不同种株利什曼原虫前鞭毛体体外向无鞭毛体转化的研究

Study on Promastigote Transformation to Amastigotes in Different Leishmania spp. in vitro

目的观察不同种株利什曼原虫前鞭毛体在不同体外培养条件下向无鞭毛体的转化。方法将6个对数生长期的不同种株利什曼原虫前鞭毛体接种于含M199和RPMI1640复合培养液的24孔板内,在不同温度、pH值、血清、CO2下培养,第3天和第6天分类计数不同形态的原虫。结果当培养温度为26℃,而其他培养条件发生变化时,利什曼原虫的增殖速度发生变化,大多数原虫保持前鞭毛体典型性形态,运动活跃。当培养温度上升至37℃,不论其他培养条件如何变化,原虫均沉积于培养孔底,停止增殖,运动减缓,显著地向中间体和无鞭毛体转化,酸性pH值和5%CO2可促进前鞭毛体转化为无鞭毛体,新生小牛血清可延长无鞭毛体的成活。但相同的培养条件下,杜氏利什曼原虫转化效率低于其他种株利什曼原虫。结论温度是影响利什曼原虫前鞭毛体转化为无鞭毛体的基本因素,酸性pH值与CO2可协同温度促进转化,不同种株利什曼原虫的遗传异质性可影响转化效率。

Objective To investigate the different Leishmania spp. promastigote transformation to amastigotes in different cultivated conditions in vitro. Methods Six strains of Leishmania spp. promastigotes in the logarithm growth period were inoculated in 24 well plates containing M199 or RPMI 1640 medium. The protozoa were cultivated under different tempera- tures, pH values, newborn calf blood serum, and 5% CO2 , quantified and the different protozoan shapes classified after 3 days and 6 days. Results When the promastigotes were cultivated at 26℃, whatever other conditions changed, the multi- plication speed of the protozoa changed. Most promastigotes maintained a typical shape and were active. When the promas- tigotes were cultivated at 37℃, the protozoa deposited to the well bottom, stopped muhiplying, their activity level de- creased, and transformed to the intermediate or the amastigote form. The acidic pH value and 5% CO2 may promote promas- tigote transformation to amastigote form and the newborn calf serum may lengthen the amastigotes survival time. Under the same conditions, the Leishmania donovani transfer efficiency was lower than other Leishmania spp. Conclusion Tempera- ture was the essential element to determine promastigote transformation to amastigotes, while the acidic pH value and CO2 may assist in promoting transfer efficiency. Different Leishrnania spp. also affected the transfer efficiency.