Lu Zhigang, Quack Thomas, Hahnel Steffen, Gelmedin Verena, Pouokam Ervice, Diener Martin, Hardt Martin, Michel Gabriela, Baal Nelli, Hackstein Holger, Grevelding Christoph G
BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany.
Institute for Veterinary Physiology and Biochemistry, Justus-Liebig-University, Giessen, Germany.
Int J Parasitol. 2015 Aug;45(9-10):663-72. doi: 10.1016/j.ijpara.2015.04.002. Epub 2015 May 1.
In the emerging era of post-genomic research on schistosomes, new methods are required to functionally analyse genes of interest in more detail. Among other tools, schistosome cell lines are needed to overcome present research constraints. Based on a recently established organ isolation protocol for adult Schistosoma mansoni, we report here on the successful enrichment of vitellarium tissue and isolation of vitelline cells. Morphological analyses performed by bright field, fluorescence, scanning and transmission electron microscopy showed typical features of S1 to S4 stage vitelline cells. In addition, molecular analyses using reverse transcription-PCR confirmed the identity of vitelline cells. Cytological and physiological studies included staining experiments with viability dyes and a neutral lipid stain, as well as calcium (Ca2+) imaging. Together they demonstrated cell viability, the possibility to define the differentiation stage of individual vitelline cells, and the suitability to investigate Ca(2+)-associated processes herein. Finally, fluorescence-activated cell sorting was shown to be a convenient way to separate and enrich S1 to S4 stage vitelline cells. In summary, these results demonstrate the expedience of the organ isolation protocol to obtain vitellarium tissue. Importantly, the protocol allows vitelline cells representing defined differentiation stages to be purified, which can be cultured in vitro and used to investigate diverse aspects of schistosome reproductive biology in the post-genomic era.
在血吸虫基因组后研究的新兴时代，需要新的方法来更详细地对感兴趣的基因进行功能分析。除了其他工具外，还需要血吸虫细胞系来克服当前的研究限制。基于最近建立的曼氏血吸虫成虫器官分离方案，我们在此报告了成功富集卵黄腺组织并分离卵黄细胞的情况。通过明场、荧光、扫描和透射电子显微镜进行的形态学分析显示了S1至S4阶段卵黄细胞的典型特征。此外，使用逆转录PCR进行的分子分析证实了卵黄细胞的身份。细胞学和生理学研究包括用活力染料和中性脂质染料进行染色实验，以及钙（Ca2+）成像。这些研究共同证明了细胞活力、确定单个卵黄细胞分化阶段的可能性以及在此研究Ca(2+)相关过程的适用性。最后，荧光激活细胞分选被证明是分离和富集S1至S4阶段卵黄细胞的便捷方法。总之，这些结果证明了器官分离方案在获取卵黄腺组织方面的便利性。重要的是，该方案允许纯化代表特定分化阶段的卵黄细胞，这些细胞可在体外培养并用于研究基因组后时代血吸虫生殖生物学的各个方面。
