飞秒激光角膜透镜移植联合胶原交联的生物力学效应与愈合机制研究

The improvements in safety and efficiency of myopic corneal refractive surgery concern vital interests of over 400 million myopic patients in China. The main purpose of this research is to convert the traditional “subtraction” principle of corneal refractive surgeries (to correct refractive errors by cutting or ablation corneal tissue) to a novel “addition” one (to correct refractive errors by thickening and strengthening corneal tissue). The project originally combines femtosecond laser assisted lenticule transplantation with accelerated corneal collagen cross-linking technique. By applying 3-dimensional scanning mode of femtosecond laser on rabbits’ or a monkeys’ corneas, a particular corneal lenticule can be extracted through a 2 millimeters-long side-cut from the corneal stromal layer of the donor and a corneal stromal pocket, which is able to contain the lenticule, can be created in the corneal stromal layer of the receptor. Accelerated corneal collagen cross-linking will be conducted soon after the extracted lenticule, which is obtained from the donor’s cornea, is transplanted into the receptor’s stromal pocket. An anterior segment analysis system (Pentacam HR, Oculus, Wetzlar, Germany) will be used to analyze the changes in refractive power and refractive uniformity of the acceptor’s cornea. Both the post-operative process of corneal nerve wound healing and the post-operative reaction of stromal cells will be assessed using an in vivo confocal microscopy. Transmission Electron Microscope will be applied to detect the changes in corneal ultrastructure. Cell death will be analyzed using TdT-mediated dUTP Nick-End Labeling (TUNEL). Various cytokines (TGF-β, α-SMA, IL-1, and Ki-67) and the changes in the expression of fibronectin mRNA were evaluated using Real-time PCR. Changes in the corneal biomechanical properties, including deformation amplitude, applanation time, applanation length, corneal velocity, peak distance, and radius will be assessed using Corvis Scheimpflug Technology (Corvis ST, OCULUS, Wetzlar, Germany). The project is designed to investigate the wound healing mechanism and changes in corneal mechanical properties following the new surgical procedure to improve the safety of refractive surgeries, which will be beneficial to the broad masses of the myopic patients.

近视手术的安全与质量的优化关系到我国4亿近视者的切身需求，实现角膜激光手术由削薄变弱的“减法”转为加固加厚的“加法”是本研究的主旨。本项目创新性地将透镜移植和角膜胶原交联技术联合，通过飞秒激光对兔及猴角膜进行三维扫描制透镜及制做基质囊袋，2mm切口取出透镜移植到受体眼并设置不同能量参数进行快速胶联。手术后用角膜地形图观察角膜屈光力及规则性，用活体共聚焦显微镜观察基质细胞反应、神经修复的规律；用透射电镜观察角膜超微结构变化；TUNEL法检测细胞凋亡、用 Real-time PCR方法从RNA水平测定角膜中TGF-β、α-SMA、IL-1、Ki-67及细胞外纤维连接蛋白mRNA含量及变化；用Corvis ST探讨角膜生物力学参数包括角膜形变时间、形变幅度、形变速率的变化规律。本项目拓展新型安全的屈光手术途径并探究其生物力学效应和愈合机制，将进一步推进近视手术，使广大近视患者受益 。

本项目分别完成了以实验动物及以人眼为对象的角膜组织透镜植入联合角膜交联手术，并在此基础上进行了形态学、生物力学、细胞学及分子水平上的探究，证明该手术为安全可行的治疗方式。首先利用实验动物完成了同种异体的飞秒激光基质透镜植入联合胶原交联手术，实现了角膜激光手术由削薄变弱的“减法”转为加固加厚的“加法”的动物学基础。对实验动物分别进行角膜形态学、生物力学测量、细胞水平包括共聚焦显微镜、透射电镜和分子水平的愈合反应分析，表明异体透镜移植联合交联术可增强角膜生物力学特性，增加角膜基质厚度且愈合反应在半年内达稳定。随后完成人眼自体角膜组织透镜植入手术矫正对侧远视眼手术及细胞学水平研究，提示自体SMILE角膜组织透镜在自体人眼受体中的生物相容性。并在该基础上形成了自体透镜移植屈光度与透镜参数间的量－效模型，建立了有效的透镜移植预测公式，对未来透镜移植临床应用具有一定意义。同时，本项目还探索了同种异体角膜组织透镜移植联合角膜交联手术在LASIK术后角膜扩张患者治疗上的应用，临床患者的屈光度、角膜厚度及稳定性均得到有效改善，这使该手术的临床应用范围及受益范围进一步扩大。