弓形虫速殖子与缓殖子体外转化体系的建立

Establishment of an in vitro tachyzoite-bradyzoite interconversion system for Toxoplasma gondii

目的建立弓形虫速殖子与缓殖子体外转化体系。方法纯化弓形虫RH株速殖子,按一定比例和条件接种于COS-7细胞内进行体外培养。分别在接种后第1、2、3、4、5、6天观察细胞和虫体的形态并提取TotalRNA,用RT-PCR的方法检测速殖子期特异性蛋白SAG1基因、缓殖子期特异性蛋白BAG1基因和SAG2C基因的表达。结果随着速殖子在COS-7细胞中培养时间的延续,虫体的数量逐渐增多,但增殖速度逐步减缓;虫体在细胞内的排列方式也在不断变化,由成对的弧形、玫瑰花形或簇形、变成半圆形最后形成圆形的类包囊样结构。RT-PCR检测显示:速殖子期特异性蛋白SAG1基因在培养的第1～6天均有表达,且表达量呈递增趋势;缓殖子期特异性蛋白BAG1基因从第2天开始出现表达,随时间延续表达量逐渐增加;缓殖子期特异性蛋白SAG2C基因从第5天开始表达,第6天表达量增加。以上结果表明有越来越多的速殖子转化为缓殖子。改变培养条件,缓殖子也能向速殖子转化。结论成功构建弓形虫速殖子与缓殖子体外相互转化体系,为其转化机制的研究奠定了基础。

Objective To establish an tachyzoite-brachyzoite interconversion system for Toxoplasma gondii RH strain in vitro.Methods COS-7 cells were inoculated with purified tachyzoites of T.gondii RH strain and cultured in vitro.The morphology of the cultured cells and parasites was observed and the total cellular RNA extracted on days 1 to 6 following the inoculation for detecting the expression of tachyzoite-specific protein （SAG1） and bradyzoite-specific proteins （BAG1 and SAG2C） using RT-PCR.Results With the passage of time,the number of parasites in COS-7 cells increased but the proliferation rate was lowered gradually.The intracellular tachyzoites proliferated by means of budding and binary fission,which led to the changes in the alignment of the parasites in the cells from curved pairs,rosette or clustered,and semi-circular patterns to spherical encapsulation-like structures.These changes indicated the gradual transformation of the tachyzoites into bradyzoites.The expressions of the tachyzoite-specific SAG1 gene were detected throughout the 6 days of in vitro culture.The expression of the bradyzoite-specific BAG1 gene had been detected since the second day after the inoculation and SAG2C gene since the fifth day.Alteration of the culture condition resulted in gradual transformation of the bradyzoites into tachyzoites.Conclusion An in vitro tachzoites-bradyzoite interconversion system for T.gondii has been successfully established,which provides the basis for further study of the mechanism of interconversion.