Contreras Víctor T, Navarro María C, De Lima Ana R, Arteaga Rosa, Duran Francy, Askue José, Franco Yunaimy
Laboratorio de Protozoolog a, Centro BioMolP, Facultad de Ciencias de la Salud, Universidad de Carabobo, Valencia, Carabobo, Venezuela.
Mem Inst Oswaldo Cruz. 2002 Dec;97(8):1213-20. doi: 10.1590/s0074-02762002000800025. Epub 2003 Jan 20.
Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.
迄今为止，在体外重现克氏锥虫所有发育阶段的尝试仅取得了部分成功。例如，在组织培养中有可能产生锥鞭毛体，并在无共生条件下获得循环后期锥鞭毛体。尽管已知克氏锥虫无鞭毛体可从锥鞭毛体和循环后期锥鞭毛体分化而来，但仅能从组织培养衍生的锥鞭毛体在体外产生无鞭毛体。触发循环后期锥鞭毛体转化为无鞭毛体所需的因素和培养条件尚未确定。我们在此表明，将循环后期锥鞭毛体在37摄氏度的培养（MEMTAU）培养基中预孵育48小时足以使寄生虫进入转化过程。在新鲜的MEMTAU培养基中孵育72小时后，90%的循环后期寄生虫分化为形态上与正常无鞭毛体无法区分的形态。SDS-PAGE、蛋白质印迹和PAABS分析表明，无共生循环后期锥鞭毛体向无鞭毛体的转化与蛋白质、糖蛋白和抗原修饰有关。这些数据表明：（a）可从循环后期锥鞭毛体大量无菌获得克氏锥虫无鞭毛体；（b）如此获得的无鞭毛体在形态、生物学和抗原性上与细胞内无鞭毛体相似。因此，该实验系统可能有助于直接在体外评估使克氏锥虫循环后期锥鞭毛体在哺乳动物宿主细胞中转化为无鞭毛体的机制。
