背景 硫酸软骨素(CS)是从动物软组织中提取的一种具有高黏滞性和高弹性的酸性黏多糖类物质,因具有广泛的生物活性而用于眼科临床.中期保存液中含有CS时,对角膜内皮细胞(CECs)具有显著的保护作用.然而,CS对长期甘油冷冻保存的CECs是否起到保护作用尚待研究. 目的 研究CS在甘油冷冻保存角膜中对CECs的保护作用,并与透明质酸钠(SH)比较,探索长期保存CECs活性的新方法.方法 获取52只Wistar雌性大鼠角膜片104只,采用随机数字表法随机分为4个组,正常对照组为新鲜角膜直接进行实验,质量分数3％ CS处理组、质量分数1％ SH处理组的角膜分别在内皮面黏附3％ CS、1％SH后放入甘油中冷冻保存,单纯甘油保存组角膜直接装入甘油中-25℃冷冻保存,每组26只角膜片.96只雌性SD大鼠作为受体,角膜植片保存2个月后各组取24只植片行同种异体穿透角膜移植术(PKP),CECs锥虫蓝-茜素红染色分别评价保存的角膜片和移植术后14d的角膜植片上CECs的存活率；透射电子显微镜下观察不同方法保存的角膜植片的超微结构.角膜移植术后1d通过裂隙灯显微镜检查各组角膜排斥反应指数(RI)和植片存活时间,分别于术后7、14、30 d制备角膜标本行常规组织病理学检查,免疫组织化学法检测各时间点植片中转化生长因子β1(TGF-β1)和细胞间黏附因子1(ICAM-1)的表达水平.结果 正常对照组大鼠角膜植片CECs细胞结构正常,未见锥虫蓝-茜素红蓝染细胞,3％ CS处理组偶见细胞蓝染,1％ SH处理组和单纯甘油保存组蓝染CECs数增加.与3％ CS处理组比较,1％ SH处理组和单纯甘油保存组的细胞密度均明显下降,差异均有统计学意义(t=2.214、4.068,P＜0.05),细胞活性率表现出同细胞密度同样的趋势.CECs超微结构检查表明,3％ CS处理组仪表现为内质网的轻度肿胀,而1％SH处理组和单纯甘油保存组细胞内质网肿胀程度较重.3％ CS处理组术后14d时植片淋巴细胞浸润及新生血管明显少于1％ SH处理组和单纯甘油保存组.临床评分表明,与1％ SH处理组和单纯甘油保存组比较,3％ CS处理组的R1明显降低而CECs生存时间明显延长,差异均有统计学意义(P＜0.05).免疫组织化学检测显示,在各时间点,3％ CS处理组植片中TGF-β1的表达量(A值)明显高于1％ SH处理组和单纯甘油保存组(P＜0.01)；而ICAM-1的表达量低于1％ SH处理组和单纯甘油保存组(P＜0.05),3％ CS处理组植片中TGF-β1和ICAM-1的表达量接近于正常对照组表达水平.结论 3％ CS处理后的甘油保存方法能长期维持CECs的活性,同种异体PKP结果显示3％ CS处理后的甘油保存方法效果优于1％ SH处理后的甘油保存法.%Background Chondroitin sulfate(CS) is a highly viscous and elastic acid mucopolysaccharide extracted from animal soft tissues,with a wide range of biological activity for use in clinical ophthalmology.Interim preservation solution containing CS has a significant protective effect on corneal endothelial cells (CECs).However,the protective effect played by CS in long-term glycerol cryopreservation of CECs remains to be studied. Objective This study is mainly attempted to investigate the protective effect of CS on graft CECs after cryopreservation by glycerin,and to compare the preserving outcome with that of sodium hyaluronate (SH). Methods One hundred and four eyes of fifty-two female Wistar rats were divided into four groups randomly.The cornea grafts were evenly anointed on the surface of the endothelium by 3％ CS and 1％ SH,respectively,and then cryopreserved in glycerol in the 3％ CS group and 1％ SH group,and the corneas cryopreserved only in glycerin were assigned to the glycerin only group.The fresh corneas of matched rats were used as the normal control group.Ninety-six female SD rats were appointed as recipients to receive allogeneic penetrating keratoplasty (PKP) 2 months after the graft cryopreservation.The viability of CECs on the corneas cryopreserved for 20 days and grafts of 14 days following surgery was assessed using trypan blue & alizarin red stain.The ultrastructure of cryopreserved corneas was examined under a transmission electron microscope.The rejection index (RI) and graft survival time were evaluated by inflammatory scoring under the slit lamp 1 day after operation,and regular pathological examination was performed after 7,14 or 30 days.The expression of transforming growth factor-β1 ( TGF-β1 ) and intercellular adhesion molecule-1 ( ICAM-1 ) in corneas and grafts were detected by immunochemistry. Results CECs morphology was normal in fresh cornea in normal control group,presenting no blue stained cells after trypan blue staining.In the 3％ CS group,few blue-stained cells were seen:however,the number of blue-stained cells noticeably increased in the 1％ SH group and glycerin only group.The viability and density of CECs were significantly lower in the 1％ SH group and glycerin only group,compared with the 3％ CS group (P＜0.05).Edema of the endoplasmic reticulum,inflammatory infiltration and neovascularization were more severe in the 1％ SH group and glycerin only group than the 3％ CS group 14 days after PKP.Compared with the 1％ SH group and glycerin only group,the R1 of the 3％ CS group was significantly reduced and the CECs survival time was delayed after PKP ( P＜0.05 ).Immunochemistry revealed that the expression of TGF-β1 in grafts (A value)was up-regulated and that of ICAM-1 was down-regulated in the 3％ CS group,compared with the 1％ SH group and glycerin only group ( P＜0.05). Conclusions The combination of 3％ CS with glycerol to cryopreserve corneal donor can maintain the viability of CECs for a longer duration and improve the effectiveness of PKP.
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