新疆驴成纤维细胞原代培养及鉴定  

Primary culture and identification of the fibroblasts of Xinjiang donkeys

作　　者：朱春灏 王艳萍[1] 赵玉坤 张文倩 曾维斌[1] ZHU Chunhao;WANG Yanping;ZHAO Yukun;ZHANG Wenqian;ZENG Weibin(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003

出　　处：《黑龙江畜牧兽医》2023年第1期113-116,121,132,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31760643);石河子大学创新发展专项(CXFZ202007)。

摘　　要：为了获得纯度较高的驴皮成纤维细胞并建立成纤维细胞特征鉴定方法,试验采用组织块贴壁法进行原代细胞培养,用胰蛋白酶联合组织块进行差时消化法分离、纯化成纤维细胞,并通过细胞形态观察、PCR扩增鉴定vimentin、免疫荧光染色法鉴定分离的细胞并计算纯度,用CCK-8法检测细胞增殖能力,台盼蓝染色检测冻存前后的细胞活率。结果表明:分离纯化后的细胞呈梭形或纺锤形,具备典型的成纤维细胞形态;PCR扩增可清晰检测到vimentin的表达条带;细胞免疫荧光显示vimentin染色为阳性,DAPI核染呈椭圆形,以上方法均鉴定为成纤维细胞;体外培养的驴皮第3代成纤维细胞48 h后进入对数生长期,其生长曲线呈“S”型;冻存前和复苏后细胞活率差异不显著(P>0.05)。说明经体外分离、纯化成功获得了驴皮成纤维细胞。In order to obtain donkey skin fibroblasts with high purity and to establish a method for characteristics characterization of the fibroblasts, in this experiment, primary cell culture was carried out by using the tissue block adherence method,separation and purification of fibroblasts by trypsin combined with tissue block differential adherence method. Vimentin was identified by cell morphology observation and PCR amplification. The isolated cells were identified by immunofluorescence staining and the purity was calculated. CCK-8 method was used to detect cell proliferation. Trypan Blue staining was used to detect cell viability before and after cryopreservation. The results showed that the isolated and purified cells were spindle or spindle shaped, with typical fibroblast morphology. PCR clearly detected the expression band of vimentin;cellular immunofluorescence showed positive vimentin staining and oval-shaped DAPI nuclear staining, which were identified as fibroblasts by the above methods. In vitro cultured donkey skin third-generation fibroblasts entered the logarithmic growth phase after 48 h, and their growth curve was “S” shaped. The differences in cell survival rates before freezing and after recovery were not significant(P>0. 05). The results indicated that donkey skin fibroblasts were successfully obtained by in vitro isolation and purification.

关 键 词：驴皮 成纤维细胞 组织块培养 纯化 波形蛋白 

分 类 号：S822[农业科学—畜牧学] Q813.11[农业科学—畜牧兽医]