摘要
目的观察光化学组织黏合(photochemical tissue bonding,PTB)技术用于修复角膜缘干细胞缺失的可行性及其对角膜愈合的影响。方法取新西兰大白兔角膜缘组织,采用组织块法培养角膜缘干细胞,取第2代细胞种植于完全刮除上皮细胞的人羊膜上,复合培养3周,制备复合培养移植物。3～4月龄雌性新西兰大白兔24只,体重1.5～2.0kg;用0.5mol/LNaOH灼烧法于左眼制备角膜缘干细胞缺失模型,分别采用PTB技术(PTB组,n=12)及传统缝线(缝合组,n=12)缝合复合培养移植物与角膜创面。术后大体观察角膜情况,并于3d及28d取角膜行组织学及免疫组织化学染色,观察炎性反应及血管生成情况;ELISA法检测IL-1β、IL-6、TNF-α蛋白含量。结果实验动物均存活至实验完成。术后3d两组角膜透明度无明显差异;28d时PTB组角膜透明度较缝合组高,新生血管形成少。组织学观察示,术后3d两组复合培养移植物与角膜基底间均有大量炎性细胞浸润,PTB组程度略轻,无新生血管形成;28d时PTB组炎性细胞明显较缝合组减少,PTB组新生血管数为(2.0±0.8)个/HP,缝合组为(6.3±1.3)个/HP,比较差异有统计学意义(t=7.966,P=0.002)。术后3d两组角膜组织中IL-1β、IL-6、TNF-α蛋白含量均较高,组间比较差异均无统计学意义(P>0.05);28d时PTB组以上指标均较缝合组明显降低(P<0.05)。结论 PTB技术可用于固定兔角膜移植物;与传统缝线缝合方法相比,PTB技术可减轻组织炎症、减少角膜血管新生,从而改善角膜缘干细胞缺失修复效果。
Objective To investigate the feasibility of photochemical tissue bonding（PTB） technique in repairing limbal stem cell（LSC） deficiency and the effect on cornea wound healing.Methods LSCs were isolated from limbus of New Zealand rabbits by tissue block culture method,and then the LSCs of 2nd passage were cultured on de-epithelialized human amniotic membrane（HAM） for 3 weeks to prepare the HAM/LSC grafts.The LSC deficiency models of the left eyes were established by 0.5 mol/L NaOH in 24 New Zealand female rabbits,aged 3-4 months and weighing 1.5-2.0 kg.HAM/LSC grafts were used to repair the cornea wounds by sutures（suture group,n=12） or by PTB technique（PTB group,n=12）.The gross was observed including the corneal transparency,erythema,and new blood vessel formation after surgery.At 3 and 28 days,the inflammatory cytokine of interleukin 1β（IL-1β）,IL-6,and tumor necrosis factor α（TNF-α） were assayed by ELISA method;and the amount of new blood vessels were quantified by immunohistochemistry staining at 28 days.Results All animals survived to the end of the experiment.At 3 days,there was no obvious difference in the corneal transparency between 2 groups;at 28 days,the corneal transparency of PTB group was higher than that of suture group,and new blood vessels decreased.HE staining showed that mass inflammatory cells infiltrated between graft and cornea basal layer at 3 days,and no new blood vessel formed.inflammatory cells infiltration significantly decreased at 28 days in PTB group;the amount of new blood vessels was（2.0 ± 0.8）/HP in PTB group and was（6.3 ± 1.3）/HP in suture group,showing significant difference（t=7.966,P=0.002）.At 28 days,the concentrations of inflammatory cytokine of IL-1β,IL-6,and TNF-α in suture group were significantly higher than those in PTB group（P 〈 0.05）;however,no significant differences were observed between 2 groups at 3 days（P 〉 0.05）.Conclusion PTB technique can be used to fix HAM/LSC grafts,which can decrease inflammatory cell infiltration and new vessel formation,and improve the outcomes when compared with suture technique
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2011年第9期1110-1114,共5页
Chinese Journal of Reparative and Reconstructive Surgery
基金
上海浦江人才计划资助项目(10PJ1407000)
上海交通大学医学院科技基金项目(09XJ21009)
上海交通大学国防基金资助项目(11GF-53)~~
关键词
光化学组织黏合技术
角膜缘干细胞
角膜修复
兔
Photochemical tissue bonding technique Limbal stem cell Cornea repair Rabbit
