目的 建立刚地弓形虫（简称弓形虫）RH株速殖子体外感染小鼠巨噬细胞RAW264.7的模型,确定体外快速高效培养速殖子的方法。方法 弓形虫RH株速殖子经昆明小鼠传代3次后,经5 μm滤膜纯化计数后,与小鼠巨噬细胞RAW264.7、人包皮成纤维（HFF）细胞共培养。RAW264.7细胞与虫体数量比例分别为20 : 1、10 : 1、1 : 1、1 : 5,HFF细胞与虫体比例为1 : 1,设无虫对照组。分别于培养后10 min、0.5 h、1 h、2 h、4 h、8 h、12 h、24 h、32 h、48 h时间点取样,高倍镜下观察共培养情况,瑞-吉氏染色观察速殖子的黏附入侵细胞情况。将速殖子以1.5 × 10⁶个/瓶接种于RAW264.7 T25细胞培养瓶中共培养,待80%假包囊破裂后,收集速殖子并计数,计算速殖子产率。用0.4%台盼蓝染液染色后,计算培养体系的活虫率。回收的弓形虫速殖子继续感染新的RAW264.7细胞,将每代细胞中收集的速殖子以1 × 10⁶个/只接种5只健康雌性昆明小鼠,同时以等量的第3代速殖子接种5只健康雌性昆明小鼠作为对照,记录每代小鼠的存活时间。应用SPSS 21.0统计学软件进行统计分析,组间比较采用单因素方差分析。结果 当RAW264.7细胞与虫体比例大于10 : 1时,共培养24 h时可见细胞边界不清,胞质增加,出现大量小泡;共培养30 h时细胞大量死亡消失,未观察到游离弓形虫;当细胞与虫体比例小于或等于1 : 1时,共培养24 h时内胞质可见细密小颗粒,未见小泡;共培养30 h时假包囊破裂达90%以上,大量速殖子被释放至培养液中,可观察到速殖子体外发育的完整过程。速殖子与RAW264.7细胞以1 : 1的比例共培养0.5~1 h,约80%的速殖子侵入细胞,虫体可进入胞质和胞核,2~4 h后假包囊开始形成,8 h可见菊花状假包囊;24 h后假包囊开始破裂,32 h后细胞悬浮不贴壁,大部分假包囊破裂并释放出游离速殖子,虫体形态好,每瓶收获弓形虫速殖子6 × 10⁸个,平均活虫率在92%以上。速殖子与HFF细胞共培养,2~4 h虫体基本入侵细胞;8 h后速殖子开始分裂增殖;共培养24 h,可见速殖子分裂成菊花状;36 h后细胞开始涨破,释放出的速殖子开始新一轮入侵HFF细胞。体外培养各代弓形虫速殖子对昆明小鼠的平均致死时间为3.94~4.10 d,经与昆明小鼠体内传代复毒虫株相比毒力未见减弱。结论 采用小鼠巨噬细胞RAW264.7可在体外培养快速获得大量弓形虫RH株速殖子,产率为体内培养方法的400倍,且虫体毒力与体内培养方法相当,培养时间和产率上也均优于HFF细胞系。

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.