胚胎干细胞微环境增强人角膜缘干细胞的干性和抑制凋亡的机制  

Mechanism of embryonic stem cells microenvironment enhancing stemness and inhibiting apoptosis of human limbal stem cells

作　　者：刘志平[1] 沙翔垠[1] 王智崇[2] 李朝阳[2] 刘颖[2] 周瑾[2] 

机构地区：[1]广州医科大学附属第二医院眼科,510260 [2]中山大学中山眼科中心眼科学国家重点实验室,广州510060

出　　处：《中华实验眼科杂志》2015年第5期389-399,共11页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学青年基金项目（81300732）;广东省医学科研基金项目（B2013305）

摘　　要：背景 成体干细胞的命运与其生存的微环境密切相关.研究表明,胚胎干细胞（ESCs）微环境能增强人角膜缘干细胞（LSCs）的干性,其作用机制是研究热点. 目的 探讨干细胞微环境增强LSCs的干性及其抑制细胞凋亡的分子机制.方法将129小鼠ES细胞株（E14细胞）分别用CnT-20培养液和CnT-20＋体积分数20％ ES（ESC-CM）培养液进行培养和传代.采用新鲜供体人角膜缘组织块培养法,分别用CnT-20培养液和ESC-CM培养液培养和传代LSCs,细胞经固定后于光学显微镜下观察细胞克隆形态并计算克隆形成率（CFE）.ESC-CM培养组细胞分别转染端粒酶逆转录酶（TERT） siRNA（19-25nt siRNA）或siRNA（sc37007）,流式细胞仪检测转染前后细胞凋亡、线粒体膜电位的改变;采用免疫荧光技术和流式细胞技术检测siRNA转染前后细胞中端粒酶的表达和活性氧簇（ROS）的产生情况;采用RT-PCR、免疫荧光技术和Western blot法检测细胞中干细胞标志物p63、三磷酸腺苷结合转运蛋白G超家族成员2（ABCG2）、整合素β1（integrin β1）和细胞分化标志物细胞角蛋白3（CK3） mRNA及其蛋白的表达;采用Western blot法检测细胞中黏着斑激酶（FAK）、Akt和糖原合成酶激酶3β（GSK3β）及其磷酸化蛋白以及p21蛋白的改变. 结果 ESC-CM培养组LSCs可传至第8代,CFE为（7.6±0.6）％,CnT-20培养组传至第6代,CFE为（5.6±0.6）％,差异有统计学意义（t=4.454,P=0.011）;ESC-CM培养组第2、3、4、5、6代LSCs的凋亡百分比均明显低于CnT-20培养组,差异均有统计学意义（均P＜0.05）;siRNA-F转染细胞的凋亡率为（7.7±1.3）％,明显低于siRNA-TERT转染细胞的（32.3±3.1）％,差异有统计学意义（t=-12.588,P=0.000）.ESC-CM培养组与CnT-20培养组的原代LSCs 中干细胞标志物mRNA及其蛋白、TERT蛋白的相对表达量差异均无统计学意义（均P＞0.05）,但ESC-CM培养组CK3 mRNA及其蛋白的相对表达量�Background The fate of adult stem cells is associated with its surrounding microenviroment.Our previous work found that embryonic stem cells （ESCs） micro-environment enhance the stemness of human limbal stem cells （LSCs）,but its mechanism has not been elucidated.Objective This study was to explore the molecular mechanism of ESC micro-environment enhancing the stemness and inhibiting the apoptosis of LSCs.Methods Human LSCs were cultured by explant culture method with CnT-20 medium and CnT-20＋20% ES culture supernatant （ESC-CM）,respectively.Colony formation assay was used to analyze the proliferation ability of cells.Telomerase reverse transcriptase （TERT） siRNA （19-25nt siRNA） or siRNA （sc-37007） was transfected into the cells of ESCCM group.Apoptosis and mitochondrial membrane potential were assayed by flow cytometry,and the expressions of telomerase and reactive oxygen species （ROS） in TERT siRNA-or siRNA-F-transfected cells by immunofluorescence and flow cytomery.RT-PCR,immunofluorescence staining and Western blot were employed to determine the expressions of p63,ATP-binding cassette transporer G2 （ABCG2）,integrin β1 mRNA and proteins and cytokeratin 3 （C K3） in the cells.The levels of focal adhesion kinase （FAK）,Akt,glycogen synthase kinase 3β （GSK3β） and p21 protein and phosphorylation proteins in the cells were detected by Western blot.Results The LSCs presented an increased proliferative capacity and passaged to the eighth generation with the colony-forming efficiency （CFE） of （7.6±0.6） % in ESC-CM group,but the cells to the sixth generation with the CFE of （5.6±0.6）%,showing a significant difference between them （t =4.454,P =0.011）.The apoptotic rates of the cells from 2 through 6 generations were lower in the ESC-CM group than those in the CnT-20 group （all at P〈0.05）.The apoptotic rate of the cells was （7.67± 1.31）% in the siRNA-F transfected group,which was significantly lower than （32.33 ±3.13）%in the siRNA-TERT transfe

关 键 词：胚胎干细胞 微环境 端粒酶 角膜缘干细胞 信号通路 凋亡 克隆形成单位 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]