中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
10期
882-887
,共6页
徐舒怡%侯光辉%吴静%徐锦堂
徐舒怡%侯光輝%吳靜%徐錦堂
서서이%후광휘%오정%서금당
人脐带间充质干细胞%猪角膜基质%组织工程角膜%上皮%细胞分化
人臍帶間充質榦細胞%豬角膜基質%組織工程角膜%上皮%細胞分化
인제대간충질간세포%저각막기질%조직공정각막%상피%세포분화
Human umbilical cord mesenchymal stem cell%Porcine corneal matrix%Tissue engineered cornea%Epithelium%Cell differentiation
背景 眼表疾病导致的角膜盲已成为全球致盲性角膜疾病中的主要原因之一.随着组织工程技术的发展和进步,组织工程角膜为眼表疾病的治疗开辟了新的途径. 目的 观察体外培养的人脐带间充质干细胞(UC-MSCs)移植到兔角膜基质后的分化发育情况,探讨人UC-MSCs分化为角膜上皮细胞以及治疗兔角膜损伤的可行性.方法 获取人脐带组织,采用Ⅳ型胶原酶消化法分离纯化人UC-MSCs并传代,取第3代细胞用于扩增和实验.流式细胞仪检测细胞的免疫表型及诱导成骨分化鉴定.24只新西兰大白兔按随机数字表法随机分为2个组,将人UC-MSCs接种于去上皮的猪角膜基质上,培养4d后行实验组兔左眼板层角膜移植；对照组以相同的手术方法单纯移植去上皮猪角膜基质.术后对角膜定期行活体激光共焦显微镜检查,并分别于术后2、4、8周摘除各组实验眼行组织病理学和免疫荧光检查,评价移植到兔角膜基质的人UCMSCs的存活、分化以及移植局部的反应等；应用免疫荧光技术检测移植后角膜上皮细胞中角蛋白3(CK3)、CK12以及转运蛋白G超家族成员(ABCG2)的表达. 结果 消化培养的人UC-MSCs呈圆形,细胞胞体较大,贴壁后细胞呈长梭形.培养获得的人UC-MSCs的细胞表型CD105 +/CD29+/CD44+/CD34-/CD45-,并可诱导分化为成骨细胞.实验组人UC-MSCs接种到去上皮猪角膜基质后贴附良好、生长迅速,术后植片在植床上存活良好,种植了人UC-MSCs的去上皮猪角膜移植到兔眼,可见实验组受体角膜较对照组透明,未见明显新生血管,在活体共焦显微镜下可见新生的角膜上皮样细胞,未发生免疫排斥反应.免疫荧光检测可见在重建的角膜上皮层检测到CK3及CK12的阳性表达,而未见ABCG2的表达. 结论 将种植了人UC-MSCs的猪角膜基质移植到损伤的兔角膜后,人UC-MSCs可以存活、增生并分化为角膜上皮样细胞,可用于修复甚至重建损伤的角膜表层.
揹景 眼錶疾病導緻的角膜盲已成為全毬緻盲性角膜疾病中的主要原因之一.隨著組織工程技術的髮展和進步,組織工程角膜為眼錶疾病的治療開闢瞭新的途徑. 目的 觀察體外培養的人臍帶間充質榦細胞(UC-MSCs)移植到兔角膜基質後的分化髮育情況,探討人UC-MSCs分化為角膜上皮細胞以及治療兔角膜損傷的可行性.方法 穫取人臍帶組織,採用Ⅳ型膠原酶消化法分離純化人UC-MSCs併傳代,取第3代細胞用于擴增和實驗.流式細胞儀檢測細胞的免疫錶型及誘導成骨分化鑒定.24隻新西蘭大白兔按隨機數字錶法隨機分為2箇組,將人UC-MSCs接種于去上皮的豬角膜基質上,培養4d後行實驗組兔左眼闆層角膜移植；對照組以相同的手術方法單純移植去上皮豬角膜基質.術後對角膜定期行活體激光共焦顯微鏡檢查,併分彆于術後2、4、8週摘除各組實驗眼行組織病理學和免疫熒光檢查,評價移植到兔角膜基質的人UCMSCs的存活、分化以及移植跼部的反應等；應用免疫熒光技術檢測移植後角膜上皮細胞中角蛋白3(CK3)、CK12以及轉運蛋白G超傢族成員(ABCG2)的錶達. 結果 消化培養的人UC-MSCs呈圓形,細胞胞體較大,貼壁後細胞呈長梭形.培養穫得的人UC-MSCs的細胞錶型CD105 +/CD29+/CD44+/CD34-/CD45-,併可誘導分化為成骨細胞.實驗組人UC-MSCs接種到去上皮豬角膜基質後貼附良好、生長迅速,術後植片在植床上存活良好,種植瞭人UC-MSCs的去上皮豬角膜移植到兔眼,可見實驗組受體角膜較對照組透明,未見明顯新生血管,在活體共焦顯微鏡下可見新生的角膜上皮樣細胞,未髮生免疫排斥反應.免疫熒光檢測可見在重建的角膜上皮層檢測到CK3及CK12的暘性錶達,而未見ABCG2的錶達. 結論 將種植瞭人UC-MSCs的豬角膜基質移植到損傷的兔角膜後,人UC-MSCs可以存活、增生併分化為角膜上皮樣細胞,可用于脩複甚至重建損傷的角膜錶層.
