树鼩永生化皮肤成纤维细胞株的建立及鉴定  

Establishment and Identification of Immortalized Skin Fibroblast Cell Line of Chinese Tree Shrew

作　　者：奎秀莹 王文广[1] 金亮子 邓伟[3] 仝品芬[1] 陆彩霞[1] 代解杰[1] KUI Xiuying;WANG Wenguang;JIN Liangzi;DENG Wei;TONG Pinfen;LU Caixia;DAI Jiejie(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650031,China;Institute of Medical Biology,Kunming Medical University,Kunming 650031,China;Institute of Medical Biology,Yunnan University,Kunming 650031,China)

机构地区：[1]中国医学科学院,北京协和医学院医学生物学研究所,云南昆明650031 [2]昆明医科大学医学生物学研究所,云南昆明650031 [3]云南大学医学生物学研究所,云南昆明650031

出　　处：《云南农业大学学报（自然科学版）》2022年第3期478-484,共7页Journal of Yunnan Agricultural University:Natural Science

基　　金：昆明市科技创新团队(2019-1-R-24483);云南省科技人才和平台项目(2018HB071,2017HC019);云南省高层次卫生计生技术人才培养(D-2018026);云南省基础研究计划面上项目(202101AT070291)。

摘　　要：【目的】建立永生化树鼩皮肤成纤维细胞,为探究真皮层中皮肤成纤维细胞和其产生的细胞外基质在其中所发挥的作用提供新的试验材料。【方法】通过酶消化法从树鼩腿部皮肤分离纯化出皮肤成纤维细胞,利用携带SV40T基因的慢病毒转染细胞,挑取单克隆后进行传代培养;对传至50代以上的细胞进行形态学观察、免疫荧光鉴定以及核型鉴定。【结果】传代培养后得到的细胞形态一致,且与原代细胞相比未发生改变,均为梭形和多角形纤维状,到第58代时细胞状态仍较好。生长曲线测定结果显示:细胞生长旺盛,第2~3天为细胞对数生长期,第5天进入平台期;免疫荧光可检测到皮肤成纤维细胞标志蛋白Vimentin和永生化SV40T强荧光信号。细胞核型分析显示:染色体数与中缅树鼩染色体一致,即所得细胞为永生化树鼩皮肤成纤维细胞。【结论】本研究成功构建1株能够稳定传代的永生化树鼩皮肤成纤维细胞,为皮肤损伤修复及其他皮肤相关疾病的研究提供了试验材料。[Purpose]To provide a new experimental material to explore the role of skin fibroblasts and their extracellular matrix in dermis,the immortalized skin fibroblast line of Chinese tree shrew was established.[Methods]The skin fibroblasts were separated and purified by digestion with trypsin from leg ministry skin of Chinese tree shrew.The skin fibroblasts were immortalized by simian virus 40 large T antigen(SV40T)transduction,and the immortalized cells were identified by mor-phological observation,immunofluorescence and karyotype identification after picked monoclonal subculture and propagated to more than 50 generations of cells.[Results]The immortalized cells showed a similar morphology to that of the Chinese tree shrew primary skin fibroblasts with un-altered form,spindle and polygonal fibrous.The 58th generation cells were still in good condition.The growth curve showed immortalized cells grew vigorously.The cells were in the logarithmic growth phase on the 2nd or 3rd day,and entered the plateau phase on the 5th day.High fluorescence representing protein of tree shrew Vimentin and SV40T were detected by immunofluorescence.Ka-ryotype analysis showed that the number of chromosomes was consistent with that of Chinese tree shrew,which means the cells obtained as immortalized tree shrew skin fibroblasts.[Conclusion]A tree shrew skin fibroblasts cell line was successfully established,which could provide an experiment-al material for the study of damages and repairing about skin and other skin related diseases.

关 键 词：树鼩 皮肤成纤维细胞 分离 永生化细胞株 

分 类 号：S865.19[农业科学—野生动物驯养]