后部板层角膜供体材料的应用实验研究

发布时间：2018-03-08 15:53

本文选题：板层角膜移植　切入点：前板层角膜供体　出处：《南昌大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：建立大鼠同种异体无缝线前、后板板层角膜移植(LKP)动物模型。观察角膜上皮细的生长及组织学特点。方法：选取Wistar大鼠27只54眼为供体制备前板（前弹力层+部分基质层）和后板（后弹力层+部分基质层）角膜植片；雌性SD大鼠108只为受体分为2组，每组54只，实验组行后板LKP，对照组行前板LKP，两组大鼠均右眼施行LKP手术，左眼为正常对照。用猪源纤维蛋白粘合剂（Porcine Fibrin Sealant Kit）替代角膜缝线行LKP，术毕行睑缘缝合术[1]。两组术后第l天拆除眼睑缝合线，前3天滴用0.5%左氧氟沙星滴眼液（参天制药可乐必妥）和妥布霉素地塞米松滴眼液（Alcon公司典必殊）4次/日，以后滴眼药水2次/日，1月后停药。两组术后3、7、l4、28、42及56天裂隙灯下观察术眼并行排斥反应指数(RI)评分，荧光素钠染色观察角膜缺损并用photoshop软件计算角膜上皮生长面积。各观察时间点处死实验组和对照组大鼠各9只，取术眼角膜制备HE染色标本、透射电镜标本观察角膜上皮各层细胞形态、层数同时行角膜K12免疫荧光检测角蛋白表达水平。结果：选入的108只SD大鼠在实验过程中因麻醉过度死亡占14只，术后植片出现感染占7只，均被清除后得到及时补充，108只SD大鼠全部进入最终结果分析。裂隙灯下观察：实验组和对照组角膜植片混浊水肿过程相同， RI图表曲线几乎重叠，其峰值及整体曲线走向大体一致，整体RI指数呈下降趋势。荧光素钠染色：两组着色范围缩小时间相同。photoshop画图软件辅助分析角膜上皮生长面积观察到两组术后各时间点角膜上皮爬行面积差异甚小。对RI和Fls评分数据及上皮生长面积，采取两样本t检验，以a=0.05，p0.05为数据有统计学意义，计算得出各时间点p0.05，差异均无统计学意义。HE染色切片光镜观察：两组术后均从早期上皮生长不典型到后期上皮结构均长出，，形态接近正常上皮细胞。透射电镜观察：早期两组角膜表皮细胞微绒毛均稀疏，至后期五层上皮细胞结构均长出，微绒毛生长致密。同时观察到角膜上皮基底膜与后弹力层连接有间隙，而与前弹力层连接紧密。K12免疫荧光检测：两组术后免疫荧光检测可见上皮细胞从早期的弱荧光到后期的强荧光。结论：1、首次建立大鼠同种异体无缝线前、后板LKP动物模型。 2、通过各项指标检测，观察到两组在不同时间点上皮爬行的速率、面积及生长形态大致相同，证实后板同样能作为角膜上皮细胞生长的载体，为后板进一步应用提供了实验和理论依据。 3、通过透射电镜观察前、后板LKP术后角膜上皮移行中超微结构的差异，为后续科研工作提供了重要参考。 4、猪源纤维蛋白粘合剂成功应用于大鼠板层角膜移植，达到无缝线手术目的，为其进一步应用于无缝线角膜移植手术提供了实验基础。
[Abstract]:Aim: to establish a rat model of LKP in anterior and posterior lamellar keratoplasty (LKP) and to observe the fine growth and histological characteristics of corneal epithelium. Methods: Twenty-seven Wistar rats (54 eyes) were selected as donors to prepare corneal grafts of anterior plate (partial stromal layer of anterior elastic layer) and posterior plate (partial stromal layer of posterior elastic layer), and 108 female SD rats were divided into 2 groups, 54 rats in each group. The posterior plate of the experimental group was treated with LKP and the control group with the anterior plate of LKP.The two groups of rats underwent LKP operation on the right eye. LKP was performed with porcine Fibrin Sealant Sealant instead of corneal suture, and eyelid margin suture was performed in both groups. The eyelid suture was removed on the 1st day after operation in both groups. 0.5% levofloxacin eye drops and tobramycin dexamethasone eye drops were used 4 times a day. Eye drops were given twice a day later and stopped after January. The patients in the two groups were observed under slit lamp for the score of rejection index (RI) on the 3rd and 56th day after operation. Cornea defect was observed by fluorescein sodium staining and photoshop software was used to calculate the corneal epithelial growth area. Nine rats of the experimental group and the control group were killed at each observation time point. The morphology of corneal epithelial cells was observed by transmission electron microscope (TEM), and keratin expression was detected by keratin expression with keratinocyte K12 immunofluorescence assay at the same time. Results: during the experiment, 14 SD rats died of excessive anaesthesia, and 7 rats were infected with grafts after operation. All 108 SD rats were removed and all entered the final result analysis. Observation under slit lamp: the process of turbid edema of corneal grafts in the experimental group and control group was the same, and the RI curve almost overlapped. The peak value and the trend of the whole curve are roughly the same. The overall RI index showed a downward trend. Fluorescein sodium staining: the two groups had the same color range reduction time. Photoshop software assisted the analysis of corneal epithelial growth area observed that there was little difference in corneal epithelial crawling area between the two groups at different time points after operation. RI and Fls score data and epithelial growth area, Using two samples t test, the data of aq.05 and p0.05 were statistically significant. The results showed that the difference was not statistically significant at each time point. The HE staining sections were observed under light microscope: after operation, both groups grew from atypical early epithelial growth to late epithelial structure, and the results showed that there was no significant difference between the two groups in the early stage of epithelium growth and in the late stage of epithelium structure. The morphology was close to normal epithelial cells. Transmission electron microscope observation: the microvilli of corneal epidermis cells in the early stage were sparse, and the structure of the five layers of epithelial cells grew up in the late stage. At the same time, there was a gap between the basal membrane of corneal epithelium and the posterior elastic layer. In the two groups, the epithelial cells were detected by immunofluorescence from early weak fluorescence to late strong fluorescence. ConclusionThe LKP model of seamless anterior and posterior plate of allograft in rats was established for the first time. 2. The rate, area and morphology of epithelial crawling at different time points in the two groups were approximately the same, which proved that the posterior plate could also be used as the carrier of corneal epithelial cell growth. It provides the experimental and theoretical basis for the further application of the back plate. 3. The difference of ultrastructure in corneal epithelial transition after LKP was observed by transmission electron microscope, which provided an important reference for further scientific research. 4. Porcine fibrin adhesives were successfully used in lamellar keratoplasty of rats, which provided experimental basis for further application of seamless keratoplasty.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.65

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