中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
5期
389-399
,共11页
刘志平%沙翔垠%王智崇%李朝阳%刘颖%周瑾
劉誌平%沙翔垠%王智崇%李朝暘%劉穎%週瑾
류지평%사상은%왕지숭%리조양%류영%주근
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
胚胎榦細胞%微環境%耑粒酶%角膜緣榦細胞%信號通路%凋亡%剋隆形成單位
배태간세포%미배경%단립매%각막연간세포%신호통로%조망%극륭형성단위
Embryonic stem cells%Niche%Telomerase%Limbal stem cells%Signaling pathway%Apoptosis%Colony-forming units
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均明显低于CnT-20培养组,差异均有统计学意义(均P＜0.01);ESCCM培养组第2代细胞中干细胞标志物蛋白及TERT蛋白表达量均明显高于CnT-20培养组,差异均有统计学意义(均P＜0.01).siRNA-TERT转染组细胞端粒酶活性为(4.83±0.67)％,明显低于siRNA-F转染组的(46.71±1.22)％,差异有统计学意义(t=52.116,P=0.000).ESC-CM培养组培养液中添加了FAK抑制剂和GSK3β抑制剂以及转染TERT-siRNA后,细胞中pFAK、pAkt、pGSK3β的表达明显减弱,而p21的表达增强.ESC-CM培养组第2代LSCs及siRNA-F转染组细胞的线粒体膜电位明显高于CnT-20培养组和siRNA-TERT 转染组,差异均有统计学意义(均P＜0.01);ESC-CM培养组原代和第2代细胞中及siRNA-F转染组细胞中ROS表达比例均明显低于CnT-20培养组及siRNA-TERT转染组,差异均有统计学意义(均P＜0.01). 结论 ESC-CM培养体系可促进LSCs干性的维持,抑制细胞凋亡,其机制可能与端粒酶-p21-线粒体通路及FAK/Wnt 信号通路的激活有关.
揹景 成體榦細胞的命運與其生存的微環境密切相關.研究錶明,胚胎榦細胞(ESCs)微環境能增彊人角膜緣榦細胞(LSCs)的榦性,其作用機製是研究熱點. 目的 探討榦細胞微環境增彊LSCs的榦性及其抑製細胞凋亡的分子機製.方法將129小鼠ES細胞株(E14細胞)分彆用CnT-20培養液和CnT-20+體積分數20％ ES(ESC-CM)培養液進行培養和傳代.採用新鮮供體人角膜緣組織塊培養法,分彆用CnT-20培養液和ESC-CM培養液培養和傳代LSCs,細胞經固定後于光學顯微鏡下觀察細胞剋隆形態併計算剋隆形成率(CFE).ESC-CM培養組細胞分彆轉染耑粒酶逆轉錄酶(TERT) siRNA(19-25nt siRNA)或siRNA(sc37007),流式細胞儀檢測轉染前後細胞凋亡、線粒體膜電位的改變;採用免疫熒光技術和流式細胞技術檢測siRNA轉染前後細胞中耑粒酶的錶達和活性氧簇(ROS)的產生情況;採用RT-PCR、免疫熒光技術和Western blot法檢測細胞中榦細胞標誌物p63、三燐痠腺苷結閤轉運蛋白G超傢族成員2(ABCG2)、整閤素β1(integrin β1)和細胞分化標誌物細胞角蛋白3(CK3) mRNA及其蛋白的錶達;採用Western blot法檢測細胞中黏著斑激酶(FAK)、Akt和糖原閤成酶激酶3β(GSK3β)及其燐痠化蛋白以及p21蛋白的改變. 結果 ESC-CM培養組LSCs可傳至第8代,CFE為(7.6±0.6)％,CnT-20培養組傳至第6代,CFE為(5.6±0.6)％,差異有統計學意義(t=4.454,P=0.011);ESC-CM培養組第2、3、4、5、6代LSCs的凋亡百分比均明顯低于CnT-20培養組,差異均有統計學意義(均P＜0.05);siRNA-F轉染細胞的凋亡率為(7.7±1.3)％,明顯低于siRNA-TERT轉染細胞的(32.3±3.1)％,差異有統計學意義(t=-12.588,P=0.000).ESC-CM培養組與CnT-20培養組的原代LSCs 中榦細胞標誌物mRNA及其蛋白、TERT蛋白的相對錶達量差異均無統計學意義(均P＞0.05),但ESC-CM培養組CK3 mRNA及其蛋白的相對錶達量均明顯低于CnT-20培養組,差異均有統計學意義(均P＜0.01);ESCCM培養組第2代細胞中榦細胞標誌物蛋白及TERT蛋白錶達量均明顯高于CnT-20培養組,差異均有統計學意義(均P＜0.01).siRNA-TERT轉染組細胞耑粒酶活性為(4.83±0.67)％,明顯低于siRNA-F轉染組的(46.71±1.22)％,差異有統計學意義(t=52.116,P=0.000).ESC-CM培養組培養液中添加瞭FAK抑製劑和GSK3β抑製劑以及轉染TERT-siRNA後,細胞中pFAK、pAkt、pGSK3β的錶達明顯減弱,而p21的錶達增彊.ESC-CM培養組第2代LSCs及siRNA-F轉染組細胞的線粒體膜電位明顯高于CnT-20培養組和siRNA-TERT 轉染組,差異均有統計學意義(均P＜0.01);ESC-CM培養組原代和第2代細胞中及siRNA-F轉染組細胞中ROS錶達比例均明顯低于CnT-20培養組及siRNA-TERT轉染組,差異均有統計學意義(均P＜0.01). 結論 ESC-CM培養體繫可促進LSCs榦性的維持,抑製細胞凋亡,其機製可能與耑粒酶-p21-線粒體通路及FAK/Wnt 信號通路的激活有關.
