Isolation And Culture Of Swine Umbilicus Veins Endothelial Cells And Cytopathogencity Of Shimen Virulent Strain Of CSFV In Endothelial Cells Culture

Classical swine fever virus (CSFV) could infect multiple animals such as cattle, sheepand rabbit without symptom,but is only pathogenic to swine. CSFV could replicate andproliferate in multiple animals' tissue cells, but not cause the cytopathic effect(CPE) ingeneral condition. Because of CSFV's affinity to mesoblastic tissue including hematopoieticand blood vessel tissue;pathogenic swines appearing degeneration and necrosis of endothelialcells of capillary and small vascular after inoculatedion of CSFV,but other cells not occuringthe CPE, we establish in vitro culture method of swine Umbilicus veins endothelial cells.shimen virulent strain of CSFV were inoculated endothelial cells,and observation ofpathologic change of inoculated cells were done. 1.Swine umbilicus veins were washed by PBS. 0.1% callagenase I was poured intoumbilicus vein and digested.Endothelial cells obtained was collected by centrifugation,cultured in M199+20% fetal calf serum. The plating efficiency to plastic was 70%～75% for24h in coolagenase dispersed cells.A confluent monolayer was former by 4～5 days.Theresult showed the endothelial cells looked like cobblestones and were stained positively forfactor Ⅷ relative antigen .It shows cells cultured are endothelial cells. 2. The 2~3th passage endothelial cells were inoculated by shimen virulent strain settingas treatment group;endothelial cells were inoculated by C virulent strain of CSFV, PK-15 cellwere inoculated by Shimen virulent strain of CSFV and endothelial cells, PK-15 cells (notinoculated )respectively as parallel groups. We observesed inoculated cells and controlcells(not inoculated) at 12h,24h,36h,48h and 72h respectively. Results showed endothelialcells inoculated by shimen virulent strain of CSFV have no obvious change after 12h,24h;At36h,cells become be elongate and appearanced defluvium;At 48h,a few of cells started tobecome round and intercellular space become auxesis;At 72h, intercellular space becomemore auxesis, defluvium of cells was acute. PK-15 cells inoculated by shimen virulent strainof CSFV,endothelial cells inoculated by C strain of CSFV and cells(not inoculated ) appearedno obvious change at 12～72h. 3. Cells inoculated by shimen virulent strain and C strain of CSFV and control cells(notinoculated)at 24h,56h were observesed by transmission electron microscopy after fixationwith methyl aldehyde respectively. The results showed,in cells inoculated shimen virulentstrain ,the number of lysosomes of cells become more, endoplasmic reticulum mild expandedIV 英文摘要and mitochondria appeared degeneration. Control and inoculated C strain cells appeared noobvirus change except the number of lysosomes of cells inoculated C strain become more. 4. In oder to verify that CPE in endothelial cells was caused by shimen virulent strain ofCSFV, in this experiment RT-PCR and nPCR assay was used to identify viral E2 gene in theendothelial cell inoculated shimen virulent strain of CSFV. The results showed a specificalband which elongateth was about 1170bp was amplificated at 24h,48h and 72h;control cellsappeared negative. Conclusion: a method of segeration and culture of swine umbilicus veins endothelialcells was established;multiplication of shimen virulent strain of CSFV in swine umbilicusveins endothelial cells can cause the CPE which was reported in domestic and foreigh.