosoma japonicum in mice. We collected adult worms which are infected 15, 20,25,30,35 days separately in mixed sexual infection and adult worms which infected 35 days in unisexual infection. Then the adult worms are fixed, stained, clarified, dehydrated, and finally observing the morphology development of the worms in different stages in mixed infection and the morphology of the worms which are infected 35 days in unisexual infection. We measure many parameters such as length, width of body; length, width, area of testicular lobes and ovary. The results of the observation and statistic analysis show every organ developed maturely, the structure of the testicular lobes,gynaecophoric canal, seminal vesicle, genital pore and the ovary,vitelline glands,vitelline duct,ootype,mehli' gland, uterus are clear under the CLSM.In each development stages, the morphology changes significantly are worms which were infected after 15-25 days. On the fifeenth day ,33% of male worms had formed a distinct gynaecophoric canal ,8% of them had testicular lobes with free areas around the few germinative cells inside. Of females, 13% had an incipient ovary lacking cellular differentiation. On the twentieth day, most worms had developed reproductive systems .Of male worms, 86% had 5-7 testicular lobes where more germinative cells could be observed, the border of the reproduction cell in this stage is not clear adequately. In female worms, vitelline glands around the digestive canal were composed of several anomalous cells which were sparsely arranged. The ovaries had more immature cells without cellular differentiation. On the twenty-fifth day, all worms had developed complete reproductive systems. In females, vitelline cell numbers were increased while the vitelline glands were irregular arranged closely. Ovaries were differentiated. 90% of them had presented few eggs in the uterus .The worms showed fully developed organs from day 30 onwards. The adult worms which were infected after 35 days in unisexual infection are smaller than in mixed infection, and develop immature especially the female. The adult worms which were infected 35 days in unisexual infection have significant differences from the mixed infection in length of body; length, width, area of testicular lobes and ovary through SPSS13.0 statistics software package.
According to in vitro dsRNA synthesis kit demand, we amplified Mago nashi gene from schistosomulum cDNA library. Then we amplified the positive and anti-sense strand with promoter T7 by PCR then transcript into ssRNA and purification, finally syntheses dsRNA with a couple of ssRNA.We use the gene provided by the kit in control group. We electroporated with dsRNA to schistosomulum at 125V for 20ms,1 pulse using an Electro Square PoratorTM ECM830(BTX).Aliquots of parasites were harvested respectively in day 1,3,5 after electroporation.Total RNA,DNA and proteins were isolated using TRIZOL Reagent according to the manufacturer's guidelines. Levels of Mago nashi mRNA and proteins were determined by RT-PCR and Western blotting analysis. The results showed that SjMago mRNA levels were decreased by22%,69%,80% in test group,respectively,at day 1,3,5 after electroporation.The SjMago protein expression levels were decreased by12%,39%,56% in test group,respectively,at day 1,3,5 after electroporation.The results suggested the dsRNA could inhibit the expression of the target gene and the protein specifically and efficiently. The schistosomula which were electroporated with dsRNA were injected into the mice,and we made them into specimen through a procession of methods after 6 weeks,then oberserved the morphology characteristics of each organ and measured the length and width of the worms,the length,width and area of the testicular lobes,the length,width and area of the ovaries under the confocal laser scanning microscopy.The results show there are many spermatozoa in testicular lobes of six worms of eight male in SjMago group and no changes in ovary and vitelline gland of female. The adult worms which were infected 6 weeks in SjMago dsRNA group have significant differences from control group in width of the worms, the length, width and area of the testicular lobes, the length, width and area of the ovaries through SPSS13.0 statistics software package. So we concluded that Mago nashi is the gene associated with reproduction in schistosomiasis japonicum.
引文
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2.周继凯,李焕德.激光扫描共聚焦显微技术在医药研究中的应用[J].中南药学,2007;5 (1):66-68.
3.张向阳,赵磊.激光扫描共聚焦显微镜的基本功能及在医学各领域中的应用[J].邯郸医学高等专科学校学报,2002;15(3):369-370.
