通过流式细胞术鉴定和评估伯氏疟原虫裂殖子和细胞周期。

Identification and Assessment of Plasmodium berghei Merozoites and Cell Cycle by Flow Cytometry.

机构信息

Experimental Therapeutics Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910-7500, USA.

出版信息

Mil Med. 2021 Jan 25;186(Suppl 1):108-115. doi: 10.1093/milmed/usaa272.

DOI:10.1093/milmed/usaa272

PMID:33499463

Abstract

BACKGROUND

The asexual blood stages of the Plasmodium berghei life cycle including merozoites are attractive targets for transmission blocking vaccines and drugs. Improved understanding of P. berghei life cycle stage growth and development would provide new opportunities to evaluate antimalarial vaccines and drugs.

METHODS

Blood stage samples from C57BL/6 albino mice infected with P. berghei sporozoites were singly stained with a high binding affinity deoxyribonucleic acid dye, YOYO-1, and measured by flow cytometry (FCM). Duplicate slides were made from samples and stained with diluted Giemsa's and YOYO-1, respectively. Correlated results were compared by FCM, light microscopy, and fluorescent microscopy.

RESULTS

Complete life cycle stage determination and analysis by FCM is reported to include merozoites, ring forms, trophozoites, immature, and mature schizonts. FCM demonstrated a clear separation between each stage using their unique fluorescence distribution. When compared to light microscopy, a strong correlation (r 2 = 0.925 to 0.974) was observed in determining the ring forms, trophozoites, and schizonts phases, but only a moderate correlation (r 2 = 0.684 to 0.778) was observed for merozoites. The identification and measurement of merozoites suggest that FCM is a useful technique to monitor the entire life stage of the parasite. Initial stage-specific data demonstrated that mefloquine has a mode of action on mature parasite forms, and artesunic acid was rapidly effective against merozoites and other immature and mature parasite forms with higher killing.

CONCLUSION

Blood stage parasites in each individual life stage, including merozoites, are reliably identified and quantified quickly by FCM, making this technique an ideal alternative to microscopy. This integrated whole life stage model, particularly with confirmed determination of merozoite population, could widely be used for drug and vaccine research in malaria therapy and prophylaxis.

摘要

背景

疟原虫无性血期包括裂殖子，是传播阻断疫苗和药物的有吸引力的靶标。对疟原虫生活史阶段生长和发育的深入了解将为评估抗疟疫苗和药物提供新的机会。

方法

用高亲和力脱氧核糖核酸染料 YOYO-1 单独染色感染疟原虫孢子的 C57BL/6 白化鼠的血期样本，并用流式细胞术（FCM）进行测量。从样品中制作两份载玻片，分别用稀释的吉姆萨氏染色法和 YOYO-1 染色。通过 FCM、相差显微镜和荧光显微镜比较相关结果。

结果

据报道，通过 FCM 进行完整的生活史阶段测定和分析包括裂殖子、环型、滋养体、未成熟和成熟裂殖体。FCM 利用其独特的荧光分布清楚地分离出每个阶段。与相差显微镜相比，在确定环型、滋养体和裂殖体阶段时观察到很强的相关性（r2=0.925 至 0.974），但在确定裂殖子阶段时仅观察到中度相关性（r2=0.684 至 0.778）。裂殖子的鉴定和测量表明，FCM 是监测寄生虫整个生活史阶段的有用技术。初步的阶段特异性数据表明，甲氟喹对成熟寄生虫形式具有作用方式，而青蒿琥酯对裂殖子和其他未成熟和成熟寄生虫形式迅速有效，杀伤率更高。