目的 观察刚地弓形虫排泄分泌抗原（TgESA）刺激后的NK细胞对小鼠B16F10黑色素瘤生长的作用。方法 取弓形虫RH株体外培养12 h后,从培养上清收集获得弓形虫ESA。取10只C57BL/6小鼠随机分为2组,每组5只,每只小鼠右腋窝皮下接种B16F10黑素瘤细胞2 × 10⁵个。接种后第7天,随机取1组小鼠腹腔注射100 μl TgESA,另1组注射等量的PBS。接种后第14天,无痛处死小鼠,无菌条件下制备小鼠脾细胞悬液,分离2组荷瘤鼠的NK细胞,分别记为NK_B16F10和NK_ESA,用于后续过继转输实验。取30只C57BL/6小鼠随机分为对照组、NK_B16F10组和NK_ESA组,每组10只。3组小鼠均右腋窝皮下接种B16F10细胞2 × 10⁵个/鼠。其中NK_B16F10组和NK_ESA组小鼠同时分别尾静脉注射2 × 10⁵个NK_B16F10和NK_ESA。观察瘤体生长情况以及各组小鼠死亡数和死亡时间,共观察35 d。结果 NK细胞过继转输后,NK_ESA组和NK_B16F10组小鼠平均出瘤时间分别为（14.70 ± 0.95）、（12.60 ± 0.70）d,均晚于对照组的（8.50 ± 0.85）d（P < 0.05）。3组小鼠出瘤后,瘤体均不断增长,至第35天,NK_B16F10组和NK_ESA组的平均瘤体面积分别为（686.53 ± 17.84）和（577.79 ± 49.70）mm²,均小于对照组的（787.84 ± 19.94）mm²（P < 0.05）。对照组、NK_B16F10组和NK_ESA组小鼠分别于接种B16F10细胞后第24、27和30天开始出现死亡,至第35天,存活的小鼠数分别为3、4和6只。结论 弓形虫ESA刺激的NK细胞过继转输小鼠后可较为明显地抑制B16F10黑色素瘤瘤体的生长。

Objective To observe the therapeutic effect of natural killer (NK) cells stimulated by Toxoplasma gondii excretory/secretory antigens (TgESA) on the growth of B16F10 melanoma in mice. Methods TgESA was obtained from the culture of T. gondii tachyzoites in vitro for 12 hours. The tumor-bearing animal models were established by direct injection of melanoma B16F10 cells into the right axillary of ten mice (2 × 10⁵/mouse). Five of the melanoma-inoculated mice were intraperitoneally treated with TgESA(100 μl/mouse) 7 days after inoculation of B16F10 cells, the rest 5 mice were treated with PBS only as control. One week after the treatment, all mice were euthanized, and the NK cells were isolated from the splenocytes of two groups (named NK_B16F10 and NK_ESA). Total 30 naïve C57BL/6 mice were randomly divided into 3 groups with 10 mice each group. Each mouse was inoculated with 2 × 10⁵ B16F10 cells subcutaneously into axillary. One week after melanoma inoculation, one group of mice were passive transferred with 2 × 10⁵ of NK_B16F10 cells through tail vein injection (group of NK_B16F10). Another group of mice were transferred with the same number of NK_ESA cells (group of NK_ESA). The third group of mice were given with PBS only as control. After passive transfer, the tumor growth, mortality and survival rate in the transferred mice were observed and recorded for 35 days. Results The visible time of tumor in mice of NK_ESA group [(14.70 ± 0.95) d] was significantly longer than that in NK_B16F10 group [(12.60 ± 0.70) d] and in control group [(8.50 ± 0.85) d] (P < 0.05). The tumor size in NK_ESA group [(577.79 ± 49.70) mm²] was also significantly smaller than that in NK_B16F10 group [(686.53 ± 17.84) mm²] and in control group [(787.84 ± 19.94) mm²] at D35. The delayed appearance of tumor and smaller tumor size in group of NK_B16F10 was also significant compared to control group. Mice began to die at D24 for control group, D27 for NK_B16F10 group and D30 for NK_ESA group after tumor inoculation, and the number of mice remain alive was 3, 4 and 6 respectively at terminal time D35 with significant difference between NK_ESA group and control group.Conclusion Passive transfer of NK cells stimulated by TgESA conferred therapeutic effect on B16F10 melanoma in recipient mice.