4. Basch PF. Cultivation of Schistosoma mansoni in vitro.Ⅱ. Production of infertile eggs by worm pairs cultured from cercariae, J Parasitol, 1981; 67: 186-189.
5. Basch PF.Why do schistosomes have separate sexes [J].Parasitol Today, 1990; 6(5):160-163.
6. Kunz W.Schistosome male-female interaction: induction of germ-cell differentiation [J].Trends Parasitol, 2001; 17(5):227-231.
7. Matzke MA, Birchler JA. RNAi-mediated pathways in the nucleus. Nat Rev Genet, 2005; 6 (1):24-35 .
8. Micklem DR, Dasgupta R, Elliott H, et al. The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Current Biology, 1997; 7: 468-478.
9. Pozzoli O, Gilardelli CN, Sordino P, et al. Identification and expression pattern of mago nashi during zebrafish development. Gene Expression Pattern, 2004; 5: 265-272.
10. Fire A, Xu S, Montgomery MK, et, al. Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans. Nature, 1998; 391: 806-811.
11. Zhang W, Yang H, Kong X, et al. Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat Med, 2005; 11(1):56-62.
12. Fowler T, Bamberg S, Moller P, et al. Inhibition of Marburg virus protein expression and viral release by RNA interference. J Gen Virol, 2005; 86:1181-1188.
13. Kodama Y, Asai N, Kawai K, et al. The RET proto-oncogene: A molecular therapeutic target in thyroid cancer. Cancer Sci, 2005; 96(3):143-148.
14. Singer E. New technologies deliver in treating neurological diseases. Nat Med, 2004; 10(12):1267.
15. Timmons, L.and A.Fire.Specific interference by ingested dsRNA.Nature, 1998; 395(6705):854.
16.毛守白.血吸虫生物学与血吸虫病的防治[M].第1版.北京:人民卫生出版社,1990:16-21.
17.周述龙,杨孟祥,孔楚豪,等.日本血吸虫精子发育与支持细胞超微结构的观察[J].中国寄生虫学与寄生虫病杂志,1993;11(1):50-52.
18.王晓楠,吴正升,杨枫,等.光学显微镜下日本血吸虫成虫生殖系统形态学观察[J].中国血吸虫病防治杂志,2007;19(5):374-376.
19. Neves RH,Machado-Silva JR,Carvalho JJ,et al.A new description of the reproductive system of Schistosoma mansoni (Trematoda: Schistosomatidae) analyzed by confocal laser scanning microscopy. Parasitol Res, 2005; 95:43–49.
20. Biolchini CL,Neves RH,Hulstijn M,et al.Development of Schistosoma mansoni worms in mice analyzed by bright field and confocal microscopy.Mem Inst Oswaldo Cruz, 2006; 101 (1): 261-265.
21. Ribeiro-Paes JT, Rodrigues V. Sex determination and female reproductive development in the genus Schistosoma: a review. Rev Inst Med Trop S?o Paulo 1997; 39: 337-344.
22. Kunz W. Schistosome male-female interaction: induction of germ-cell differentiation. Trends Parasitol 2001; 17: 227-231.
23. Grevelding CG, Sommer G, Kunz W. Female-specific gene expression in Schistosoma mansoni is regulated by pairing. Parasitology, 1997; 115: 635-640.
24. Napolic, C., C.Lemirux, R.Jorgensen. Introduction of a chalacome synthase gene into Petunia results in reversible cosuppression of homologous genes in trans.Plant Cell, 1990; 2:279-289.
25. Bernstein, E., A.A.Caudy, S.M.Hammond et al.Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature, 2001; 409:363-366.
26. Tabara, H., A.Grishok, C.C.Mello.RNA in C.elegans: Soaking in the genome sequence.Science, 1998; 282(5388):430-431.
27. Timmons, L., D.L.Court, A.Fire.Ingestion of bacterially expressed dsRNA can produce specific and potent genetic interference in Caenorhabditis elegans.Gene, 2001; 263(1-2):103-112.
