冰冻对弓形虫GT1速殖子体外感染力的影响  

Effect of the freezing followed by melting at RT on the infection ability of Toxoplasma gondii GT1 tachyzoites in vitro

作　　者：郭海婷 谭洁 何玉林 梁雪 李中原 GUO Hai-ting;TAN Jie;HE Yu-lin;LIANG Xue;Li Zhong-yuan(Guangxi Key Laboratory of Brain and Cognitive Neuroscience,Basic Medicine College,Guilin Medical University,Guilin,Guangxi 541199,China)

机构地区：[1]桂林医学院基础医学院,广西脑与认知神经科学重点实验室,广西桂林541199

出　　处：《中国病原生物学杂志》2022年第10期1145-1149,共5页Journal of Pathogen Biology

基　　金：广西自然科学基金项目(No.2019GXNSFBA185031);广西研究生教育创新计划项目(No.JGY2022198);广西高等学校千名中青年骨干教师培育计划第4期资助项目(No.2020-58);桂林医学院新引进博士科研启动项目(No.31304018021)。

摘　　要：目的探讨冰冻不同时间对弓形虫GT1速殖子体外感染力的影响。方法用HFF细胞传代GT1速殖子3次,刮净培养物,取2.0 mL充分破碎后计数。将剩余培养物中速殖子调至10000个/mL,分装至1.5 mL离心管(100μL/管),并随机分成3组。上述样品放置于4℃,分别在0、12、24、36、48、60 h时取出样品,5管/组,再置于4℃、-20℃或-80℃保存。待首批冰冻72 h时取出全部样品,于室温(约22℃)完全融化后全量加入预先铺有HFF细胞的6孔培养板内,继续培养。于10倍物镜下随机选取3个视野,观察并记录感染3、5、7 d时的噬斑形成数,若噬斑总数为0,则进行全视野扫描检测。盲传感染7 d仍未检测到噬斑形成的培养物,用全视野扫描盲传7 d时各瓶内的噬斑。结果随机观察3个视野,复苏前4℃放置的GT1速殖子仅在感染3 d时形成的噬斑清晰可辨,且各孔间无明显不同;-20℃和-80℃冰冻均能减弱GT1速殖子的体外感染力。盲传后观察,经-20℃和-80℃冰冻24 h的GT1速殖子仍能在感染HFF细胞7 d时形成噬斑,且-80℃较-20℃冰冻组形成的噬斑数更多;冰冻36 h及以上的GT1速殖子未形成噬斑。结论以1000个速殖子/孔的剂量感染6孔培养板内的HFF细胞,4℃放置不超过72 h对GT1速殖子体外毒力无显著影响,-20℃或-80℃冰冻36 h及以上能减弱弓形虫GT1速殖子体外感染力。Objective To explore the effect of the freezing followed by melting at room temperature(RT)on the infection ability of Toxoplasma gondii GT1 tachyzoites in vitro.Methods T.gondii GT1 tachyzoites were passaged for three times using HFF cells,and the cultures at the last time were scraped,2.0 mL of which were broken and subsequently used for counting parasite.The residual cultures in which the number of parasites was switched to 10000 per milliliter,were loaded into 1.5 mL tube,100μL each.After divided into three groups randomly,all the samples were simultaneously placed at 4℃for freshness,and then the time was as 0 h.Five tubes each group were taken out at 0,12,24,36,48 and 60 h post placement,and followed to store at 4℃,-20℃,or-80℃.All the samples were fully melted at RT about 22℃when the first ones were frozen for 72 h,and were completely added into 6-well plates to continuously co-culture with HFF cells.The plaques within three visual fields were totally recorded under 10 objective lens at 3,5,and 7-day post infection(dpi),respectively.If the total number was zero,the full-vision scanning would be performed,which was also used to identify the plaques at 7 d post the blind passage of the cell cultures with no plaque formation detected at 7 dpi.Results The results from the three random visual fields showed that the plaques formed by the GT1 tachyzoites stored at 4℃were distinguished only at 3 dpi,and no statistic difference was detected among all the wells containing these parasites.Moreover,the infection abilities of T.gondii GT1 tachyzoites were both significantly reduced by the freezing at-20℃and-80℃followed by melting at RT in vitro.The data from the blind passages showed that the plaques of the GT1 tachyzoites were still detected at 7 dpi,which were frozen at-20℃or-80℃for 24 h followed by melted at RT before,and the number of plaques formed at-80℃was more than that of-20℃.Noteworthily,the plaque cannot be detected during passaging the cell cultures in which the parasites were frozen

关 键 词：弓形虫 GT1 冰冻 感染 

分 类 号：R382.5[医药卫生—医学寄生虫学]