高强度聚焦超声辐照活体兔眼角膜对眼前节的影响

发布时间：2018-06-08 19:51

本文选题：高强度聚焦超声 + 角膜　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:利用高强度聚焦超声(High Intensity Focused Ultrasound,HIFU)的热效应,使角膜周边胶原纤维收缩,增加屈光力。探讨将其作为一种治疗老视的新方法,辐照活体兔眼角膜基质后,对眼前节的影响,评价其生物安全性。方法:第一部分:HIFU以圆环状定位辐照18只新西兰大白兔右眼角膜周边基质,左眼不接受辐照为对照组。术后不同时间段(术后1、7、15、30、60、90天)裂隙灯下观察角膜透明情况并行荧光素钠染色,取材后对角膜组织切片行HE染色,免疫组织化学方法检测各时间点Ⅰ型胶原(Collagen type I)、基质金属蛋白酶-2(Matrix Metalloproteinase-2,MMP-2)、基质金属蛋白酶-9(Matrix Metalloproteinase-9,MMP-9)在角膜内的表达,角膜细胞凋亡情况用TUNEL法检测,并用透射电子显微镜(Transmission Electron Microscope,TEM)观察角膜显微结构改变。第二部分:HIFU以圆环状定位辐照18只新西兰大白兔右眼角膜周边基质,左眼不接受辐照为对照组。术后不同时间段(术后1、7、15、30、60、90天)裂隙灯下观察晶状体透明度及位置,取材后对晶状体组织切片行he染色,检测超氧化物歧化酶(superoxidedismutase,sod)及丙二醛(malonaldehyde,mda)在晶状体内的含量,晶状体上皮细胞凋亡情况用tunel法检测。结果:第一部分:1.裂隙灯观察结果:各时间段均未见角膜感染、穿孔。hifu角膜基质术后1天,角膜周边可见白色焦域环,后逐渐变淡,术后90天基本消失。2.角膜荧光素钠染色:术后1天hifu辐照环角膜部分上皮着染,术后7天仅有少量着染,术后15天角膜未见着染。3.角膜组织切片he染色:术后1天部分角膜基质深染,可见生物学焦域;术后7天,焦域变淡,基质纤维排列紊乱,辐照区上皮增生;术后15天至60天,焦域消失,纤维排列逐渐整齐,辐照区上皮增生逐渐减轻;术后90天组织结构恢复正常。4.免疫组化检测:术后1、7天焦域内无角膜Ⅰ型胶原表达,15天后明显增加,30天时恢复正常。术后1天角膜上皮mmp-2少量表达,7天时表达增加,15天、30天时平均光密度达到最大,60天时表达明显下降,90天时回到正常水平。术后7天角膜上皮mmp-9少量表达,15天时平均光密度达到最大,30至60天表达明显下降,90天时回到正常水平。5.tunel检测角膜凋亡细胞:术后1天角膜焦域内出现大量凋亡细胞,术后7天、15天、30天角膜凋亡细胞明显减少,术后60天、90天无角膜凋亡细胞。6.电镜观察结果:术后1天可见辐照处角膜基质细胞肿胀、破裂,基质纤维断裂;术后7天至术后60天,角膜基质细胞内线粒体肿胀、溶解,粗面内质网数量增加,基质纤维紊乱扭曲;术后90天基质细胞呈正常形态,基质纤维整齐排布。第二部分:1.裂隙灯观察结果:HIFU角膜基质术后各时间段晶状体均透明,无混浊。2.晶状体组织切片HE染色:术后各时间段晶状体囊膜完整,晶状体上皮细胞排列整齐,位于前囊膜下并延续至赤道,晶状体纤维呈长纤维,排列紧密而有序。3.晶状体SOD、MDA检测:术后各时间段实验组晶状体SOD、MDA含量与对照组比较均无明显差异(P0.05)。4.TUNEL检测晶状体上皮凋亡细胞:术后各时间段均未见晶状体上皮凋亡细胞。结论:1.HIFU角膜基质术后未出现角膜感染、穿孔,术后1天至60天角膜焦域内组织形态改变,术后90天恢复正常,表明HIFU应用于角膜改变角膜曲率是安全可靠的。2.术后一段时间内角膜Ⅰ型胶原减少、MMP-2与MMP-9活化、部分角膜细胞凋亡、电镜下角膜显微结构改变,说明HIFU辐照角膜基质产生热效应,在一段时间内引起新生胶原修复重塑,角膜成纤维细胞活化,角膜细胞凋亡,均在术后90天恢复正常,表明HIFU角膜基质术具有较好的安全性。3.术后各时间段,晶状体均保持透明,组织形态无改变,SOD及MDA含量正常,无晶状体上皮凋亡细胞,表面HIFU角膜基质术能精确、安全地改变角膜曲率,不会对晶状体产生影响。
[Abstract]:Objective: to make use of the thermal effect of High Intensity Focused Ultrasound (HIFU) to constriction the collagen fibers around the cornea and increase the diopter. The corneal peripheral matrix of 18 New Zealand white rabbits was irradiated in the right eye, and the left eye was not irradiated as the control group. The corneal transparency was observed at different time periods (1,7,15,30,60,90 days after operation) and fluorescein staining was observed under the slit lamp. The corneal tissue sections were stained with HE, and the time point type I was detected by immunohistochemical method. The expression of collagen (Collagen type I), matrix metalloproteinase -2 (Matrix Metalloproteinase-2, MMP-2), matrix metalloproteinase -9 (Matrix Metalloproteinase-9, MMP-9) in the cornea, the corneal cell apoptosis was detected by TUNEL method, and the corneal microstructure changes were observed by transmission electron microscopy. The second part: HIFU was used to irradiate the corneal peripheral matrix of 18 New Zealand white rabbits in the right eye, and the left eye did not receive irradiation as the control group. The transparency and location of the lens were observed under the slit lamp at different time periods (1,7,15,30,60,90 days after the operation). The he staining of the lens tissue sections was performed to detect the superoxide dismutase (superoxide). The content of dismutase, SOD) and malondialdehyde (malonaldehyde, MDA) in the crystalline body and the apoptosis of lens epithelial cells were detected by TUNEL. Results: the first part: 1. the results of the slit lamp observation: no corneal infection was found at all time, 1 days after the perforated.Hifu corneal stroma, the white focal region around the cornea was visible, then gradually dilute, and 90 days after the operation. The disappearing.2. cornea fluorescein sodium staining: 1 days after the operation, HIFU irradiated corneal epithelium was stained, only a small amount of staining was observed on the 7 day after operation. The cornea was not stained with.3. corneal tissue section on the 15 day after the operation. The corneal stroma was deeply dyed on the 1 day after the operation, and the biological focal area was seen; the focal region became pale, the matrix fiber was arranged in disorder, and the irradiated area increased on the 7 day after the operation. 