中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
9期
797-801
,共5页
文晔%沙翔垠%宋莉%刘志平%彭娟%谢莉菲
文曄%沙翔垠%宋莉%劉誌平%彭娟%謝莉菲
문엽%사상은%송리%류지평%팽연%사리비
人羊膜间充质干细胞%共培养%诱导%分化%眼表细胞
人羊膜間充質榦細胞%共培養%誘導%分化%眼錶細胞
인양막간충질간세포%공배양%유도%분화%안표세포
Human amnion mesenchymal stem cell%Co-culture%Induce%Differentiation%Ocular surface cell
背景 人羊膜间充质干细胞(hAMSCs)在体外能诱导分化为多种体细胞,但目前有关hAMSCs分化为眼表细胞的研究鲜有报道. 目的 探讨体外共培养诱导hAMSCs向眼表细胞分化的可能性及其分化机制.方法 本研究经广州医科大学第二附属医院伦理委员会批准,在健康产妇的知情同意下从人胎盘中分离hAMSCs并进行培养,采用流式细胞术计数分析CD44、CD45、CD73、CD90在培养细胞中的表达以鉴定细胞,此外通过成骨诱导分化实验和成脂诱导分化实验对培养细胞进行体外分化鉴定.在患者知情同意下获取眼科手术中弃用的人眼球结膜组织,用组织块培养法分离和培养人球结膜成纤维细胞(hBCFs),将hAMSCs与hBCFs在Transwell培养体系中进行共培养,分为hAMSCs培养组和hAMSCs与hBCFs共培养组,用含质量分数5％胎牛血清(FBS)的DMEM高糖/F12培养基培养7d,采用免疫荧光技术检测hAMSCs中对上皮细胞角蛋白19(CK19)的表达,评价hAMSCs向眼表细胞分化的程度,并检测hAMSCs中α-平滑肌肌动蛋白(α-SMA)的表达,评价其分化过程与间质上皮化转换(MET)过程的关系.结果 传代至3～7代的hAMSCs均为细长形,但随着传代增加,细胞体积增大,细胞中CD44、CD73、CD90表达呈强阳性,但不表达CD45.经成骨或成脂诱导分化后3～4周,细胞中茜素红S(ARS)染色或油红O染色阳性.hAMSCs与hBCFs共培养后1周,hAMSCs形态由细长形变为类上皮细胞型,共培养组的部分细胞中CK19表达阳性,所有细胞α-SMA表达呈微弱阳性,而hAMSCs细胞培养组可见呈绿色荧光的α-SMA阳性细胞,但不表达CK19. 结论 通过体外共培养可诱导hAMSCs向人眼表细胞分化,其分化机制可能与MET过程相关.
揹景 人羊膜間充質榦細胞(hAMSCs)在體外能誘導分化為多種體細胞,但目前有關hAMSCs分化為眼錶細胞的研究鮮有報道. 目的 探討體外共培養誘導hAMSCs嚮眼錶細胞分化的可能性及其分化機製.方法 本研究經廣州醫科大學第二附屬醫院倫理委員會批準,在健康產婦的知情同意下從人胎盤中分離hAMSCs併進行培養,採用流式細胞術計數分析CD44、CD45、CD73、CD90在培養細胞中的錶達以鑒定細胞,此外通過成骨誘導分化實驗和成脂誘導分化實驗對培養細胞進行體外分化鑒定.在患者知情同意下穫取眼科手術中棄用的人眼毬結膜組織,用組織塊培養法分離和培養人毬結膜成纖維細胞(hBCFs),將hAMSCs與hBCFs在Transwell培養體繫中進行共培養,分為hAMSCs培養組和hAMSCs與hBCFs共培養組,用含質量分數5％胎牛血清(FBS)的DMEM高糖/F12培養基培養7d,採用免疫熒光技術檢測hAMSCs中對上皮細胞角蛋白19(CK19)的錶達,評價hAMSCs嚮眼錶細胞分化的程度,併檢測hAMSCs中α-平滑肌肌動蛋白(α-SMA)的錶達,評價其分化過程與間質上皮化轉換(MET)過程的關繫.結果 傳代至3～7代的hAMSCs均為細長形,但隨著傳代增加,細胞體積增大,細胞中CD44、CD73、CD90錶達呈彊暘性,但不錶達CD45.經成骨或成脂誘導分化後3～4週,細胞中茜素紅S(ARS)染色或油紅O染色暘性.hAMSCs與hBCFs共培養後1週,hAMSCs形態由細長形變為類上皮細胞型,共培養組的部分細胞中CK19錶達暘性,所有細胞α-SMA錶達呈微弱暘性,而hAMSCs細胞培養組可見呈綠色熒光的α-SMA暘性細胞,但不錶達CK19. 結論 通過體外共培養可誘導hAMSCs嚮人眼錶細胞分化,其分化機製可能與MET過程相關.
