口蹄疫病毒前导蛋白(Lpro)致牛肾细胞(MDBK)作用的形态学观察

Morphological observation of Bovine Kidney (MDBK) cells effected by foot-and-mouth disease virus Lpro

摘要:

为探讨口蹄疫病毒Lpro致MDBK细胞病变效应中的形态学变化, 本实验在成功构建可稳定表达口蹄疫病毒Lpro 目的基因的MDBK细胞系的基础上, 人工诱导Lpro表达后, 采用光学显微镜观察、Hoechst 33258染色、AO-EB染色、DNA Ladder等进行检测, 研究口蹄疫病毒Lpro致MDBK细胞的病变效应。结果显示, MDBK细胞系在诱导表达口蹄疫病毒Lpro 24 h后, 光学显微镜下细胞形态表现为细胞体积缩小、核浓缩、细胞周围出现透明圈等现象; Hoechst 33258染色检测呈现典型的细胞核浓缩和梅花状核碎裂; 诱导表达Lpro 36 h后, AO-EB染色显示早期病变细胞核染亮绿色呈致密斑块或碎片状, 晚期病变细胞核染橘黄色呈致密斑块; DNA凝胶电泳显示可见的DNA Ladder“梯状”条带。证明口蹄疫病毒Lpro 在体外可诱导MDBK细胞发生凋亡。

Abstract:

In order to explore the morphological changes of Bovine Kidney (MDBK) cells induced by foot-and-mouth disease virus (FMDV) L protease, we induced the expression of FMDV L protease in bovine kidney cells (MDBK) artificially. All work is carried out on the basis of a stable MDBK cell line inducibly expresses the Lab gene under the control of tetracycline. We use cell morphology, Hoechst 33258 staining, AO-EB staining, and DNA Ladder abstraction to research the morphological changes of MDBK cells. 24 hours after FMDV L protease were induced and expressed in MDBK cells, cells shown the diminish of cell size, nuclear enrichment and the appearance of transparency circle under the light microscope. Apoptosis characteristics of nuclear condensation, fragmentation, accompanied by apoptotic bodies formation (Hoechst 33258 staining). 36 hours after the expression, nuclear staining of early lesions showed bright green plaque or debris-like dense, and advanced lesions showed Orange and dense plaques (AO-EB staining). 48 hours after the expression, DNA gel electrophoresis showed visible DNA ladder. Results indicate that FMDV L protease can induce apoptosis of MDBK and apoptosis plays an important role in the cytopathogencity effect of FMDV.