Li J, Zhu J D, Appiah A, McCutchan T F, Long G W, Milhous W K, Hollingdale M R
Second Military Medical University, Shanghai, People's Republic of China.
Exp Parasitol. 1991 May;72(4):450-8. doi: 10.1016/0014-4894(91)90091-a.
A stage-specific ribosomal RNA probe has been used to quantitate exoerythrocytic development of Plasmodium berghei in primary cultures of mouse hepatocytes. Parasite rRNA could be detected as soon as 6 hr after sporozoite invasion and was increased during schizogony to a maximum at 48 hr, when mature schizonts were identified by microscopy. As few as 10 exoerythrocytic schizonts could be detected by filter blot hybridization, followed by autoradiography and liquid scintillation counting. By hybridizing the culture rRNA samples with either parasite-specific or universal rRNA probes, the in vitro tissue schizonticidal activity and hepatotoxicity of primaquine, two of its analogues, and pyrimethamine, could be assessed. After a 48-hr exposure of the culture to serial dilutions of each drug, a quantitative relationship was demonstrated between the decrease of the parasite rRNA and the increase of the drug concentrations. No significant parasite-specific rRNA could be detected at the concentration achieving complete inhibition of schizont formation but causing no cytotoxic effects on host hepatocytes. In contrast to microscopic-based assays, this molecular approach provides an objective and quantitative in vitro method for rapid screening and evaluation of tissue schizonticidal antimalarials.
一种阶段特异性核糖体RNA探针已被用于定量伯氏疟原虫在小鼠肝细胞原代培养物中的红细胞外期发育。子孢子入侵后6小时即可检测到寄生虫rRNA，在裂体增殖期间rRNA增加，至48小时达到最大值，此时通过显微镜鉴定出成熟裂殖体。通过斑点杂交、放射自显影和液体闪烁计数，可检测到低至10个红细胞外裂殖体。通过将培养的rRNA样品与寄生虫特异性或通用rRNA探针杂交，可评估伯氨喹及其两种类似物以及乙胺嘧啶的体外组织裂殖体杀灭活性和肝毒性。在将培养物暴露于每种药物的系列稀释液48小时后，寄生虫rRNA的减少与药物浓度的增加之间呈现出定量关系。在达到完全抑制裂殖体形成但对宿主肝细胞无细胞毒性作用的浓度下，未检测到明显的寄生虫特异性rRNA。与基于显微镜的检测方法不同，这种分子方法提供了一种客观、定量的体外方法，用于快速筛选和评估组织裂殖体杀灭性抗疟药。
