基于三维细胞培养的多房棘球蚴感染模型的建立

Alveolar echinococcosis is a zoonosis caused by the larval forms of Echinococcus multilocularis, and can lead to serious impact on human and animal health. For Echinococcosis study, the in vitro models, which can mimic the in vivo microenvironment of the host is not yet well established. Recently, using three-dimensional (3D) cell culture models to investigate host-pathogen interactions have provided a new method for the in vitro study of disease. In this research, we will focus on the development of a 3D cell culture model of hepatocytes co-cultured with bone marrow mesenchymal stem cells, based on the collagen scaffold platform to mimic the in vivo 3D environment. Furthermore, this 3D co-culture cell model will be used to establish in vitro Echinococcus infection models, in order to study the growth and proliferation of in vitro cultured protoscolices, to detect the factors, protein secretion and gene expression during their proliferation, and to analyze the hepatocytes-parasites interaction. This research would offer references for the control and treatment of the Echinococcosis, and also provide a new method to study the host-parasites interactions.

多房棘球蚴病是由棘球属多房棘球绦虫的幼虫（多房棘球蚴）寄生于动物体及人体内所致的一种严重影响人和动物健康的人兽共患寄生虫病，目前对该病的研究缺乏可模拟体内微环境的体外宿主-寄生虫研究模型。基于三维（3D）细胞培养的宿主-病原相互作用研究是当前的研究热点，为疾病的体外研究提供了新方法。本研究使用胶原蛋白为原料制备的3D多孔支架结构作为培养支架，实现原代肝实质细胞与骨髓间充质干细胞的3D共培养；进一步建立基于3D肝实质细胞-骨髓间充质干细胞的体外原头蚴的生长、发育的肝脏感染模型，检测可调节原头蚴的发育、增殖的相关的因子和蛋白质的分泌及基因的表达，分析细胞-原头蚴之间相互作用和影响。本研究为棘球蚴病的控制及治疗提供依据，为体外研究宿主-寄生虫相互作用提供新方法。

本研究利用三维（Three-dimensional, 3D）胶原支架实现了肝实质细胞和骨髓间充质干细胞共培养，并基于该3D肝细胞共培养系统来研究多房棘球绦虫的幼虫——原头蚴的发育，模拟中间宿主体内寄生虫寄生于肝脏的组织模型。研究分析了3D培养肝细胞特异性代谢能力的维持和基因的表达。结果表明， 3D培养肝实质细胞的特异性代谢能力，包括白蛋白分泌、尿素合成、7乙氧基-O-脱乙基酶活性、CYP450酶功能的维持优于2D肝实质细胞培养；并且，3D肝细胞模型的肝细胞功能基因Cldn-3，Bsep，AFP，G6P，α1-AT，CYP3A4和NR1I3表达高于2D肝细胞培养。进一步，将原头蚴与上述建立的3D肝实质细胞-骨髓间充质干细胞进行共培养，观察到原头蚴培养8 w内迅速发育为包囊，且该包囊具有感染性，而无细胞培养的对照组中培养8w后，原头蚴及原头蚴发育的小包囊死亡率为100%。对3D共培养的包囊和原头蚴的转录组分析，鉴定得到807个差异表达基因。差异基因的GO和KEGG富集分析表明，包囊具有更高的代谢能力和更强的耐受性。基于上述体外棘球蚴培养模型，对体外培养原头蚴发育而成包囊的囊壁和囊液，以及小鼠体内发育的包囊的囊壁和囊液进行了样品分离及蛋白质ITRAQ分析，结果表明：体外培养包囊囊液与小鼠体内包囊囊液比较有722个差异蛋白；体外培养包囊壁与小鼠体内包囊囊壁比较有778个差异蛋白。本研究首次建立的体外3D棘球蚴培养及包虫病模型，它将为体外寄生虫-宿主相互作用的研究提供基础。