摘要
目的建立弓形虫RH株体外培养及速殖子与缓殖子相互转化体系。方法纯化弓形虫RH株速殖子,接种于NIH3T3细胞单层,进行体外培养。接种4h后,换用pH8.1的碱性培养基进行速殖子向缓殖子的诱导转化培养,RT-PCR检测缓殖子蛋白1(BAG1)基因的表达。将诱导的缓殖子重新用pH7.2的培养基在常规环境下培养,诱导缓殖子向速殖子转化。为探索温度对速殖子向缓殖子转化的影响,将接种RH株速殖子的NIH3T3细胞分别放在37℃、39℃、41℃、43℃中诱导转化培养,RT-RCR检测BAG1基因的表达。结果弓形虫RH株能在NIH3T3细胞单层中生长增殖,更换高pH值碱性培养环境后,能诱导弓形虫速殖子转化为缓殖子,RT-PCR检测出缓殖子期特异蛋白BAG1基因的表达,并且随着诱导时间的延长,BAG1基因mRNA的转录水平逐渐提高,表明有更多的速殖子转化为缓殖子。恢复pH7.2的常规培养条件,碱性诱导的缓殖子能转化为速殖子。在41℃诱导条件下,能诱导RH株速殖子转化为缓殖子。结论成功构建弓形虫体外培养及速殖子与缓殖子相互转化体系,为转化机制的研究奠定基础。
To develop a cell culture system for the study of tachyzoite to brachyzoite interconversion of Toxoplasma gondii RH strain in vltro,the purified tachyzoites of the RH strain were cultivated in the NIH3T3 cell line monolayers. After 4 hours cultivation,the differentiation of T. gondii from tachyzoite to bradyzoite was induced by changing the pH to 8. 1 with alkaline medium and the induced bradyzoites were again cultured in routine culture medium of pH 7. 2 under normal condition. To investigate the influence of temperature on the tachyzoite to bradyzoite inter conversion, the NIH3T3 cell monolayers with tachyzoites of the RH strain were cultivated in incubators at 37 ℃ ,39 ℃ ,41 ℃ and 43 ℃ respectively,for 48 hrs. and observe the effect on interconversion. Meanwhile, the bradyzoite-specific BAG1 mRNA was identified during differentiation with RT- PCR. It was found that the RH strain of T. gondii grew well in NIH3T3 cell monolayers and the interconversion from tachyzoites to bradyzoites could be induced by changing the pH of culture medium to 8.1. The expression of the bradyzoite-specific BAG1 gene could be detected by RT-PCR,and the transcriptive level of BAG1 gene elevated along with the prolongation of induction time. Only under the cultural temperature at 41 ℃ ,the conversion from tachzoites to bradyzoites was possible. Thus a cell culture system for interconversion from tachzoites to bradyzoites was successively established and this system can be used for the further study of this interconversion of T. gondii in vitro.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2008年第7期612-615,620,共5页
Chinese Journal of Zoonoses
基金
国家自然科学基金(30671837)
高等学校博士学科点专项科研基金(20069981003)
广东省医学科学研究基金(B2005078)资助项目
关键词
弓形虫
体外培养
速殖子
缓殖子
转化
Toxoplasma gondii
cell culture
tachyzoite
bradyzoite
differentiation
