中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
Chinese Journal of Experimental Ophthalmology
2015年
9期
787-792
,共6页
王瑶%段豪云%杨玲玲%曲明俐%周庆军
王瑤%段豪雲%楊玲玲%麯明俐%週慶軍
왕요%단호운%양령령%곡명리%주경군
Rho相关激酶/拮抗剂&抑制剂%细胞培养%角膜缘/细胞学%干细胞%角膜上皮%细胞生存/药物作用%角膜保存%兔
Rho相關激酶/拮抗劑&抑製劑%細胞培養%角膜緣/細胞學%榦細胞%角膜上皮%細胞生存/藥物作用%角膜保存%兔
Rho상관격매/길항제&억제제%세포배양%각막연/세포학%간세포%각막상피%세포생존/약물작용%각막보존%토
Rho-associated kinases/antagonists & inhibitors%Cells cultured%Limbus cornea/cytology%Stem cells%Epithelium,cornea%Cell survival/drug effects%Cornea preservation%Rabbit
背景 角膜缘干细胞(LSCs)对维持角膜上皮的稳定和角膜组织透明性有重要作用.Rho关联卷曲螺旋蛋白激酶(ROCK)抑制剂Y-27632能够促进人胚胎干细胞、角质上皮细胞等的增生,减少细胞凋亡.目的 探讨Y-27632对离体兔角膜保存和体外LSCs扩增能力的影响. 方法 在细胞培养基MEM中加入质量分数12.5％硫酸软骨素、质量分数10.0％低分子右旋糖酐、20.0 mg/L地塞米松、100 mg/L妥布霉素注射液、9.5 g/L Hepes,使用时添加0.375 mg/L L-谷氨酰胺,制备成角膜活性保存液.将新西兰大白兔的角膜组织片分别置于含或不含Y-27632的角膜活性保存液中保存4、7、14d,采用质量分数0.25％锥虫蓝和质量分数0.2％茜素红染色法观察角膜内皮细胞密度及形态,采用Giemsa染色法并使用Image J图像分析软件计数克隆球数量和LSCs活性,计算LSCs存活率和克隆形成效率. 结果 角膜片保存4d时,Y-27632保存液组和单纯角膜中期保存液组角膜内皮细胞形态无明显差别,角膜片保存7d时,Y-27632保存液组角膜内皮细胞形态仍保持规则的六边形,而单纯角膜中期保存液组角膜内皮细胞的细胞膜轻微皱缩,少数细胞体积变大.角膜片保存14 d时单纯角膜中期保存液组角膜内皮细胞可见较多的茜素红斑.单纯角膜中期保存液组角膜内皮细胞计数为(2 262±75)/mm2,Y-27632保存液组角膜内皮细胞计数为(2 425 ±95)/mm2,差异有统计学意义(P＜0.001).新鲜角膜片和角膜片保存4d时,Y-27632保存液组和单纯角膜中期保存液组LSCs的克隆球均较大,其内细胞数较多,而保存7d和14 d时,与Y-27632保存液组比较,单纯角膜中期保存液组LSCs克隆球直径明显缩小.新鲜分离的角膜片和角膜片保存4d时,Y-27632保存液组与单纯角膜中期保存液组LSCs的克隆形成率和角膜上皮细胞存活率的差异均无统计学意义(均P＞0.05);角膜片保存7d和14d时,角膜缘上皮细胞的活性率分别为(73.00±2.12)％和(56.00±0.71)％,明显高于单纯角膜中期培养液组的(66.00±4.00)％和(49.00±0.71)％,差异均有统计学意义(t=3.098,P=0.018;t=9.798,P=0.000);角膜片保存7d和14 d时,Y-27632保存液组与单纯角膜中期保存液组LSCs的克隆形成效率分别为(11.05±0.21)％和(3.10±1.97)％,明显高于单纯角膜中期保存液组中的(2.05±1.20)％和(0.40±0.14)％,差异均有统计学意义(t=18.107,P=0.000;t=3.184,P=0.017).结论角膜保存液中添加Y-27632可明显提高离体角膜保存的效果,同时可维持LSCs的活性及克隆形成能力.Y-27632可作为角膜保存液的有效添加成分.