28. Issa, Z., W.N.Grant, S.Stasiuk et al.Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubrformis.International J.Parasitol. , 2005; 35(9):935-940.
29. Kotze, A.C.and N.H.Begnall.RNA interference in Haemonchus contortus: Suppression of beta-tubulin gene expression in L3, L4 and adult worms in vitro. Mol.Biochem.Parasitol, 2006; 145:101-110.
30. Jason M. Correnti, Paul J. Brindley, Edward J. Pearce. Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth Mol.Biochem.Parasitol, 2005; 143:209–215.
1. Jorgensen R. Altered gene expression in plants due to trans-interactions between homologous genes [J].Trends Biotechnol, 1990, 8 (12): 340-344.
2. Cogoni C, Macino G. Gene silencing in neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase [J]. Nature, 1999, 399: 166.
3. Fire A, Xu S, MontgomeryMK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391: 806.
4. Elbashir SM, Harborth J, LendeckelW, et al. Dup lexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411: 494.
5. Fire A, Xu S, Montgomery MK, et al .Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegan [J] .Nature, 1998, 391: 806-811.
6. Montgomery MK, Xu S , Fire A , et al .RAN as a target or double - stranded RNA mediated genetic interference in Caenorhabditis elegan [J].Proc Natl Acad Sci US , 1998 ,95 (26) : 15502-15507.
7. Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J]. Nature, 2001, 409 (6818): 363.
8. Elbashir SM, LendeckelW, Tuschl T. RNA interference is mediated by 21-and22-nucleotide RNAs [J]. GenesDev, 2001, 15 (2): 188.
9. Hannon G J. RNA interferences [J]. Nature, 2002, 418 (6894): 244.
10. Trevor Stokes. RNAi-Based gene expression manipulation [J]. Genetic Engineering News, 2007, 27 (2): 28.
11. Boyle JP, Wu XJ, Shoemaker CB, et al. Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts. Mol Biochem Parasitol, 2003; 128(2):205-215.
12. Skelly PJ, Da’dara A, Ham DA. Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference. Int J Parasitol, 2003; 33(4):363-369.
13. Malhotra P, Dasaradhi PV, Kumar A, et al. Double-stranded RNA-mediated gene silencing of cysteine proteases (falcipain-1 and -2) of Plasmodium falciparum. Mol Microbiol, 2002;45(5): 1245-1254.
14. McRobert L, McConkey GA. RNA interference inhibits growth of Plasmodium falciparum. Mol Biochem Parasitol, 2002; 119(2):273-278.
15. Hao Ran,Yang Ya-ping,Yang Jing,et al.Primary investigations on the functions of TS87 gene of Trichinella Spiralis using RNA interference.Acta Parasitologica ET Medica Entomologica Sinica,2008;15(1):26-32.
16. Martinez M A, Clotet B, Este J A. RNA interference of HIV replication [J] .Trends Immunol, 2002, 23 (12):559-561.
17. Jacque J M, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference [J] .Nature, 2002, 418 (6896): 435-438.
18. Lee N S , Dohhjima T , Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells [J].Nat Biotechnol , 2002 , 20(19) : 500-505.
19. Novina C D , Murray M F , Dykxhoorn D M , et al. siRNA2 directed inhibition of HIV-1 infection [J].Nat Med , 2002 , 8 (7) : 681-686.
20. Qiu S, Adema CM, Lane T. A computational study of off-target effects of RNA interference [J]. Nucleic Acids Res, 2005, 33 (6): 1834.
21. Hannon GJ, Conklin DS. RNA interference by short hairp in RNAs expressed in vertebrate cells [J]. Methods Mol Biol, 2004, 257: 255.
2.周继凯,李焕德.激光扫描共聚焦显微技术在医药研究中的应用[J].中南药学,2007;5 (1):66-68.
