Excellent Center for Drug Discovery (ECDD), Faculty of Science, Mahidol University, Bangkok, Thailand.
Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
Malar J. 2018 Jan 25;17(1):50. doi: 10.1186/s12936-018-2198-4.
Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay.
A novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay.
imHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening.
imHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.
由于间日疟原虫休眠的肝脏期（hypnozoite）能够导致患者复发，因此疟疾的根除较为困难。为了更好地了解间日疟原虫休眠期的生物学特性并设计预防复发的策略，研究人员付出了诸多努力，但由于缺乏一种可靠的体外培养肝脏期寄生虫的模型，这些努力受到了阻碍。虽然 HC-04 肝癌细胞系被用于培养肝脏期疟原虫，但这些细胞会不受限制地增殖，并在几天后从培养皿中脱落，这限制了它们在长期休眠期检测中的应用。
评估了一种新型永生化肝样细胞系（imHC）支持间日疟原虫孢子感染的能力。首先，检测了 imHC 中基本肝细胞标志物和所有主要疟原虫孢子相关宿主受体的表达情况。接下来，通过间接免疫荧光法检测了 imHC 中的体外肝细胞感染性和孢子的细胞内发育情况。还测量了细胞色素 P450 同工酶的活性，以确定 imHC 代谢药物的能力。最后，使用抗肝脏期药物伯氨喹来测试该模型的药物敏感性。
imHCs 维持着主要的肝脏功能，并表达了 CD81、SR-BI 和 EphA2 等必需因子，这些因子是寄生虫在肝脏中入侵和发育所必需的。imHCs 可以在单层中长期维持，不会过度生长，因此是支持间日疟原虫肝脏期，包括休眠期的入侵和生长的良好基质。观察到的高药物代谢活性以及肝脏期寄生虫对伯氨喹的强烈反应突出了该 imHC 模型在抗疟药物筛选中的潜在用途。
imHCs 保持着肝细胞表型和药物代谢酶的表达，是体外疟原虫肝脏期研究的替代宿主，特别是针对间日疟原虫休眠期生物学的研究。它们可能为筛选针对肝脏期寄生虫的药物提供一种新颖、强大的模型。