揹景 角膜緣榦細胞(LSCs)對維持角膜上皮的穩定和角膜組織透明性有重要作用.Rho關聯捲麯螺鏇蛋白激酶(ROCK)抑製劑Y-27632能夠促進人胚胎榦細胞、角質上皮細胞等的增生,減少細胞凋亡.目的 探討Y-27632對離體兔角膜保存和體外LSCs擴增能力的影響. 方法 在細胞培養基MEM中加入質量分數12.5％硫痠軟骨素、質量分數10.0％低分子右鏇糖酐、20.0 mg/L地塞米鬆、100 mg/L妥佈黴素註射液、9.5 g/L Hepes,使用時添加0.375 mg/L L-穀氨酰胺,製備成角膜活性保存液.將新西蘭大白兔的角膜組織片分彆置于含或不含Y-27632的角膜活性保存液中保存4、7、14d,採用質量分數0.25％錐蟲藍和質量分數0.2％茜素紅染色法觀察角膜內皮細胞密度及形態,採用Giemsa染色法併使用Image J圖像分析軟件計數剋隆毬數量和LSCs活性,計算LSCs存活率和剋隆形成效率. 結果 角膜片保存4d時,Y-27632保存液組和單純角膜中期保存液組角膜內皮細胞形態無明顯差彆,角膜片保存7d時,Y-27632保存液組角膜內皮細胞形態仍保持規則的六邊形,而單純角膜中期保存液組角膜內皮細胞的細胞膜輕微皺縮,少數細胞體積變大.角膜片保存14 d時單純角膜中期保存液組角膜內皮細胞可見較多的茜素紅斑.單純角膜中期保存液組角膜內皮細胞計數為(2 262±75)/mm2,Y-27632保存液組角膜內皮細胞計數為(2 425 ±95)/mm2,差異有統計學意義(P＜0.001).新鮮角膜片和角膜片保存4d時,Y-27632保存液組和單純角膜中期保存液組LSCs的剋隆毬均較大,其內細胞數較多,而保存7d和14 d時,與Y-27632保存液組比較,單純角膜中期保存液組LSCs剋隆毬直徑明顯縮小.新鮮分離的角膜片和角膜片保存4d時,Y-27632保存液組與單純角膜中期保存液組LSCs的剋隆形成率和角膜上皮細胞存活率的差異均無統計學意義(均P＞0.05);角膜片保存7d和14d時,角膜緣上皮細胞的活性率分彆為(73.00±2.12)％和(56.00±0.71)％,明顯高于單純角膜中期培養液組的(66.00±4.00)％和(49.00±0.71)％,差異均有統計學意義(t=3.098,P=0.018;t=9.798,P=0.000);角膜片保存7d和14 d時,Y-27632保存液組與單純角膜中期保存液組LSCs的剋隆形成效率分彆為(11.05±0.21)％和(3.10±1.97)％,明顯高于單純角膜中期保存液組中的(2.05±1.20)％和(0.40±0.14)％,差異均有統計學意義(t=18.107,P=0.000;t=3.184,P=0.017).結論角膜保存液中添加Y-27632可明顯提高離體角膜保存的效果,同時可維持LSCs的活性及剋隆形成能力.Y-27632可作為角膜保存液的有效添加成分.
배경 각막연간세포(LSCs)대유지각막상피적은정화각막조직투명성유중요작용.Rho관련권곡라선단백격매(ROCK)억제제Y-27632능구촉진인배태간세포、각질상피세포등적증생,감소세포조망.목적 탐토Y-27632대리체토각막보존화체외LSCs확증능력적영향. 방법 재세포배양기MEM중가입질량분수12.5％류산연골소、질량분수10.0％저분자우선당항、20.0 mg/L지새미송、100 mg/L타포매소주사액、9.5 g/L Hepes,사용시첨가0.375 mg/L L-곡안선알,제비성각막활성보존액.장신서란대백토적각막조직편분별치우함혹불함Y-27632적각막활성보존액중보존4、7、14d,채용질량분수0.25％추충람화질량분수0.2％천소홍염색법관찰각막내피세포밀도급형태,채용Giemsa염색법병사용Image J도상분석연건계수극륭구수량화LSCs활성,계산LSCs존활솔화극륭형성효솔. 결과 각막편보존4d시,Y-27632보존액조화단순각막중기보존액조각막내피세포형태무명현차별,각막편보존7d시,Y-27632보존액조각막내피세포형태잉보지규칙적륙변형,이단순각막중기보존액조각막내피세포적세포막경미추축,소수세포체적변대.각막편보존14 d시단순각막중기보존액조각막내피세포가견교다적천소홍반.단순각막중기보존액조각막내피세포계수위(2 262±75)/mm2,Y-27632보존액조각막내피세포계수위(2 425 ±95)/mm2,차이유통계학의의(P＜0.001).신선각막편화각막편보존4d시,Y-27632보존액조화단순각막중기보존액조LSCs적극륭구균교대,기내세포수교다,이보존7d화14 d시,여Y-27632보존액조비교,단순각막중기보존액조LSCs극륭구직경명현축소.신선분리적각막편화각막편보존4d시,Y-27632보존액조여단순각막중기보존액조LSCs적극륭형성솔화각막상피세포존활솔적차이균무통계학의의(균P＞0.05);각막편보존7d화14d시,각막연상피세포적활성솔분별위(73.00±2.12)％화(56.00±0.71)％,명현고우단순각막중기배양액조적(66.00±4.00)％화(49.00±0.71)％,차이균유통계학의의(t=3.098,P=0.018;t=9.798,P=0.000);각막편보존7d화14 d시,Y-27632보존액조여단순각막중기보존액조LSCs적극륭형성효솔분별위(11.05±0.21)％화(3.10±1.97)％,명현고우단순각막중기보존액조중적(2.05±1.20)％화(0.40±0.14)％,차이균유통계학의의(t=18.107,P=0.000;t=3.184,P=0.017).결론각막보존액중첨가Y-27632가명현제고리체각막보존적효과,동시가유지LSCs적활성급극륭형성능력.Y-27632가작위각막보존액적유효첨가성분.
