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

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。

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

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.