山羊原代口腔上皮细胞的分离培养及鉴定  

Isolation,Culture and Identification of Primary Oral Epithelial Cells of Goats

作　　者：李蓉 富国文 朱红林 刘泽武 王雅 王瑾艺 四朗玉珍[2] 樊月圆 LI Rong;FU Guo-wen;ZHU Hong-lin;LIU Ze-wu;WANG Ya;WANG Jin-yi;SILANG Yu-zhen;FAN Yue-yuan(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China;Institute of Animal Husbandry and Veterinary Medicine,Tibet Academy of Agricultural and Animal Husbandry Sciences,Lhasa,Xizang 850000,China)

机构地区：[1]云南农业大学动物医学院,云南昆明650201 [2]西藏自治区农牧科学院畜牧兽医研究所,西藏拉萨850000

出　　处：《动物医学进展》2025年第4期72-77,共6页Progress In Veterinary Medicine

基　　金：西藏自治区“十四五”重大科技专项(XZ202101ZD0001N)。

摘　　要：建立一种高效制备山羊原代口腔上皮细胞的体外分离培养方法,为后续研究山羊口腔疾病提供细胞材料。试验采用0.1μg/mL分散酶Ⅱ和300 U/mL胶原酶Ⅰ组合消化法、组织贴壁法分离纯化山羊口腔上皮细胞,用含10%胎牛血清和10 ng/mL表皮生长因子(EGF)完全培养基培养口腔上皮细胞,观察其形态特征并记录生长曲线,经过特异性蛋白PCR扩增、免疫荧光、质粒pGEFP-N1转染进行口腔上皮细胞的鉴定。结果表明,组合酶消化法后细胞基本贴壁,细胞形态正常,呈铺路石状,可见有成纤维细胞夹杂生长;组织贴壁法48 h后逐渐爬出细胞,呈铺路石状,无杂细胞夹杂生长;原代口腔上皮细胞分离后采用CCK-8试剂盒对细胞活力进行检测,表明1~3 d增殖速度缓慢,培养3~5 d细胞增殖速度加快,活性增强,进入对数生长期,培养5~7 d后细胞增殖速率下降,原代口腔上皮细胞生长曲线呈S形;使用角蛋白、肌间线蛋白、波形蛋白特异性引物对分离到的山羊口腔上皮细胞进行PCR扩增,扩增结果表明所分离纯化得到的细胞是口腔上皮细胞;免疫荧光染色结果显示角蛋白抗体免疫阳性率达98%以上;质粒pEGFP-N1转染口腔上皮细胞12 h荧光较弱,24 h荧光增强,阴性无荧光,表明山羊原代口腔上皮细胞可整合外源基因并作为表达外源基因的生物反应器。建立了山羊原代口腔上皮细胞体外分离培养方法。To establish an in vitro isolation and culture method for preparing goat primary oral epithelial cells,and to provide cell materials for the follow-up study of goat oral diseases.Goat oral epithelial cells were isolated and purified by the combined digestion method of 0.1μg/mL disperse enzymeⅡand 300 U/mL collagenaseⅠ,and tissue adhesion method.Oral epithelial cells were cultured in complete medium containing 10%fetal bovine serum and 10 ng/mL epidermal growth factor(EGF).Morphological characteristics of oral epithelial cells were observed and growth curves were recorded.Oral epithelial cells were identified by specific protein PCR amplification,immunofluorescence and plasmid pGEFP-N1 transfection.Results showed that,after combination enzyme digestion,the cells were basically adherent to the wall,the morphology of the cells was normal,and there was fibrocyte inclusion growth.After 48 h of tissue adhesion method,the cells gradually crawled out,showing a paving stone shape,and there was no impurity cell inclusion growth.The proliferation rate of primary oral epithelial cells was slow at 1 to 3 days after isolation,and accelerated at 3 to 5 days after culture,with enhanced activity,and entered the logarithmic growth phase.After 5 to 7 days of culture,the proliferation rate of primary oral epithelial cells decreased,and the growth curve of primary oral epithelial cells was S-shaped.The cultured goat oral epithelial cells were amplified by PCR with specific primers of keratin,myoglobin and vimentin.The results showed that the purified cells were oral epithelial cells.The immunofluorescence staining results showed that the positive rate of keratin antibody was more than 98%.After transfection with pEGFP-N1,the fluorescence of oral epithelial cells was weak at 12 h,and increased at 24 h,indicating that the primary oral epithelial cells of goats could integrate foreign genes and act as bioreactors for expressing foreign genes.Conclusion:the method of isolation and culture of goat primary oral epithelial cells in vit

关 键 词：山羊口腔上皮细胞 原代培养 分离鉴定 胶原酶消化法 

分 类 号：S852.654[农业科学—基础兽医学] S858.27[农业科学—兽医学]