【作者】 洪海霞；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2004， 硕士

【摘要】 猪瘟病毒（CSFV）只能专一性对猪致病，但可以使牛、羊、兔等多种动物发生无症状性感染。CSFV 可在多种动物的组织细胞中复制，但一般不产生致细胞病变效应(CPE)。由于猪瘟病毒对中胚层组织，特别是造血和血管组织具有特殊的亲和力，CSFV感染猪后使小血管和毛细血管内皮细胞发生变性和坏死，而不能使其他细胞发生病变。为了给研究CSFV对猪专一性致病机理和对猪血管内皮细胞致病变作用机理提供细胞材料，于是我们开展了猪脐静脉血管内皮细胞体外培养方法的研究，并观察了猪瘟病毒石门强毒株对猪血管内皮细胞的致病变作用，获得了以下结果： 1. 无菌采取猪脐带，用 0.1%胶原酶消化分离内皮细胞，于 37℃，5％CO2 下培养。对培养细胞生长特性进行检测，并对细胞进行第Ⅷ 因子相关抗原(F-ⅧRAg)和扫描电镜形态学鉴定。结果表明，70%细胞在 24h 内贴壁，4～5d 长成单层。培养的细胞呈单层贴壁生长，典型铺路石状，第Ⅷ因子相关抗原(F-ⅧRAg)检测阳性。证明分离到了猪血管内皮细胞。 2. 取 2～3 代生长良好的猪血管内皮细胞，待长至 80%单层时接种 CSFV 石门株脾毒，加维持液（含 2%无牛病毒性腹泻病毒抗体的胎牛血清）培养。同时设猪血管内皮细胞接种 CSFV C 株（弱毒疫苗株）对照、PK-15 细胞上接种 CSFV 石门株对照和阴性（不接毒）对照。分别于 12h，24h，36h，48h，72h 和 96h 观察细胞形态。结果表明，血管内皮细胞于接种 CSFV 石门株后 12h，24h 无明显变化，至 36h 时，细胞开始拉长，个别细胞开始脱落；至 48h 时，部分细胞变圆，脱落，细胞间隙增大；至 72h，细胞拉长呈长梭状，细胞间隙很大，细胞脱落严重。3 个对照组的细胞均未出现明显变化。 3. 取接种 CSFV 石门株脾毒、C 株后 24h 和 56h 的血管内皮细胞和对照血管内皮细胞，经甲醛固定后进行透射电镜观察。结果表明，接种石门株的细胞在 24h 时，细胞溶酶体增多，内质网轻度扩张，线粒体变小，皱缩。在 56h 时，溶酶体显著增多，内质网扩张明显，线粒体变性，皱缩；而接种 C 株的内皮细胞除溶酶体增多外，其余无明显变化。阴性对照血管内皮细胞无明显变化。 4.为了确证血管内皮细胞 CPE 是否由 CSFV 石门株引起，将 CSFV 石门株接种于培养的猪血管内皮细胞，从接种后 24h，48h 和 72h 的血管内皮细胞和阴性对照血管内皮细胞提取细胞 RNA，经 RT-PCR 和 nPCR 扩增 CSFV 的 E2 基因。结果表明，接种后 24h，48h 和 72h，血管内皮细胞内均能扩增出约为 1170bp 的特异性片段，经核苷酸序列分析，证明与 GenBank 中 CSFV 石门株的 E2 基因序列相符。而阴性对照细胞则无条带出现。<WP=6>II 中文摘要 以上结果表明，本文成功的建立了猪脐静脉血管内皮细胞体外分离培养的方法；CSFV 石门株能够使猪血管内皮细胞产生细胞病变效应（CPE），这在国内外均属首次报道。更多还原

【Abstract】 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 expanded<WP=8>IV 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.更多还原