15 to 60 days after operation, the focal area disappeared, the fiber arrangement was neatly arranged, the epithelial hyperplasia in the irradiated area was gradually reduced, and the tissue structure was restored to normal.4. immunohistochemistry on the 90 day after the operation: there was no expression of type I collagen in the focal area at 1,7 days after the operation, obviously increased in 15 days, and returned to normal at 30 days. The corneal epithelium was expressed in a small amount of MMP-2 and expression at 7 days after the operation. The average light density reached the maximum at 15 days and 30 days, the expression decreased obviously at 60 days and returned to the normal level at 90 days. The corneal epithelial MMP-9 was expressed in a small amount in the 7 day after the operation, the average light density reached the maximum at 15 days, the expression of the cornea was significantly decreased in 30 to 60 days, and the corneal apoptotic cells were detected at the normal level by.5.tunel at 90 days, and the corneal focal area appeared 1 days after the operation. A large number of apoptotic cells, 7 days, 15 days, 30 days after operation, corneal apoptotic cells significantly decreased, 60 days after operation, 90 days without corneal apoptotic cells.6. electron microscope observation results: 1 days after the operation, corneal stroma cells swelling, rupture, matrix fiber fracture, 7 days to 60 days after the operation, corneal stroma cells in the mitochondria swelling, dissolved, rough endoplasmic reticulum number. The matrix fibers were distorted and the matrix fibers were in normal shape after 90 days of operation, and the matrix fiber was arranged neatly. The second part: 1. slit light observation results: the crystalline bodies were all transparent after HIFU corneal stroma, without the cloudy.2. lens tissue section, HE staining: the lens capsule was complete and the lens epithelial cells were arranged and arranged after the operation. Qi, located under the anterior capsule and continued to the equator, lens fiber was long fiber, arranged closely and orderly.3. lens SOD, MDA detection: the experimental group of lens SOD after the operation, MDA content compared with the control group no significant difference (P0.05).4.TUNEL detection of lens epithelial apoptotic cells: no lens epithelial apoptosis at all time after operation Conclusion: there was no corneal infection and perforation after 1.HIFU corneal stroma, and the morphological changes in the corneal focal area were changed from 1 to 60 days after operation. It was restored to normal 90 days after the operation. It showed that HIFU was used in the corneal change of corneal curvature to be a safe and reliable.2. after a period of time, MMP-2 and MMP-9 were activated and some of the corneal cells were apoptotic. The change of corneal microstructure under electron microscope shows that HIFU irradiates the corneal stroma to produce heat effect. It causes the remolding of the new collagen in a period of time, the activation of corneal fibroblasts and the apoptosis of the cornea cells, all of which are restored to normal 90 days after the operation, indicating that the HIFU corneal stroma has good safety in every time after.3., and the lens is all transparent. There is no change in the tissue morphology, the content of SOD and MDA is normal, the apoptotic cells of the lens epithelium, the surface HIFU corneal stroma can accurately and safely change the curvature of the cornea, and do not affect the lens.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R77

【参考文献】