一种快速获得恶性疟原虫不同生长周期的体外同步化方法及其在双氢青蒿素处理3D7后差异基因表达谱中的应用

Method for rapid synchronization of different growth cycles of Plasmodium falciparum in vitro and application in differential gene expression profile of 3D7 after dihydroartemisinin treatment

疟原虫体外培养体系常用作抗疟药物评价模型,但体外培养的恶性疟原虫生长趋向于无序化,影响体外抗疟药物机制研究与评价。该文通过磁珠分离法和山梨醇同步法联合构建了一种能够快速获得恶性疟原虫不同生长周期的同步化方法,短时间内获得大量窗口期较窄的疟原虫。该研究进一步利用双氢青蒿素(DHA)干预恶性疟原虫3D7早期滋养体期4 h后,提取mRNA,并进行RNA-seq分析药物处理后mRNA的差异表达情况,得到差异表达谱。按照|log2FC|>1,P<0.05的筛选标准对差异基因进行表达分析,其中上调有262条,下调有77条;对所有差异基因进行了GO功能富集,主要富集的条目包括细胞膜、丝氨酸/苏氨酸激酶活性、转运体(transporter)、茂式点(Maurer′s clefts,MCs)、rhoptry、抗原变异和免疫逃避等;KEGG通路富集包含了疟疾,脂肪酸降解、过氧化物酶体等通路;关键基因的蛋白相互作用(PPI),其中关联度(degree)较大的模块中下调基因包括rhoptry,myosin complex,transporter等与疟原虫入侵及免疫逃逸的重要生命活动相关的基因,上调基因主要与疟原虫各种毒性输出蛋白如MCs的PfSBP1相关。进行qRT-PCR验证部分基因表达水平,大部分与测序结果相近,SBP1水平明显上调,而部分抗原性蛋白表达水平下调。综上分析,DHA作用的关键分子主要集中在疟原虫的rhoptry、transporter、抗原性变异、疟原虫输出蛋白,这为进行青蒿素抗疟机制研究提供了研究方向,为开发新的抗疟药物提示了一种新的思路。

Plasmodium culture in vitro is often used as an antimalarial drug evaluation model, but the lifecycle of P. falciparum culture in vitro tends to be disordered, which affects the research and evaluation of antimalarial drug mechanism in vitro. By combining magnetic bead separation method with sorbitol synchronization method, a synchronization method was constructed to quickly acquire different lifecycles of P. falciparum and obtain large amounts of parasite with a narrow synchronization window in a short period. Furthermore, the dihydroartemisinin(DHA) was used to treat the early trophozoite phase of P. falciparum 3 D7 for 4 h. Then mRNA was extracted and RNA-seq was conducted to analyze the differential expression of mRNA after drug treatment and obtain the differential gene expression profile. Differential expression of up-regulated genes and down-regulated genes was analyzed according to the screening criteria of |log2FC|>1 and P<0.05. There, 262 genes were up-regulated and 77 genes were down-regulated. GO functional enrichment analysis of all the differentially expressed genes showed that the enrichment items mainly included cell membrane components, transporter activity, serine/threonine kinase activity, Maurer′s clefts(MCs), rhoptry, antigen variation and immune evasion. The enrichment of KEGG pathway included malaria, fatty acid metabolism and peroxisome. Protein-protein interaction(PPI) analysis showed that the down-regulated genes in the modules with high degree of association included rhoptry, myosin complex, transporter and other genes related to the important life activities of malaria invasion and immune escape;the up-regulated genes were mainly related to various toxic exportins of malaria, such as PfSBP1 of MCs. qRT-PCR was used to verify the expression level of some genes, and most of the results were the same as the sequencing results. SBP1 was significantly up-regulated, while some antigenic protein expression levels were down-regulated. Above all, key molecules of DHA therapy were mainly involved in the parasites′ rhoptry, transporter, antigenic variation, plasmodium exportin. These results offer us many hints to guide the further studies on mechanism of artemisinin and provide a new way for development of new antimalarial drugs.