3.张向阳,赵磊.激光扫描共聚焦显微镜的基本功能及在医学各领域中的应用[J].邯郸医学高等专科学校学报,2002;15(3):369-370.
4. Basch PF. Cultivation of Schistosoma mansoni in vitro.Ⅱ. Production of infertile eggs by worm pairs cultured from cercariae, J Parasitol, 1981; 67: 186-189.
5. Basch PF.Why do schistosomes have separate sexes [J].Parasitol Today, 1990; 6(5):160-163.
6. Kunz W.Schistosome male-female interaction: induction of germ-cell differentiation [J].Trends Parasitol, 2001; 17(5):227-231.
7. Matzke MA, Birchler JA. RNAi-mediated pathways in the nucleus. Nat Rev Genet, 2005; 6 (1):24-35 .
8. Micklem DR, Dasgupta R, Elliott H, et al. The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Current Biology, 1997; 7: 468-478.
9. Pozzoli O, Gilardelli CN, Sordino P, et al. Identification and expression pattern of mago nashi during zebrafish development. Gene Expression Pattern, 2004; 5: 265-272.
10. Fire A, Xu S, Montgomery MK, et, al. Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans. Nature, 1998; 391: 806-811.
11. Zhang W, Yang H, Kong X, et al. Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat Med, 2005; 11(1):56-62.
12. Fowler T, Bamberg S, Moller P, et al. Inhibition of Marburg virus protein expression and viral release by RNA interference. J Gen Virol, 2005; 86:1181-1188.
13. Kodama Y, Asai N, Kawai K, et al. The RET proto-oncogene: A molecular therapeutic target in thyroid cancer. Cancer Sci, 2005; 96(3):143-148.
14. Singer E. New technologies deliver in treating neurological diseases. Nat Med, 2004; 10(12):1267.
15. Timmons, L.and A.Fire.Specific interference by ingested dsRNA.Nature, 1998; 395(6705):854.
16.毛守白.血吸虫生物学与血吸虫病的防治[M].第1版.北京:人民卫生出版社,1990:16-21.
17.周述龙,杨孟祥,孔楚豪,等.日本血吸虫精子发育与支持细胞超微结构的观察[J].中国寄生虫学与寄生虫病杂志,1993;11(1):50-52.
18.王晓楠,吴正升,杨枫,等.光学显微镜下日本血吸虫成虫生殖系统形态学观察[J].中国血吸虫病防治杂志,2007;19(5):374-376.
19. Neves RH,Machado-Silva JR,Carvalho JJ,et al.A new description of the reproductive system of Schistosoma mansoni (Trematoda: Schistosomatidae) analyzed by confocal laser scanning microscopy. Parasitol Res, 2005; 95:43–49.
20. Biolchini CL,Neves RH,Hulstijn M,et al.Development of Schistosoma mansoni worms in mice analyzed by bright field and confocal microscopy.Mem Inst Oswaldo Cruz, 2006; 101 (1): 261-265.
21. Ribeiro-Paes JT, Rodrigues V. Sex determination and female reproductive development in the genus Schistosoma: a review. Rev Inst Med Trop S?o Paulo 1997; 39: 337-344.
22. Kunz W. Schistosome male-female interaction: induction of germ-cell differentiation. Trends Parasitol 2001; 17: 227-231.
23. Grevelding CG, Sommer G, Kunz W. Female-specific gene expression in Schistosoma mansoni is regulated by pairing. Parasitology, 1997; 115: 635-640.
24. Napolic, C., C.Lemirux, R.Jorgensen. Introduction of a chalacome synthase gene into Petunia results in reversible cosuppression of homologous genes in trans.Plant Cell, 1990; 2:279-289.
25. Bernstein, E., A.A.Caudy, S.M.Hammond et al.Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature, 2001; 409:363-366.
