中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
9期
819-822
,共4页
谭美华%陈建苏%陈剑%吴静%招志毅%戴应%李善义
譚美華%陳建囌%陳劍%吳靜%招誌毅%戴應%李善義
담미화%진건소%진검%오정%초지의%대응%리선의
量子点%标志物%诱导多能干细胞%示踪
量子點%標誌物%誘導多能榦細胞%示蹤
양자점%표지물%유도다능간세포%시종
Quantum dot%Marker%Induced pluripotent stem cell%Trace
背景 近年来,诱导多能干细胞(iPSCs)的多向分化特性为各种临床疾病的细胞替代治疗提供了新的种子来源,在眼科领域,iPSCs已成为人眼变性类疾病发病机制研究的良好模型,而更好的iPSCs识别和鉴定技术是对其体内生物学特性进行研究的基础.目的 探讨量子点标记人iPSCs的可行性及其最佳浓度,并观察量子点标记人iPSCs的光稳定性,为iPSCs应用于眼部细胞移植的实验研究和临床研究提供一种新的示踪技术.方法 用无饲养层细胞培养法培养和扩增脐带间充质基质细胞-人iPSCs系,并用免疫荧光法对其干细胞特性进行鉴定；选取不同浓度CdSe/ZnS核壳结构的量子点对扩增培养的iPSCs进行标记,用三维去卷积实时活细胞成像系统观察标记效果.将量子点标记后的iPSCs继续培养,分别于1次传代后1周和2次传代后1周观察荧光标记的稳定性.结果 倒置显微镜下观察可见培养和扩增的iPSCs呈克隆样生长,免疫荧光检测显示iPSCs中OCT4和Nanog两种干细胞标志蛋白均阳性表达.5.0、7.5、10.0 nmol/L三种浓度的量子点标记后均可见iPSCs中的橘红色荧光,随着量子点浓度的增加,iPSCs中标记的橘红色颗粒明显增加,10.0 nmol/L量子点标记后iPSCs中的橘红色荧光最强,荧光颗粒最多.iPSCs 2次传代1周后仍可观察到量子点的橘红色荧光,但与传代前和1次传代后1周相比荧光强度减弱.结论 量子点标记技术可示踪iPSCs,其荧光标记效果呈浓度依赖性.本研究实验条件下量子点标记的荧光至少可维持2周.
揹景 近年來,誘導多能榦細胞(iPSCs)的多嚮分化特性為各種臨床疾病的細胞替代治療提供瞭新的種子來源,在眼科領域,iPSCs已成為人眼變性類疾病髮病機製研究的良好模型,而更好的iPSCs識彆和鑒定技術是對其體內生物學特性進行研究的基礎.目的 探討量子點標記人iPSCs的可行性及其最佳濃度,併觀察量子點標記人iPSCs的光穩定性,為iPSCs應用于眼部細胞移植的實驗研究和臨床研究提供一種新的示蹤技術.方法 用無飼養層細胞培養法培養和擴增臍帶間充質基質細胞-人iPSCs繫,併用免疫熒光法對其榦細胞特性進行鑒定；選取不同濃度CdSe/ZnS覈殼結構的量子點對擴增培養的iPSCs進行標記,用三維去捲積實時活細胞成像繫統觀察標記效果.將量子點標記後的iPSCs繼續培養,分彆于1次傳代後1週和2次傳代後1週觀察熒光標記的穩定性.結果 倒置顯微鏡下觀察可見培養和擴增的iPSCs呈剋隆樣生長,免疫熒光檢測顯示iPSCs中OCT4和Nanog兩種榦細胞標誌蛋白均暘性錶達.5.0、7.5、10.0 nmol/L三種濃度的量子點標記後均可見iPSCs中的橘紅色熒光,隨著量子點濃度的增加,iPSCs中標記的橘紅色顆粒明顯增加,10.0 nmol/L量子點標記後iPSCs中的橘紅色熒光最彊,熒光顆粒最多.iPSCs 2次傳代1週後仍可觀察到量子點的橘紅色熒光,但與傳代前和1次傳代後1週相比熒光彊度減弱.結論 量子點標記技術可示蹤iPSCs,其熒光標記效果呈濃度依賴性.本研究實驗條件下量子點標記的熒光至少可維持2週.
배경 근년래,유도다능간세포(iPSCs)적다향분화특성위각충림상질병적세포체대치료제공료신적충자래원,재안과영역,iPSCs이성위인안변성류질병발병궤제연구적량호모형,이경호적iPSCs식별화감정기술시대기체내생물학특성진행연구적기출.목적 탐토양자점표기인iPSCs적가행성급기최가농도,병관찰양자점표기인iPSCs적광은정성,위iPSCs응용우안부세포이식적실험연구화림상연구제공일충신적시종기술.방법 용무사양층세포배양법배양화확증제대간충질기질세포-인iPSCs계,병용면역형광법대기간세포특성진행감정；선취불동농도CdSe/ZnS핵각결구적양자점대확증배양적iPSCs진행표기,용삼유거권적실시활세포성상계통관찰표기효과.장양자점표기후적iPSCs계속배양,분별우1차전대후1주화2차전대후1주관찰형광표기적은정성.결과 도치현미경하관찰가견배양화확증적iPSCs정극륭양생장,면역형광검측현시iPSCs중OCT4화Nanog량충간세포표지단백균양성표체.5.0、7.5、10.0 nmol/L삼충농도적양자점표기후균가견iPSCs중적귤홍색형광,수착양자점농도적증가,iPSCs중표기적귤홍색과립명현증가,10.0 nmol/L양자점표기후iPSCs중적귤홍색형광최강,형광과립최다.iPSCs 2차전대1주후잉가관찰도양자점적귤홍색형광,단여전대전화1차전대후1주상비형광강도감약.결론 양자점표기기술가시종iPSCs,기형광표기효과정농도의뢰성.본연구실험조건하양자점표기적형광지소가유지2주.
Background The multipotent differentiation features of induced pluripotent stem cells (iPSCs) offer a new option for cell replacement therapy of many clinical diseases.In ophthalmology,iPSCs are a good model in studying the pathogenic mechanism of degenerative ocular diseases.A better identification method for iPSCs is critical for analyzing the in vivo biological characteristics of iPSCs.Objective This study was to investigate the feasibility and stability of labeling iPSCs with quantum dots.Methods Human umbilical mesenchymal stromal cells-iPSC lines were cultured and amplified on matrigel,and the characteristics of iPSCs were evaluated by immunofluorescence.Different concentrations (5.0,7.5 and 10.0 nmol/L) of quantum dots with a CdSe/ZnS nuclear shell structure were used to label iPSCs after passaging and proliferation.The labeling outcome was observed with a three-dimensional deconvolution real-time live cells imaging system.The labeled iPSCs were subsequently cultivated,and then changes in fluorescence intensity were examined 7 days after the first and the second passaging of iPSCs.Results iPSCs were observed to grow in a clonal manner under the inverted microscope.The iPSC markers,OCT4 and Nanog,were detected by immunofluorescence.With increasing concentrations of quantum dots,the fluorescence intensities representing the levels of OCT4 and Nanog in iPSCs were gradually elevated,with optimal levels of fluorescence observed at a concentration of 10 nmol/L of quantum dots.The fluorescent labeling of OCT4 and Nanog in iPSCs remained and weakened gradually till day 7 even after the second passage.Conclusions Quantum dots labeling could be used to track iPSCs in a dose-independent manner.The fluorescent signal from the quantum dots labeling the iPSCs lasts 2 weeks at least.
