小鼠鼻黏膜上皮细胞的原代培养和鉴定  

Primary culture and identification of mouse nasal mucosal epithelial cells

作　　者：常换换[1] 崔沐 王洁[1] CHANG Huanhuan;CUI Mu;WANG Jie(Otolaryngology Department,Children's Hospital Affiliated to Xi'an Jiaotong University,Xi'an 710003;School of Nursing,Xi'an Medical University,Xi'an 710021,China)

机构地区：[1]西安交通大学附属儿童医院耳鼻喉科,陕西西安710003 [2]西安医学院护理系,陕西西安710021

出　　处：《临床医学研究与实践》2020年第35期1-3,共3页Clinical Research and Practice

基　　金：国家自然科学基金资助项目(No.82000959);陕西省自然科学基础研究计划(No.2019JQ-434);陕西省卫生健康科研基金项目(No.2020JM-606)。

摘　　要：目的探讨酶消化分离法原代培养小鼠鼻黏膜上皮细胞的实验步骤和技术要点,提高原代培养成功率,为后续实验提供良好的培养模型。方法小鼠鼻黏膜组织块中加入约组织块5倍体积的0.1%Ⅰ型胶原酶并混合均匀,后置于37℃孵箱中孵育10 min,加入适量FBS终止消化反应。吸除酶液后,继续加入等量的0.25%胰蛋白酶并混合均匀,后置于37℃孵箱中孵育2 h,加入适量FBS终止消化反应。200目细胞过滤筛过滤细胞液,过滤液移置离心管,1000 r/min离心10 min弃上清。沉淀液中加入HBSS漂洗1次,1000 r/min离心5 min弃上清,余细胞加入含血清的DMEM/F12培养基中混合均匀。培养皿置于37℃、5%CO2、95%O2培养箱中,2~3 d换液。倒置显微镜下观察细胞形态及生长状况,免疫荧光鉴定细胞性质及纯度。结果原代培养的鼻黏膜上皮细胞第7天可见,第14天大约可以长满培养皿的50%,培养第21天可以长满培养皿;鼻黏膜上皮细胞边缘清晰,数量大,活性好,可供下一步实验研究应用;CK18鉴定为上皮源性细胞,鼻黏膜上皮细胞纯度高达90%。结论本培养方法简单,鼻黏膜上皮细胞增殖好、纯度高。Objective To explore the experimental steps and technical points of enzyme digestion and isolation for primary culture of mouse nasal mucosal epithelial cells,so as to improve the success rate of primary culture,and provide a good culture model for subsequent experiments.Methods 0.1%collagenase of typeⅠ(about 5 times the volume of the tissue block)was added into the nasal mucosa tissue block of mice and mixed evenly,incubated in 37℃incubator for 10 min,and appropriate amount of FBS was added to terminate the digestion reaction.After the enzyme solution was removed,the same amount of 0.25%trypsin was added and mixed evenly,incubated in 37℃incubator for 2 h,and appropriate amount of FBS was added to terminate the digestion reaction.The 200 mesh cell filter was used to filter the cell liquid,the filtrate was put into a centrifuge tube,centrifuged at 1000 r/min for 10 min,and then the supernatant was discarded.HBSS was added into the precipitate,washed once,centrifuged at 1000 r/min for 5 min,and the supernatant was discarded;the remaining cells were added into DMEM/F12 medium containing serum and mixed evenly.The culture dish was placed in the incubator with 5%CO2 and 95%O2 at 37℃,the fluid was changed after 2-3 days.The morphology and growth of the cells were observed under the inverted microscope,and the nature and purity of the cells were identified by immunofluorescence.Results The primary culture nasal mucosal epithelial cells could be observed on the 7th day,cover about 50%of the culture dish on the 14th day,and cover the whole culture dish on the 21st day of culture;the nasal mucosal epithelial cells had clear edges,large numbers and good activity,which could be used for further experimental study;the cells was epithelial-derived cells by CK18 identification,and the purity of nasal mucosal epithelial cells was as high as 90%.Conclusion This culture method is simple,with good nasal mucosal epithelial cells proliferation and high purity.

关 键 词：鼻黏上皮细胞 细胞培养 抗细胞角蛋白18 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]