26. Tabara, H., A.Grishok, C.C.Mello.RNA in C.elegans: Soaking in the genome sequence.Science, 1998; 282(5388):430-431.
27. Timmons, L., D.L.Court, A.Fire.Ingestion of bacterially expressed dsRNA can produce specific and potent genetic interference in Caenorhabditis elegans.Gene, 2001; 263(1-2):103-112.
28. Issa, Z., W.N.Grant, S.Stasiuk et al.Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubrformis.International J.Parasitol. , 2005; 35(9):935-940.
29. Kotze, A.C.and N.H.Begnall.RNA interference in Haemonchus contortus: Suppression of beta-tubulin gene expression in L3, L4 and adult worms in vitro. Mol.Biochem.Parasitol, 2006; 145:101-110.
30. Jason M. Correnti, Paul J. Brindley, Edward J. Pearce. Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth Mol.Biochem.Parasitol, 2005; 143:209–215.
1. Jorgensen R. Altered gene expression in plants due to trans-interactions between homologous genes [J].Trends Biotechnol, 1990, 8 (12): 340-344.
2. Cogoni C, Macino G. Gene silencing in neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase [J]. Nature, 1999, 399: 166.
3. Fire A, Xu S, MontgomeryMK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391: 806.
4. Elbashir SM, Harborth J, LendeckelW, et al. Dup lexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411: 494.
5. Fire A, Xu S, Montgomery MK, et al .Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegan [J] .Nature, 1998, 391: 806-811.
6. Montgomery MK, Xu S , Fire A , et al .RAN as a target or double - stranded RNA mediated genetic interference in Caenorhabditis elegan [J].Proc Natl Acad Sci US , 1998 ,95 (26) : 15502-15507.
7. Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J]. Nature, 2001, 409 (6818): 363.
8. Elbashir SM, LendeckelW, Tuschl T. RNA interference is mediated by 21-and22-nucleotide RNAs [J]. GenesDev, 2001, 15 (2): 188.
9. Hannon G J. RNA interferences [J]. Nature, 2002, 418 (6894): 244.
10. Trevor Stokes. RNAi-Based gene expression manipulation [J]. Genetic Engineering News, 2007, 27 (2): 28.
11. Boyle JP, Wu XJ, Shoemaker CB, et al. Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts. Mol Biochem Parasitol, 2003; 128(2):205-215.
12. Skelly PJ, Da’dara A, Ham DA. Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference. Int J Parasitol, 2003; 33(4):363-369.
13. Malhotra P, Dasaradhi PV, Kumar A, et al. Double-stranded RNA-mediated gene silencing of cysteine proteases (falcipain-1 and -2) of Plasmodium falciparum. Mol Microbiol, 2002;45(5): 1245-1254.
14. McRobert L, McConkey GA. RNA interference inhibits growth of Plasmodium falciparum. Mol Biochem Parasitol, 2002; 119(2):273-278.
15. Hao Ran,Yang Ya-ping,Yang Jing,et al.Primary investigations on the functions of TS87 gene of Trichinella Spiralis using RNA interference.Acta Parasitologica ET Medica Entomologica Sinica,2008;15(1):26-32.
16. Martinez M A, Clotet B, Este J A. RNA interference of HIV replication [J] .Trends Immunol, 2002, 23 (12):559-561.
17. Jacque J M, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference [J] .Nature, 2002, 418 (6896): 435-438.
18. Lee N S , Dohhjima T , Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells [J].Nat Biotechnol , 2002 , 20(19) : 500-505.
19. Novina C D , Murray M F , Dykxhoorn D M , et al. siRNA2 directed inhibition of HIV-1 infection [J].Nat Med , 2002 , 8 (7) : 681-686.
20. Qiu S, Adema CM, Lane T. A computational study of off-target effects of RNA interference [J]. Nucleic Acids Res, 2005, 33 (6): 1834.
21. Hannon GJ, Conklin DS. RNA interference by short hairp in RNAs expressed in vertebrate cells [J]. Methods Mol Biol, 2004, 257: 255.