Background: Malassezia pachydermatis is the main causative agent of canine otitis and also of a myriad of dermatological problems in companion animals;its interaction mechanisms with host cells are still unclear. Obje...Background: Malassezia pachydermatis is the main causative agent of canine otitis and also of a myriad of dermatological problems in companion animals;its interaction mechanisms with host cells are still unclear. Objectives: To establish an in vitro infection model of M. pachydermatis-exposed RK13 cells in order to evaluate cell morphological changes as well as changes in the structure of actin filaments. Methods: Cultures of RK13 cells were infected with M. pachydermatis, alterations caused by the yeast were evaluated by optical and fluorescence microscopy. Results: M. pachydermatis adheres itself to the cell and produces the formation of multiple agglomerates that cause changes in cell morphology, formation of cell aggregates in overlays, presence of syncytia and destruction of cell culture structure. The damaged cells presented changes in the actin filaments consisting of thickening of the cell cortex and loss of stress fibers. On the other hand, the formation of perinuclear actin rings in the yeasts was observed. Conclusions: An in vitro infection model was established with M. pachydermatis and alterations in cell morphology were observed consisting of changes in the structure of the actin filaments, overgrowth of the cells and the presence of syncytia.展开更多
Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could a...Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could also be detected by IFA. The titre of the recovered virus is 104.6/mL. Immune electron micro-scopic observation of the virus particles revealed that the particles were rotund with a diameter of about 30 nm. Besides, virus titre quantification obtained by qRT-PCR showed a correlation between time from infection and virus titre. All these results showed that we have recovered RHDV from RK13 cells by re-verse genetics technology successfully, and this would be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and new type vaccines of RHDV.展开更多
为筛选兔瘟病毒在兔肾上皮细胞(rabbit kidney epithelial cells,RK13)上的受体,从RK13细胞中提取基因组总RNA,然后用链霉亲和素磁珠(SA-PMP)纯化mRNA,再用SMART(Switching methanism at 5 end ofRNA transcript)技术合成cDNA第1链。采...为筛选兔瘟病毒在兔肾上皮细胞(rabbit kidney epithelial cells,RK13)上的受体,从RK13细胞中提取基因组总RNA,然后用链霉亲和素磁珠(SA-PMP)纯化mRNA,再用SMART(Switching methanism at 5 end ofRNA transcript)技术合成cDNA第1链。采用长距离PCR(long-distance-PCR,LD-PCR)方法扩增双链cDNA,经纯化后克隆到真核表达载体pEXP-Lib中。最后,电转化宿主菌(DH5α),构建了RK13细胞的全长cDNA真核表达文库。鉴定结果表明,该文库插入的cDNA片段平均长度达到1.0 kb,库容量达到2.55×105CFU。展开更多
The apoptosis of RK13 cells induced by RHDV was investigated with DAPI staining,DNA ladder,Caspase 3 activity and flow cytometry,etc.The results showed that nuclear staining of infected cells with DAPI showed graduall...The apoptosis of RK13 cells induced by RHDV was investigated with DAPI staining,DNA ladder,Caspase 3 activity and flow cytometry,etc.The results showed that nuclear staining of infected cells with DAPI showed gradually morphological changes of the nuclei.As shown in the paper,a canonic oligonucleosome-sized DNA ladder was observed in cells harvested at 24h,48h and 72h post-infection,confirming that DNA fragmentation was induced by RHDV infection.The results of flow cytometry showed that about 63 % of cells were in apoptosis at 48h post-infection.Besides,we also demonstrated that the activation of Caspase 3 occurred during the infection process.In conclusion,our results showed that apoptosis in RHD might be determinant in the development of the pathogenesis of RHD.展开更多
文摘Background: Malassezia pachydermatis is the main causative agent of canine otitis and also of a myriad of dermatological problems in companion animals;its interaction mechanisms with host cells are still unclear. Objectives: To establish an in vitro infection model of M. pachydermatis-exposed RK13 cells in order to evaluate cell morphological changes as well as changes in the structure of actin filaments. Methods: Cultures of RK13 cells were infected with M. pachydermatis, alterations caused by the yeast were evaluated by optical and fluorescence microscopy. Results: M. pachydermatis adheres itself to the cell and produces the formation of multiple agglomerates that cause changes in cell morphology, formation of cell aggregates in overlays, presence of syncytia and destruction of cell culture structure. The damaged cells presented changes in the actin filaments consisting of thickening of the cell cortex and loss of stress fibers. On the other hand, the formation of perinuclear actin rings in the yeasts was observed. Conclusions: An in vitro infection model was established with M. pachydermatis and alterations in cell morphology were observed consisting of changes in the structure of the actin filaments, overgrowth of the cells and the presence of syncytia.
基金This work was supported by the Natural Science Foundation of Zhejiang Province(Grant No.Y305047).
文摘Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could also be detected by IFA. The titre of the recovered virus is 104.6/mL. Immune electron micro-scopic observation of the virus particles revealed that the particles were rotund with a diameter of about 30 nm. Besides, virus titre quantification obtained by qRT-PCR showed a correlation between time from infection and virus titre. All these results showed that we have recovered RHDV from RK13 cells by re-verse genetics technology successfully, and this would be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and new type vaccines of RHDV.
文摘The apoptosis of RK13 cells induced by RHDV was investigated with DAPI staining,DNA ladder,Caspase 3 activity and flow cytometry,etc.The results showed that nuclear staining of infected cells with DAPI showed gradually morphological changes of the nuclei.As shown in the paper,a canonic oligonucleosome-sized DNA ladder was observed in cells harvested at 24h,48h and 72h post-infection,confirming that DNA fragmentation was induced by RHDV infection.The results of flow cytometry showed that about 63 % of cells were in apoptosis at 48h post-infection.Besides,we also demonstrated that the activation of Caspase 3 occurred during the infection process.In conclusion,our results showed that apoptosis in RHD might be determinant in the development of the pathogenesis of RHD.
