人角膜基质间充质干细胞的分离培养及表型鉴定  

Isolation,Culture and Phenotype Identification of Human Corneal Stromal Mesenchymal Stem Cells

作　　者：刘先宁 汪耀[1] 朱秀萍[1] 程燕[1] 潘士印[1] 肖湘华[1] 银勇 安娜[1] 王亚妮[1] 刘睿 LIU Xian-ning;WANG Yao;ZHU Xiu-ping;CHENG Yan;PAN Shi-yin;XIAO Xiang-hua;YIN Yong;AN Na;WANG Ya-ni;LIU Rui(Xi’an No.1 Hospital,Shaanxi Institute of Ophthalmology,Shaanxi Key Laboratory of Ophthalmology,Clinical Research Center for Ophthalmology Diseases of Shaanxi Province,the First Affiliated Hospital of Northwest University,Xi'an 710002,China)

机构地区：[1]西安市第一医院陕西省眼科研究所陕西省眼科学重点实验室陕西省眼科疾病临床医学研究中心西北大学附属第一医院

出　　处：《现代检验医学杂志》2019年第4期28-30,34,共4页Journal of Modern Laboratory Medicine

基　　金：陕西省重点研发计划项目(2018ZDXM-SF-056);陕西省自然科学基金面上项目(2017JM8040)

摘　　要：目的建立人角膜基质间充质干细胞(HCS-MSC)体外分离培养、传代和鉴定等方法。方法无菌条件下,取眼科临床角膜移植术后剩余的供体角膜缘组织,去除上皮和内皮组织,将基质组织切成1mm×2mm的组织块,采用无动物源性血清的间充质干细胞培养液进行组织块贴壁法培养,倒置显微镜下动态观察细胞的生长形态及贴壁状态,将传代培养后的第三代细胞采用流式细胞分析法进行表型鉴定,液氮冷冻保存。结果采用人角膜缘基质组织块贴壁法培养,于培养后第7天开始向周围呈放射状长出细胞,14天左右长满培养皿,细胞形态呈均一梭形;传代培养后生长快速,于2~4天达90%融合;连续传代3次,其生长特性和形态不变,CD90,CD29和CD105阳性率>95%,CD34和CD-HLA-dr阳性率<3%。结论采用人角膜缘基质组织可分离培养出间充质干细胞,为角膜组织修复、重建及对抗免疫排斥反应等奠定基础。Objective To establish a method for isolation, culture,passage,identification and preservation of human corneal stromal mesenchymal stem cells in vitro.Methods After clinical keratoplasty, the residual donor limbal tissue was taken under sterile conditions,the epithelial and endothelial tissues were removed,and the stromal tissue was cut into 1 mm × 2 mm tissue mass.The mesenchymal stem cell culture medium without animal-derived serum was used.The tissue mass was cultured in an adherent manner,and the growth morphology and adherence state of the cells were observed dynamically under an inverted microscope.The third-generation cells were identified by flow cytometry and cryopreserved using liquid nitrogen.Results Human limbal stromal tissue was cultured using tissue mass adherent culture method.On the 7 th days,the cells began to radially grow.The culture dish was covered about 14 days after culture,and the cell morphology was uniform as a spindle.The cells were serially passaged 3 times,and their growth characteristics and morphology were unchanged.After subculture,the growth rate was rapid,reaching 90% fusion on 2 nd or 4 th days.The positive rates of CD90,CD29 and CD105 were all >95%,and the positive rates of CD34 and CD-HLA-dr were all <3%.Conclusion The results indicated that mesenchymal stemcells can be isolated and cultured from human limbal stromal tissue,which lays a foundation for corneal tissue repair and reconstruction as well as resistance to immune rejection.

关 键 词：人角膜基质间充质干细胞 体外分离培养 表型鉴定 

分 类 号：R446.8[医药卫生—诊断学]