Background Limbal stem cells (LSCs) play an important role on the stability of corneal epithelium and corneal transparency.Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor can promote cell proliferation and reduce apoptosis,such as human embryonic stem cells and karatin epithelial cells.Objective This study was to investigate the improving effect of Y-27632,a ROCK inhibitor,on the activity of rabbit LSCs in corneal preservation medium.Methods Corneal preservation solution was prepared by adding 12.5％ chondroitin sulfate,10.0％ low molecular dextran,20.0 mg/L dexamethasone,100 mg/L tobramycin sulfate,9.5 g/L Hepes and 0.375 mg/L L-glutamine in MEM.The corneas of New Zealand white rabbits were collected and preserved in the corneal preservation solution with or without Y-27632 for 4,7,14 days,and the density and morphology of corneal endothelial cells were examined by using 0.25％ trypan blue staining and 0.2％ alizarin red staining.Isolated corneal epithelial cells were seeded on 3T3 feeder layer and cultured for 7-10 days until colonies formation.Colony shape of LSCs was observed under the light microscope,and colony-formation efficiency was analyzed after Giemsa staining by Image J software.Results The morphology and density of corneal endothelial cells were normal in the corneal preservation solution with and without Y-27632 for 4 days.In the seventh day after preservation,the cells remained the regular hexagon in shape in the preservation solution with Y-27632,however,the cellular membrane was slightly shrinking with the positive staining for alizarin red in the preservation solution without Y-27632.The density of corneal endothelial cells in the corneal preservation solution without Y-27632 was (2 262-± 75) cells /mm2,while in the preservation solution with Y-27632 was (2 425 ±95) cells/mm2(P＜0.001).The cloning spheres of LSCs were similar in preservation solution both with and without Y-27632 in the freshly isolated cornea or preserved corneas and exhibited more cells inside.But in 7 days and 14 days after preservation,the cloning spheres were much smaller in the preservation solution without Y-27632 group than those in the preservation with Y-27632 group.No significant differences were found in the cloning-formation rate and survival rate of corneal epithelial cells in corneas freshly isolated or preserved for 4 days in both groups (all at P＞0.05).In 7 days and 14 days after preservation,the active rates of corneal epitheli.al cells were (73.00±2.12)％ and (56.00±0.71)％ in the preservation solution with Y27632,which were significantly higher than (66.00 ± 4.00) ％ and (49.00 ± 0.71) ％ in the preservation solution without Y-27632,showing statistically significant differences between them (t =3.098,P =0.018;t =9.798,P =0.000).In addition,the cloning-formation rates of LSCs were (11.05±0.21)％ and (3.10±1.97)％ in the preservation solution with Y-27632 in 7 days and 14 days after preservation,revealing significantly elevation in comparison with (2.05 ± 1.20) ％ and (0.40 ±0.14) ％ in the preservation solution without Y-27632 (t =18.107,P =0.000;t=3.184,P=0.017).Conclusions Y-27632 promotes the vitality and cloning-formation ability of LSCs in corneal preservation medium,suggesting its potential use during storage of cornea.
