目的 探讨胶联羊膜治疗兔角膜深层损伤的效果,分析其促进角膜损伤愈合的机制.方法 新西兰大白兔45只,均以右眼作为实验眼,实验前采用裂隙灯显微镜检查排除角膜疾病,建立兔角膜深层损伤模型.采用单纯随机抽样法,将兔分为胶联羊膜组、普通双层羊膜组及损伤对照组,每组15只.前两组行嵌合式羊膜移植.术后观察记录羊膜溶解脱落时间,角膜荧光素染色,角膜混浊及新生血管等情况,并进行临床疗效评定.术后第7、14、28天,行苏木素-伊红染色组织病理学检查;原位末端标记法检测角膜基质凋亡细胞.两个实验组和对照组各时间点参数评分以及末端脱氧核苷酸转移酶介导的脱脲苷三磷酸缺口末端标记法(TUNEL)阳性细胞表达值分别进行正态性检验,方差齐性检验,采取的统计方法为两独立样本t检验.结果 胶联羊膜覆盖于角膜创面的时间(20.00±2.43)d,明显长于普通双层羊膜(13.15±2.68)d(t=8.470,P=0.000).术后第28天,胶联羊膜组角膜创面全部恢复正常厚度,角膜上皮荧光素染色均为阴性,角膜透明度高,新生血管较少.而普通双层羊膜和对照组尚有部分动物角膜荧光素小片浅层着色,角膜混浊明显,瘢痕致密,粗大新生血管长入角膜中央.胶联羊膜组和对照组术后第14、28天,角膜混浊和新生血管总评分比较,差异有统计学意义(术后第14天:胶联羊膜组2.62,对照组5.19,t=3.986,P=0.004;术后第28天:胶联羊膜组2.87,对照组4.78,t=3.608,P=0.007).整个观测期,胶联羊膜组损伤角膜无感染穿孔;而普通双层羊膜组和对照组分别有3例和2例,因角膜上皮迁延愈合导致感染穿孔.组织病理检查显示胶联羊膜组损伤角膜全部正常上皮化,基质胶原纤维排列整齐.在各观察时间点上,胶联羊膜组角膜基质凋亡细胞均明显少于对照组(术后第7天:t=8.153,P=0.000;术后第14天:t=9.693,P=0.000;术后第28天:t=14.050,P=0.000).而普通双层羊膜组与对照组比较,仅在术后第7天,差异有统计学意义(术后第7天:t=5.474,P=0.000).结论 胶联羊膜对兔角膜深层损伤具有良好的治疗效果,可显著促进角膜损伤愈合,抑制新生血管和瘢痕形成,抑制角膜基质细胞凋亡.%Objective To treat deep layer corneal damage using fibrin glue and amniotic memrbane transplant. Methods Forty-five rabbits were given deep lamellar keratectomies to cause deep layer corneal damage in their right eyes and were then randomly divided into 3 groups. The first group was given doublelayer amniotic membrane transplants where fibrin glue was used to connect the two layers of amniotic membrane. The second group was given double-layer amniotic membrane transplants where no fibrin glue was used. The third group was given no treatment. Clinical outcome was graded by corneal integrity, opacity and neovascularization, and detachment of amniotic membrane was recorded. The expression of apoptosis was monitored to assess the changes of morphology and histology on the 7th, 14th and 28th days after surgery.Results While the double-layer amniotic membrane without fibrin glue covered the cornea for (13. 15 ±2. 68 ) d, the double-layer amniotic membrane using fibrin glue covered the surface of the cornea for (20. 00 ± 2. 43 ) d ( t = 8. 470, P = 0. 000 ). The corneas in the first group recovered smoothly and transparently, maintained normal thickness and less neovascularization, whereas the corneas in the other two groups recovered irregularly, lost their transparency, became turbid and showed higher levels of neovascularization. There were statistically significant differences between the first and third groups for corneal opacity and neovasculariazation, where the 14th day after surgery, the first group scored 2. 62 and the third group scored 5. 19 (t =3. 986, P =0. 004), and the 28th day after surgery, the first group scored 2. 87 and the third group scored 4. 78 (t =3. 608, P=0. 007). Perforation did not appear in the first group,but the second group had 3 cases and the third group had 2 cases, all due to infection. The changes of morphology and histology showed that the damaged corneas were contained normal epithelial cells and regularly arranged fibrous cells on the 28th day after surgery. The apoptosis in corneas of the first group was less than that of the third group at all observed points (7th day: t =8. 153, P =0.000; 14th day: t =9. 693, P =0. 000; 28th day: t = 14. 050, P =0. 000), however, apoptosis in corneas in the second group was only different from that in the third group on the 7th day after surgery while other observed points showed no difference (7th day: t =5.474, P=0. 000). Conclusion Using bio-engineered fibrin glue in amniotic membrane transplants can repair deep layer corneal damage, reduce neovascularization, scarring and corneal apoptosis.
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