배경 인양막간충질간세포(hAMSCs)재체외능유도분화위다충체세포,단목전유관hAMSCs분화위안표세포적연구선유보도. 목적 탐토체외공배양유도hAMSCs향안표세포분화적가능성급기분화궤제.방법 본연구경엄주의과대학제이부속의원윤리위원회비준,재건강산부적지정동의하종인태반중분리hAMSCs병진행배양,채용류식세포술계수분석CD44、CD45、CD73、CD90재배양세포중적표체이감정세포,차외통과성골유도분화실험화성지유도분화실험대배양세포진행체외분화감정.재환자지정동의하획취안과수술중기용적인안구결막조직,용조직괴배양법분리화배양인구결막성섬유세포(hBCFs),장hAMSCs여hBCFs재Transwell배양체계중진행공배양,분위hAMSCs배양조화hAMSCs여hBCFs공배양조,용함질량분수5％태우혈청(FBS)적DMEM고당/F12배양기배양7d,채용면역형광기술검측hAMSCs중대상피세포각단백19(CK19)적표체,평개hAMSCs향안표세포분화적정도,병검측hAMSCs중α-평활기기동단백(α-SMA)적표체,평개기분화과정여간질상피화전환(MET)과정적관계.결과 전대지3～7대적hAMSCs균위세장형,단수착전대증가,세포체적증대,세포중CD44、CD73、CD90표체정강양성,단불표체CD45.경성골혹성지유도분화후3～4주,세포중천소홍S(ARS)염색혹유홍O염색양성.hAMSCs여hBCFs공배양후1주,hAMSCs형태유세장형변위류상피세포형,공배양조적부분세포중CK19표체양성,소유세포α-SMA표체정미약양성,이hAMSCs세포배양조가견정록색형광적α-SMA양성세포,단불표체CK19. 결론 통과체외공배양가유도hAMSCs향인안표세포분화,기분화궤제가능여MET과정상관.
Background Recent studies indicated that human amnion mesenchymal stem cells (hAMSCs) can be induced to differentiate into multiple types of cells in vitro,but whether the hAMSCs can differentiate into ocular surface cells has not been reported yet.Objective This study was to investigate the feasibility of inducing differentiation of hAMSCs into ocular surface cells by co-culturing with human bulbar conjunctiva fibroblasts (hBCFs).Methods This study was approved by Ethic Committee of Affiliated Second Hospital of Guangzhou Medical University.HAMSCs were isolated from placenta under the informed consent of healthy delivery women.hAMSCs were cultured,passaged and identified by detecting the expressions of CD44,CD45,CD73,CD90 in the cells with flow cytometer,osteogenesis and adipogenic differentiation experiments.Human conjunctival tissue was obtained during the eye operation under the informed consent of patients and hBCFs were isolated and cultured with explant culture.The cells were divided into the hAMSCs culture group and the hAMSCs and hBCFs co-culture group and cultivated in Transwell chambers for 7 days.The expressions of cytokeratin 19 (CK19) and α-smooth muscle actin (α-SMA) in the cells were assayed by immnofluorescence technique.Results Cultured hAMSCs showed the slender shape and cell body enlarged with passage.CD44,CD73 and CD90 were expressed in the cells,and the expression of CD45 was absent.After 3-4 weeks of osteogenesis and adipogenic induce,the cells showed red staining for alizarin and oil red O.In the co-culture group of hAMSCs and hBCFs,hAMSCs presented the epithelioid cell-like in shape and showed the positive response for CK19 and weaker response for α-SMA.However,in the hAMSCs culture group,the cells showed the positive response for α-SMA and absent response for CK19.Conclusions The hAMSCs can differentiate into ocular surface cells after being induced by hBCFs.And the differentiation mechanism is possibly relevant to mesenchymal cells epithelium.
