关键词: 刚地弓形虫, 速殖子, 鼠巨噬细胞, 人包皮成纤维细胞

Abstract:

Objective To establish an infection model for Toxoplasma gondii RH strain tachyzoites infection to mouse macrophage cell line RAW264.7, and to define a rapid and efficient method to culture tachyzoites in vitro. Methods The tachyzoites of T. gondii RH strain were purified with a 5 μm filtration membrane after 3 passages in Kunming mice, and then co-cultured with mouse macrophage cell RAW264.7 (ratios to tachyzoites set at 20 : 1, 10 : 1, 1 : 1 and 1 : 5) and human foreskin fibroblasts (HFF) cells (ratio to tachyzoites 1 : 1). The cells without tachyzoites were set as the control group. The co-cultures were observed at 10 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h and 48 h under a high-power microscope, and underwent Wright-Giemsa staining to observe the adhesion invasion of tachyzoites. The tachyzoites were inoculated at 1.5 × 10⁶/vial in a RAW264.7 T25 cell culture flask for co-culture. After 80% of pseudomonts were destroyed, the tachyzoites were collected and counted, and the productivity of tachyzoites was calculated. After staining with 0.4% trypan blue dye, the tachyzoite’s survival rate was calculated in the culture system. The harvested T. gondii tachyzoites were further used to infect new RAW264.7 cells, and the tachyzoites at each passage were inoculated at 1 × 10⁶/mouse in five healthy female Kunming mice. Meanwhile, the same amount of tachyzoites at passage 3 were inoculated in five healthy female mice as the control. The survival time of mice inoculated with tachyzoites at each passage was recorded. The statistical software SPSS 21.0 was used for statistical analysis. One-way analysis of variance was used for comparison between groups. Results When the ratio of RAW264.7 cells and tachyzoites was over 10 : 1, the boundary of infected cells turned obscure after infection for 24 h, and the cytoplasm was increased, with increased number of vesicles. After infection for 30 h, a large number of cells died, with no appearance of free tachyzoites. When the ratio was less than or equal to 1 : 1, fine particles could be seen in the cytoplasm within 24 h with no vacuoles. After infection for 30 h, more than 90% of pseudocysts were ruptured, and a plenty of tachyzoites were released into the medium. The completion in vitro development process of tachyzoites could be observed under this conditions. Therefore, tachyzoites were co-cultured with RAW264.7 cells at a ratio of 1 : 1. After infection for 0.5-1 h, about 80% of tachyzoites could invade into RAW264.7 cells, and enter both cytoplasm and nucleus. After 2-4 h, pseudocysts began to form. At 8 h, a part of pseudocysts proliferated into visible chrysanthemum shape. After 24 h, partial pseudocysts began to rupture. After 32 h, the cells were suspended and unable to adhere to the wall, most of pseudocysts ruptured and free tachyzoites were released. The morphology of the newborn tachyzoites was fine. We harvested 6 × 10⁸ tachyzoites, among which, the average live rate was higher than 92%. Tachyzoites were co-cultured with HFF cells, and the tachyzoites basically invaded the cells after 2-4 h; after 8 h, tachyzoites began to divide and proliferate; after 24 h of co-culture, the tachyzoites could divide into chrysanthemum shape; After 36 h, the cells began to burst and the tachyzoites released began a new round of HFF cell attack and infection. All passages of in vitro cultured tachyzoites showed an average lethal time to mice around 3.94-4.10 days, and the virulence of the tachyzoites was not weakened compared with those harvested under in vivo conditions in mice. Conclusion Mouse macrophage RAW264.7 cells could be used to produce large amounts of T. gondii RH tachyzoites quickly in vitro, and the yields are about 400 times of that by the in vivo culture method. Further, the in vitro produced tachyzoites have a comparable virulenc to those obtained by in vivo method, and are superior to those obtained in the HFF cells in the yield time and rate.

Key words: Toxoplasma gondii, Tachyzoite, RAW264.7 cell, human foreskin fibroblasts cell