The Death Pattern Of Bovine Kidney(mdbk) Cells Induced By Foot-and-mouth Disease Virus (fmdv)L^proand Molecular Mechanism

Foot-and-mouth disease (FMD) is a highly infectious disease of domestic and wild cloven-hoofed animals, known for the rapid spread of the infection rate. The World Organization for Animal Health (OIE) will be listed as category A infectious disease. FMD has been recognized as the most important constraint to international trade in animals and animal products by the World Organization for Animal Health (OIE). Therefore, FMD is also known as "political - economic disease."Now,the death pattern to the host cell and the mechanism induced by FMDV is not clear.With the development of molecular biology-related research in the application of FMDV, researchers have got varying degrees of understanding in FMDV genome structure and function . The toxicity studies for the host cell toxic protein of FMDV has become a hot topic at home and abroad. In this study, we do research between L^pro protease and the MDBK cells,and study the death pattern of MDBK cell and its molecular mechanism induced by foot and mouth disease virus L^pro . All work is carried out on the basis of a stable MDBK cell line inducibly expresses the Lab gene under the control of tetracycline.The object of this study is the stable MDBK cell line inducibly expresses the Lab gene under the control of tetracycline. First, we identified the stability of the cell lines. After the rapid rejuvenation, MDBK-L cells were continuously cultured and passaged in DMEM containing tetracycline. Collectting the MDBK-L cells of 1,5,10,20,30 generation and the normal cells (cultured 48h)to be detected.Using PCR , we confirmed Lab gene was integrated into the genome of MDBK cells, and can be stable taken along with the host cells; Using RT-PCR, we confirmed the Lab gene can be transcribed in the absence of tetracycline circumstances; Using Wensten-blotting,we detected the expression of L^pro in MDBK. The results showed that the target protein can responses to the O-specific FMDV antisera. Second,in order to explore the morphological changes of Bovine Kidney (MDBK) cells induced by foot-and-mouth disease virus (FMDV) L^pro, we induced the expression of FMDV L^pro 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. After that,we obtained the quantitative relation to the apoptosis by flow cytometry,and labeled and identified the positive apoptotic cells by TUNEL. Finally, we studied the preliminary molecular mechanisms of the apoptosis induced by FMDV L^pro .There are two major pathway of apoptosis .They are the death receptor-mediated apoptosis pathway and the mitochondrial apoptosis pathway. Although the two upstream pathway of apoptosis events are different, they ultimately have to activate a common apoptotic effector-the specific caspase. Using RT-PCR and Western blot, We observed that Caspase-3 was activated and expressed in the transcription and translation during the apoptosis of MDBK cells which induced by foot and mouth disease virus L^pro.