배경 성체간세포적명운여기생존적미배경밀절상관.연구표명,배태간세포(ESCs)미배경능증강인각막연간세포(LSCs)적간성,기작용궤제시연구열점. 목적 탐토간세포미배경증강LSCs적간성급기억제세포조망적분자궤제.방법장129소서ES세포주(E14세포)분별용CnT-20배양액화CnT-20+체적분수20％ ES(ESC-CM)배양액진행배양화전대.채용신선공체인각막연조직괴배양법,분별용CnT-20배양액화ESC-CM배양액배양화전대LSCs,세포경고정후우광학현미경하관찰세포극륭형태병계산극륭형성솔(CFE).ESC-CM배양조세포분별전염단립매역전록매(TERT) siRNA(19-25nt siRNA)혹siRNA(sc37007),류식세포의검측전염전후세포조망、선립체막전위적개변;채용면역형광기술화류식세포기술검측siRNA전염전후세포중단립매적표체화활성양족(ROS)적산생정황;채용RT-PCR、면역형광기술화Western blot법검측세포중간세포표지물p63、삼린산선감결합전운단백G초가족성원2(ABCG2)、정합소β1(integrin β1)화세포분화표지물세포각단백3(CK3) mRNA급기단백적표체;채용Western blot법검측세포중점착반격매(FAK)、Akt화당원합성매격매3β(GSK3β)급기린산화단백이급p21단백적개변. 결과 ESC-CM배양조LSCs가전지제8대,CFE위(7.6±0.6)％,CnT-20배양조전지제6대,CFE위(5.6±0.6)％,차이유통계학의의(t=4.454,P=0.011);ESC-CM배양조제2、3、4、5、6대LSCs적조망백분비균명현저우CnT-20배양조,차이균유통계학의의(균P＜0.05);siRNA-F전염세포적조망솔위(7.7±1.3)％,명현저우siRNA-TERT전염세포적(32.3±3.1)％,차이유통계학의의(t=-12.588,P=0.000).ESC-CM배양조여CnT-20배양조적원대LSCs 중간세포표지물mRNA급기단백、TERT단백적상대표체량차이균무통계학의의(균P＞0.05),단ESC-CM배양조CK3 mRNA급기단백적상대표체량균명현저우CnT-20배양조,차이균유통계학의의(균P＜0.01);ESCCM배양조제2대세포중간세포표지물단백급TERT단백표체량균명현고우CnT-20배양조,차이균유통계학의의(균P＜0.01).siRNA-TERT전염조세포단립매활성위(4.83±0.67)％,명현저우siRNA-F전염조적(46.71±1.22)％,차이유통계학의의(t=52.116,P=0.000).ESC-CM배양조배양액중첨가료FAK억제제화GSK3β억제제이급전염TERT-siRNA후,세포중pFAK、pAkt、pGSK3β적표체명현감약,이p21적표체증강.ESC-CM배양조제2대LSCs급siRNA-F전염조세포적선립체막전위명현고우CnT-20배양조화siRNA-TERT 전염조,차이균유통계학의의(균P＜0.01);ESC-CM배양조원대화제2대세포중급siRNA-F전염조세포중ROS표체비례균명현저우CnT-20배양조급siRNA-TERT전염조,차이균유통계학의의(균P＜0.01). 결론 ESC-CM배양체계가촉진LSCs간성적유지,억제세포조망,기궤제가능여단립매-p21-선립체통로급FAK/Wnt 신호통로적격활유관.
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 transfected group (t =-12.588,P =0.000).No significant differences were seen in the expression levels of p63,ABCG2,integrin β1 mRNA and proteins and TERT protein in the primary cells between the ESC-CM group and the CnT-20 group (all at P＞0.05),but significantly declined expressions of CK3 mRNA and protein were found in the ESC-CM group compared with the CnT-20 group (all at P＜0.01).However,the expressions of p63,ABCG2,integrin β1 mRNA and proteins and TERT protein in the second generation of the cells were significantly higher in the ESC-CM group compared with the CnT-20 group (all at P＜0.01).The telomerase activity was (4.83±0.67) ％ in the siRNA-TERT transfected group,which was significantly lower than (46.71±1.22) ％ of the siRNA-F transfected group (t =52.116,P =0.000).The expression of pFAK,pAkt,pGSK3β proteins were weakened,but the expression of p21 was increased in the ESC-CM group after addition of FAK inhibitor,GSK3β inhibitor and TERT-siRNA transfected group.Mitochondrial membrane potential in the second generation of cells was elevated in the ESC-CM group in comparison with the CnT-20 group and the siRNA-TERT transfected group (all at P＜0.01),and the rates of ROS positively reaction was lower in the ESC-CM group and the siRNA-F transfected group than those of the CnT-20 group and siRNA-TERT transfected group (all at P＜0.01).Conclusions ESC-CM culture system can effectively keep the stemness of LSCs and inhibit apoptosis.ESC-CM culture system plays functions probably via telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways.
