绵羊睾丸支持细胞的分离鉴定及其在山羊痘病毒疫苗株培养中的应用

为研究绵羊睾丸支持细胞对山羊痘病毒的增殖特性,采用胶原酶Ⅳ和胰酶两步法分离绵羊睾丸支持细胞,通过倒置显微镜进行形态学观察、细胞染色鉴定以及特异性表达ABP蛋白鉴定,然后接种山羊痘病毒疫苗株。结果显示,分离的原代细胞24 h可长满单层,用0.05%的胰酶消化处理3次可得到纯度约为80%的睾丸支持细胞;油红O染色可观察到绵羊睾丸支持细胞的细胞质含有大量脂肪滴,细胞核可见双极小体;HE染色后细胞核为蓝色,细胞质呈红色或粉红色。ABP蛋白间接免疫荧光可观察到在绵羊睾丸支持细胞的细胞质和细胞核周围有绿色荧光,经显微镜下计数其纯度达80%以上。接种山羊痘病毒疫苗株后第4天有90%细胞产生病变,细胞变圆,细胞聚集成簇状,细胞收缩、细胞核出现空泡化。qPCR扩增Ct值为18.81,Ct值低于其他细胞传代数值。结论,本研究成功分离培养了绵羊睾丸支持细胞,该细胞能较好地培养山羊痘病毒疫苗株,对羊痘疫苗的生产有一定的现实意义。

Isolation and identification of sheep testis Sertoli cell and it’s application in the culture of goatpox virus vaccine strain

To study the proliferation feature of sheep testis sertoli cells for the culture of goatpox virus,collagenase IV and trypsin were used to digest sheep testis tissue to separate sheep testis sertoli cells.Testis sertoli cells were identified by using inverted microscopy,cell staining identification,specific expression of ABP protein and inoculated with goatpox virus vaccine strain.Result showed that the isolated primary cells can be overgrown for 24 hours,and partially purified by 0.05% trypsin for 3 times to obtain testis Sertoli cells with a purity of about 80%,and oil red O staining show that the cytoplasm of sheep testis sertoli cells contained many fat droplets and bipolar bodies were observed in the nucleus.HE staining showed that the nucleus was blue and the cytoplasm was red or pink,and green fluorescence were observed around the cytoplasm and nucleus of sheep testis sertoli cells by the specific expression of immunofluorescence ABP,and the purity of cell was more than 80%.After four days cultured,90% of cells were developed to be lesions,round and wire drawing,and the cell nucleus were vacuolized.The Ct value of the q PCR amplification was 18.81 that was lower than those of other cell.In conclusion:The sheep testis sertoli cells,which were successfully isolated and cultured in this experiment,were cultured goatpox virus vaccine strain better,and had certain practical significance for the production of capripoxvirus.