배경 안표질병도치적각막맹이성위전구치맹성각막질병중적주요원인지일.수착조직공정기술적발전화진보,조직공정각막위안표질병적치료개벽료신적도경. 목적 관찰체외배양적인제대간충질간세포(UC-MSCs)이식도토각막기질후적분화발육정황,탐토인UC-MSCs분화위각막상피세포이급치료토각막손상적가행성.방법 획취인제대조직,채용Ⅳ형효원매소화법분리순화인UC-MSCs병전대,취제3대세포용우확증화실험.류식세포의검측세포적면역표형급유도성골분화감정.24지신서란대백토안수궤수자표법수궤분위2개조,장인UC-MSCs접충우거상피적저각막기질상,배양4d후행실험조토좌안판층각막이식；대조조이상동적수술방법단순이식거상피저각막기질.술후대각막정기행활체격광공초현미경검사,병분별우술후2、4、8주적제각조실험안행조직병이학화면역형광검사,평개이식도토각막기질적인UCMSCs적존활、분화이급이식국부적반응등；응용면역형광기술검측이식후각막상피세포중각단백3(CK3)、CK12이급전운단백G초가족성원(ABCG2)적표체. 결과 소화배양적인UC-MSCs정원형,세포포체교대,첩벽후세포정장사형.배양획득적인UC-MSCs적세포표형CD105 +/CD29+/CD44+/CD34-/CD45-,병가유도분화위성골세포.실험조인UC-MSCs접충도거상피저각막기질후첩부량호、생장신속,술후식편재식상상존활량호,충식료인UC-MSCs적거상피저각막이식도토안,가견실험조수체각막교대조조투명,미견명현신생혈관,재활체공초현미경하가견신생적각막상피양세포,미발생면역배척반응.면역형광검측가견재중건적각막상피층검측도CK3급CK12적양성표체,이미견ABCG2적표체. 결론 장충식료인UC-MSCs적저각막기질이식도손상적토각막후,인UC-MSCs가이존활、증생병분화위각막상피양세포,가용우수복심지중건손상적각막표층.
Background Corneal blindness caused by ocular surface disease is one of the main reasons for the global blinding corneal diseases.With the development and progress of tissue engineering technology,tissueengineered cornea offers a new approach to the treatment of ocular surface disease.Objective This study was to obscrve the growth and differentiation of human umbilical cord mesenchymal stem cclls (UC-MSCs) on thc corneal stroma of receipts and investigate the feasibility of human UC-MSCs differentiated into corneal epithelium-like cells and the reparation of injury cornea.Methods Human UC-MSCs were isolated from human umbilical cord using collagenase Ⅳ digestion and passaged in DMEM/F12 containing fetal bovine serum in vitro.The immunophenotype of cultured human UC-MSCs was evaluated by flow cytometry.The differentiated osteoblasts from the human UC-MSCs by directional induce was identified.Twenty-four New Zealand albino rabbits were randomly divided into 2 groups.The human UC-MSCs were cultured on porcine corneal matrix without corneal epithelium for 4 days and then transplanted onto the 12 left eyes of 12 New Zealand albino rabbits,and porcine corneal matrix without corneal epithelium was transplanted onto the left eyes of other 12 New Zealand albino rabbits as control group.The rabbits received keratoplasty were examined using in vivo confocal microscope through focusing(CMTF).The eyeballs were taken off after 2,4 and 8 weeks,the growth and differentiation,expression of cytokeratin 3 (CK3),CK12 and ATP-binding cassette superfamily G memben 2 (ABCG2)of human UC-MSCs were observed by histopathology and immunofluorescence staining.This use of the experimental animals complied with ARVO Statement.Results Digestive human UCMSCs formed round in shape and was large in size.The attached cells displayed long-fusiform shape like fibroblasts.The cultured human UC-MSCs phenotype was CD105+/CD29+/CD44+/CD34-/CD45-and could be induced toward osteoblast differentiation under the appropriate experimental conditions.Human UC-MSCs grew well on the porcine corneal matrix.The corneal grafts survived wcll without rejection till the experiment end in experimental eyes,but the rejection of corneal graft occurred in control eyes.Confocal microscope could observe corneal epithelium-like cells.The corneal epithelium cells showed the positive response for CK3 and CK12 and absent response for ABCG2.Conclusions Human UC-MSCs with porcine corneal matrix can survive,proliferate and differentiate into corneal epithelium-like cells after transplanting onto the corneal stroma of rabbits.This result suggests that human UC-MSCs is able to repair and reconstruct the injured corneal surfaces.
