Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro.
Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro.
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DOI：
10.1371/journal.pntd.0004994
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发表时间：
2016-09
影响因子：
3.8
通讯作者：
Maule AG
中科院分区：
文献类型：
--
作者：
McCusker P;McVeigh P;Rathinasamy V;Toet H;McCammick E;O'Connor A;Marks NJ;Mousley A;Brennan GP;Halton DW;Spithill TW;Maule AG
片形吸虫病（或称肝片形吸虫病）是一种由肝片形吸虫属的肝吸虫引起的社会经济上重要的寄生虫病。抗流感病毒剂的耐药性暴露了对肝吸虫控制新药和/或疫苗的需求。一个快速改进的肝吸虫“分子工具箱”包括高质量的基因组/转录组数据集和RNA干扰平台，有助于功能基因组学方法进行药物/疫苗靶标验证。缺乏有效的培养/维持系统，将支持在体外研究少年吸虫发育/生物学，这些资源的开发受到破坏。在这里，我们报告了显着改善体外维持方法肝片吸虫，达到65%的存活率后，6个月的幼年吸虫补充50%的鸡血清的标准细胞培养基。我们发现，这种长期维持依赖于吸虫的生长，这是由类似于其他扁形虫中描述的“新生”干细胞的细胞增殖增加所支持的。生长导致幼鱼的形态发生巨大变化，包括消化道、生殖器官和外皮的发育，使其更接近成年鱼的形态。DNA合成的抑制阻止了成新细胞样细胞的增殖并抑制生长/发育。支持我们的断言，我们已经引发了发展的青少年向成人一样的吸虫，质谱分析表明，不断增长的吸虫有一个排泄/分泌蛋白质的配置文件，是不同的新excysted青少年和更接近的离体不成熟和成人的吸虫。此外，在体外维持吸虫显示过渡，在他们的运动从探测行为与迁移阶段蠕虫到一个较慢的波浪般的运动在成年人中看到。我们刺激F. hepatica为其长期体外研究奠定了第一个简单的平台，补充了最近肝吸虫资源的扩大，并促进了肝吸虫发育生物学的体外靶点验证研究。寄生蠕虫需要宿主生物体才能生存和繁殖。因此，很难在宿主之外研究它们。有些寄生虫可以用细胞培养方法在体外维持，如F.对于Hepatica，以前报道的方法是不令人满意的，因为它们难以繁殖并且不能支持长期生长和发育。在这里，我们开发了一套新的方法来维持F。离体培养的肝幼体。这些方法使用简单，常用的试剂和技术，使我们能够保持吸虫在体外存活至少6个月，以及刺激类似于成虫寄生虫的特征的发展。随着时间的推移，我们的体外吸虫显示出个体组织的结构和复杂性以及它们产生的蛋白质的变化，因此它们更像成年吸虫，而不是幼年吸虫。此外，我们证明，吸虫的生长是由类似干细胞的细胞分裂支持的，这是以前没有报道过的。肝。这项工作将支持对肝吸虫的研究，从而能够开发用于治疗人类和动物肝吸虫感染的新药和疫苗。
Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving ‘molecular toolbox’ for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the “neoblast” stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke. Parasitic worms require a host organism in order to survive and reproduce. As such, it is difficult to study them outside of a host. Some parasites can be maintained in vitro using cell culture methods; in the case of F. hepatica, previously-reported methods are unsatisfactory because they are difficult to reproduce and unable to support long term growth and development. Here we have developed a new set of methods for maintaining F. hepatica juveniles in vitro. These methods use simple, commonly available reagents and techniques, enabling us to keep fluke alive in vitro for at least 6 months, as well as stimulating the development of characteristics that resemble adult parasites. Over time, our in vitro fluke show changes in the structure and complexity of individual tissues, and the proteins they produce, such that they are more reminiscent of adult, than juvenile fluke. Additionally, we demonstrate that fluke growth is supported by the division of cells resembling stem cells, which have not been reported previously for F. hepatica. This work will support the study of liver fluke, enabling the development of new drugs and vaccines for the treatment of liver fluke infections of humans and animals.
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幼年片形吸虫属的 RNAi 动态。肝吸虫揭示了体外基因沉默的持续性。
影响因子：
3.8
作者：
McVeigh P;McCammick EM;McCusker P;Morphew RM;Mousley A;Abidi A;Saifullah KM;Muthusamy R;Gopalakrishnan R;Spithill TW;Dalton JP;Brophy PM;Marks NJ;Maule AG
通讯作者：
Maule AG
影响因子：
2
作者：
Fredensborg, BL;Poulin, R
通讯作者：
Poulin, R
影响因子：
2.1
作者：
Ndegwa, David;Krautz-Peterson, Greice;Skelly, Patrick J.
通讯作者：
Skelly, Patrick J.
影响因子：
2.4
作者：
Hodgkinson, J.;Cwiklinski, K.;Williams, D. J. L.
通讯作者：
Williams, D. J. L.
影响因子：
4.4
作者：
LaCourse, E. J.;Perally, S.;Brophy, P. M.
通讯作者：
Brophy, P. M.