CAJ | 학술논문

背景目前角膜移植手术是治疗严重角膜病变的主要方法,角膜供体来源的匮乏限制该疗法的应用.组织工程角膜为角膜疾病的治疗开辟了新的途径. 目的探讨人羊膜间充质干细胞(hAMSCs)构建组织工程角膜上皮层的可能性. 方法新鲜羊膜组织剪成6 cm×6 cm大小的组织块后用胰蛋白酶+EDTA消化液消化,将人羊膜上皮细胞(AECs)刮除干净,然后将羊膜组织剪碎,用胶原酶Ⅱ进行消化,分离原代hAMSCs并进行培养.分离新西兰白兔的角膜基质片后按随机数字表法分为2个组,实验组将培养至第3代的hAMSCs以1×105/ml的密度种植于去角膜上皮细胞的兔角膜基质片上进行培养,空白对照组为不种植细胞的空白角膜基质片.待细胞达80％～ 90％融合时再将兔角膜基质片移至插入式培养皿中进行气液界面诱导分化培养,培养至14 d时将兔角膜基质片在质量分数4％多聚甲醛中固定,制备组织切片,行苏木精-伊红染色,观察兔角膜基质片的形态.采用免疫组织化学染色和免疫荧光技术检测兔角膜基质片上诱导分化的hAMSCs中细胞角蛋白3(CK3)和CK12的表达. 结果实验组hAMSCs种植于去角膜上皮细胞的兔角膜基质片上气液界面培养14d后可形成3～5层的复层细胞,可见兔角膜基底膜的hAMSCs细胞核呈椭圆形,表层的hAMSCs细胞核呈长梭形,形态与正常角膜上皮层相似;而空白对照组兔角膜基质上无细胞生长.免疫组织化学染色显示兔角膜基底膜上的hAMSCs对CK3、CK12呈阳性反应,为细胞质中棕黄色染色,而阴性对照组未见CK3、CK12染色.免疫荧光检测显示兔角膜基底膜上的hAMSCs细胞质中可见CK3、CK12呈绿色荧光,而阴性对照片细胞质荧光缺失.结论 hAMSCs在兔角膜基质片气液界面上可成功诱导分化为角膜样上皮细胞.
배경 목전각막이식수술시치료엄중각막병변적주요방법,각막공체래원적궤핍한제해요법적응용.조직공정각막위각막질병적치료개벽료신적도경. 목적 탐토인양막간충질간세포(hAMSCs)구건조직공정각막상피층적가능성. 방법 신선양막조직전성6 cm×6 cm대소적조직괴후용이단백매+EDTA소화액소화,장인양막상피세포(AECs)괄제간정,연후장양막조직전쇄,용효원매Ⅱ진행소화,분리원대hAMSCs병진행배양.분리신서란백토적각막기질편후안수궤수자표법분위2개조,실험조장배양지제3대적hAMSCs이1×105/ml적밀도충식우거각막상피세포적토각막기질편상진행배양,공백대조조위불충식세포적공백각막기질편.대세포체80％～ 90％융합시재장토각막기질편이지삽입식배양명중진행기액계면유도분화배양,배양지14 d시장토각막기질편재질량분수4％다취갑철중고정,제비조직절편,행소목정-이홍염색,관찰토각막기질편적형태.채용면역조직화학염색화면역형광기술검측토각막기질편상유도분화적hAMSCs중세포각단백3(CK3)화CK12적표체. 결과 실험조hAMSCs충식우거각막상피세포적토각막기질편상기액계면배양14d후가형성3～5층적복층세포,가견토각막기저막적hAMSCs세포핵정타원형,표층적hAMSCs세포핵정장사형,형태여정상각막상피층상사;이공백대조조토각막기질상무세포생장.면역조직화학염색현시토각막기저막상적hAMSCs대CK3、CK12정양성반응,위세포질중종황색염색,이음성대조조미견CK3、CK12염색.면역형광검측현시토각막기저막상적hAMSCs세포질중가견CK3、CK12정록색형광,이음성대조편세포질형광결실.결론 hAMSCs재토각막기질편기액계면상가성공유도분화위각막양상피세포.
Background Corneal transplantation is an effective treatment to severe corneal diseases,but the shortage of cornea donor limits its application.Tissue-engineered cornea is being a new approach to corneal diseases.Objective This study was to investigate the possibility of construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells (hAMSCs) in vitro.Methods Fresh human amniotic membranes were obtained under the approval of Ethic Committee of Affiliated First Hospital of Jinan University and informed consent of maternal women.The 6 cm×6 cm amniotic membrane tissue explant was digested using trypsin+ EDTA,and then the amniotic epithelial cells (AECs) were scraped before putting into collagenase Ⅱ digestion medium to isolate hAMSCs.hAMSCs of passage 3 were cultured to achive 80％-90％ confluence,and then the ceils were incubated on rabbit deepithelial corneal stroma at a 1 ×105/ml density.The corneal stroma was co-cuhured with hAMSCs at an air-liquid interface till 14 days.Rabbit deepithelial corneal stroma with and without hAMSCs (experimental group and control group) were fixed in 4％ para formaldehyde, and sections were prepared for histopathological examination.Immunochemistry and immunofluorescence were empoyed to detect the expressions of cytokeratin3 (CK3) and CK12 in hAMSCs.Results hAMSCs grew well and formed a stratified epidermal structure resembling native corneal epithelium on rabbit corneal stroma in cultured 14 days in the experimental group,with the oval nucleus at basement and fusiform nucleus on the surface of corneal stroma.There was no cell structure in the control group.Immunochemistry revealed brown staining for CK3, CK12 in cytoplasm of hAMSCs on the rabbits corneal stroma,and the green fluorescence for CK3 and CK12 was also seen in the hAMSCs.However,the response for CK3 and CK12 was absent in the control sections either immunochemistry or immunofluorescence test.Conclusions hAMSCs can be induced to differentiate into corneal epithelioid cells at an air-liquid interface on the rabbit corneal stroma.