微小隐孢子虫感染HCT-8细胞模型的建立及不同阶段虫体形态的观察

Development of in vitro cultivation model of Cryptosporidium parvum in HCT-8 cells and morphological observation of different developmental stages

利用人回盲肠癌(HCT-8)细胞作为感染细胞,采用不同的接种细胞浓度和培养血清浓度进行培养,观察细胞的生长状况;在培养细胞中加入1×105个经活力检测的微小隐孢子虫卵囊,培养72h后用荧光标记的隐孢子虫卵囊、子孢子染色试剂盒Crypt-a-Glo和Sporo-Glo进行染色,观察不同发育阶段的隐孢子虫形态。结果显示,在六孔培养板中接种10×105个细胞后,24h后长成80%～100%单细胞层,可以用来接种微小隐孢子虫。培养72h后观察到了隐孢子虫各个阶段虫体的形态。结果表明,成功建立了微小隐孢子虫感染HCT-8细胞模型。

In order to develop an in vitro cultivation model for Cryptosporidium parvum and to observe the morphology of its different developmental stage,human colon cancer（HCT-8）cell was selected to culture the parasite.Different number of inoculated cells and serum concentration were used to observe the growth condition of cells.After activity detection,1×105 oocysts were added to each well of 6-well culture plate with single-cell layer of HCT-8. Morphology of different life cycle stages of C.parvum were observed in the culture system using fluorescently-labeled oocyst and sporozoite staining kit,Crypt-a-Glo and Sporo-Glo stain after 72 h culturing with 1% FBS.The results showed that the single-cell layer occupied 80%-100% area of the 6-well culture plate at 24h post cultivation,which suited for infection of C.parvum. After 72h cultivation with 1% FBS,the shapes of complete life cycle stages of C.parvum,including sporozoites,trophozoites,merontⅠ,merontⅡ,merozoites,microgamonts,macrogamonts and oocysts,were found in the culture system using the Crypt-a-Glo and Sporo-Glo stain.