紫外光-核黄素诱导羊膜胶原交联的实验研究

Crosslinking of human amniotic membrane by ultraviolet Ariboflavin

背景研究证实，紫外光一核黄素光动力疗法可通过改变角膜的理化性质控制圆锥角膜的进展，羊膜移植已广泛用于眼表疾病，羊膜胶原成分与角膜相似，目前关于紫外光一核黄素诱导的胶原交联用于羊膜组织的研究尚少见。目的探讨紫外光一核黄素对冻存羊膜的作用。方法在知情同意条件下，获取并常规处理人羊膜组织，-80℃保存。制备成2mm×15mm大小，用简单随机法中的抽签法将其分为4个组，每组6片羊膜，前3组均给予紫外光一核黄素（质量分数0．1％）处理30rain（波长为370nm，紫外光功率分别为1、2、3mW／cm^2，光源距羊膜10mm），第4组不做任何处理，为对照组。利用微力材料试验机对处理后的各组羊膜分别进行生物力学测量，记录羊膜长度改变5％、10％、15％时各组样本所需的拉力值（mN）。用环钻钻取直径7mm的羊膜，用抽签法将其分为4个组，每组5片羊膜，各组处理情况同上，将处理后的各组羊膜浸于质量分数0．1％胶原蛋白酶I溶液中进行消化，对羊膜透明度进行评分，并记录羊膜完全溶解时间。关于羊膜的组织学观察，用抽签法将羊膜分为紫外光一核黄素组、核黄素组及对照组，每组3片羊膜，各组分别给予紫外光一核黄素（0．1％）处理30rain（波长为370nm，紫外光功率为3mW／cm^2，光源距羊膜10mm）、0．1％核黄素浸泡30min和生理盐水浸泡30min，透射电子显微镜下进行观察。结果羊膜长度改变5％、10％、15％时，对照组和1、2、3roW／era。紫外光组4个组所需拉力值差异均有统计学意义（F：3．411，P=0．037；F=9．927，P=0．001；F=11．118，P=0．000）；交联后抗拉力性能明显增强，且紫外光功率越大，羊膜的抗拉性增加越明显，差异均有统计学意义（P〈0．05）。对照组羊膜完全酶解时间为（8．6±1．8）h，1、2、3mW／cm。紫外光组分别为（39．6±2．3）、（71．4±0．9）、（78．8±1．8）h，交联后抗酶解能力增强，且紫外光功率越大，羊膜的抗酶解能力增强越明显，差异有统计学意义（P〈0．01）。透射电子显微镜超微结构观察，交联后羊膜基质层胶原纤维密度增加、胶原间连接增多、胶原与上皮问的连接加强。结论紫外光-核黄素可使冻存羊膜发生胶原交联，从而使其组织结构发生变化，生物力学性质改变，抗酶解能力加强。

Background Studies confirmed that ultraviolet A （UVA）riboflavin photodynamic therapy can control keratoconus progresses by altering the physicochemical property of cornea. The collagen components of amniotic membrane transplantation is similar to that of cornea and amniotic membrane transplantation has been widely used to ocular surface reconstruction. However,the study on UVA riboflavin-induced-collagen crosslinking for amniotie tissue is less now. Objective This study was to investigate the role of UVAriboflavin on frozen-preserved human amniotie membrane. Methods Human amnions were obtained in informed consent and prepared into 2 mm×l5 mm pieces and were then divided into 4 groups using lottery method and 6 pieces for each group. The first 3 groups were treated with the photosensitizer riboflavin and UVA-irradiation （ wavelength ：370 nm ; irradiation energy ： 1,2 or 3 mW/cm^2 , distance ：10 mm） for 30 minutes,and the untreated fourth group was as control group. Biomechanica] stressstrain test was performed using a microcomputercontrolled biomateria] tester and the stress（mN） was recorded when the strains were set to 5% , 10% and 15% . The 7 mm diameter of human amniotie membrane pieces were trephined and divided into 4 groups（5 pieces for each group） with the treated method as mentioned above, and then the buttons were exposed to 0. 1% collagenase I solution. The transparency was scored and the complete dissolving time was record. In histological evaluation, three groups （3 pieces for each group） of human amniotie membranes were treated using UVA-riboflavin（ 3 mW/em2）, 0. 1% riboflavin, normal saline for 30 minutes respectively and examined under the transmission electron microscopy. This study was performed under the permission of the Ethic Commission of Beijing Tongren Hospital. Results When the strain was 5% , 10% , 15% , the stress of control group and 1 , 2, 3 mW/cm2UVA group were statistically signifeantly different （ F = 3. 411 , P = 0. 037; F = 9. 927, P = 0. 001 ; F= 11. 118,P=0. 000）. The tensile strength of human amniotic membrane cross-linked with UVA-riboflavin was statistically significantly increased in comparison to the control group（ P〈0.05 ）, and the tensile strength of human amniotie membrane became stronger as UVA power increased. The complete dissolve time was（8.6±1.8 ） hours for the control group, （ 39.6±2.3 ） hours for 1 mW/cm2 UVA group, （ 71.4± 0.9 ） hours for 2 mW/cm2 UVA group, （78.8± 1.8 ） hours for 3 mW/cm2 UVA group, showing the enhanced antienzyme ability of human amniotic membrane after cross-linking（ P〈0.01 ）. The collagen density in the UVAriboflavin treated group was increased,the connection among the collagen fibers as well as between the stroma and the epithelium became tighter than those of control group. Conclusions Collagen crosslinking with UVAriboflavin make the biomechanical strength and enzymatic resistance of human amniotic membrane enhance and ultrastructure change of human amniotic membrane.