本文選題：高強度聚焦超聲 + 角膜��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:利用高強度聚焦超聲(High Intensity Focused Ultrasound,HIFU)的熱效應(yīng),使角膜周邊膠原纖維收縮,增加屈光力。探討將其作為一種治療老視的新方法,輻照活體兔眼角膜基質(zhì)后,對眼前節(jié)的影響,評價其生物安全性。方法:第一部分:HIFU以圓環(huán)狀定位輻照18只新西蘭大白兔右眼角膜周邊基質(zhì),左眼不接受輻照為對照組。術(shù)后不同時間段(術(shù)后1、7、15、30、60、90天)裂隙燈下觀察角膜透明情況并行熒光素鈉染色,取材后對角膜組織切片行HE染色,免疫組織化學(xué)方法檢測各時間點Ⅰ型膠原(Collagen type I)、基質(zhì)金屬蛋白酶-2(Matrix Metalloproteinase-2,MMP-2)、基質(zhì)金屬蛋白酶-9(Matrix Metalloproteinase-9,MMP-9)在角膜內(nèi)的表達(dá),角膜細(xì)胞凋亡情況用TUNEL法檢測,并用透射電子顯微鏡(Transmission Electron Microscope,TEM)觀察角膜顯微結(jié)構(gòu)改變。第二部分:HIFU以圓環(huán)狀定位輻照18只新西蘭大白兔右眼角膜周邊基質(zhì),左眼不接受輻照為對照組。術(shù)后不同時間段(術(shù)后1、7、15、30、60、90天)裂隙燈下觀察晶狀體透明度及位置,取材后對晶狀體組織切片行he染色,檢測超氧化物歧化酶(superoxidedismutase,sod)及丙二醛(malonaldehyde,mda)在晶狀體內(nèi)的含量,晶狀體上皮細(xì)胞凋亡情況用tunel法檢測。結(jié)果:第一部分:1.裂隙燈觀察結(jié)果:各時間段均未見角膜感染、穿孔。hifu角膜基質(zhì)術(shù)后1天,角膜周邊可見白色焦域環(huán),后逐漸變淡,術(shù)后90天基本消失。2.角膜熒光素鈉染色:術(shù)后1天hifu輻照環(huán)角膜部分上皮著染,術(shù)后7天僅有少量著染,術(shù)后15天角膜未見著染。3.角膜組織切片he染色:術(shù)后1天部分角膜基質(zhì)深染,可見生物學(xué)焦域;術(shù)后7天,焦域變淡,基質(zhì)纖維排列紊亂,輻照區(qū)上皮增生;術(shù)后15天至60天,焦域消失,纖維排列逐漸整齊,輻照區(qū)上皮增生逐漸減輕;術(shù)后90天組織結(jié)構(gòu)恢復(fù)正常。4.免疫組化檢測:術(shù)后1、7天焦域內(nèi)無角膜Ⅰ型膠原表達(dá),15天后明顯增加,30天時恢復(fù)正常。術(shù)后1天角膜上皮mmp-2少量表達(dá),7天時表達(dá)增加,15天、30天時平均光密度達(dá)到最大,60天時表達(dá)明顯下降,90天時回到正常水平。術(shù)后7天角膜上皮mmp-9少量表達(dá),15天時平均光密度達(dá)到最大,30至60天表達(dá)明顯下降,90天時回到正常水平。5.tunel檢測角膜凋亡細(xì)胞:術(shù)后1天角膜焦域內(nèi)出現(xiàn)大量凋亡細(xì)胞,術(shù)后7天、15天、30天角膜凋亡細(xì)胞明顯減少,術(shù)后60天、90天無角膜凋亡細(xì)胞。6.電鏡觀察結(jié)果:術(shù)后1天可見輻照處角膜基質(zhì)細(xì)胞腫脹、破裂,基質(zhì)纖維斷裂;術(shù)后7天至術(shù)后60天,角膜基質(zhì)細(xì)胞內(nèi)線粒體腫脹、溶解,粗面內(nèi)質(zhì)網(wǎng)數(shù)量增加,基質(zhì)纖維紊亂扭曲;術(shù)后90天基質(zhì)細(xì)胞呈正常形態(tài),基質(zhì)纖維整齊排布。第二部分:1.裂隙燈觀察結(jié)果:HIFU角膜基質(zhì)術(shù)后各時間段晶狀體均透明,無混濁。2.晶狀體組織切片HE染色:術(shù)后各時間段晶狀體囊膜完整,晶狀體上皮細(xì)胞排列整齊,位于前囊膜下并延續(xù)至赤道,晶狀體纖維呈長纖維,排列緊密而有序。3.晶狀體SOD、MDA檢測:術(shù)后各時間段實驗組晶狀體SOD、MDA含量與對照組比較均無明顯差異(P0.05)。4.TUNEL檢測晶狀體上皮凋亡細(xì)胞:術(shù)后各時間段均未見晶狀體上皮凋亡細(xì)胞。結(jié)論:1.HIFU角膜基質(zhì)術(shù)后未出現(xiàn)角膜感染、穿孔,術(shù)后1天至60天角膜焦域內(nèi)組織形態(tài)改變,術(shù)后90天恢復(fù)正常,表明HIFU應(yīng)用于角膜改變角膜曲率是安全可靠的。2.術(shù)后一段時間內(nèi)角膜Ⅰ型膠原減少、MMP-2與MMP-9活化、部分角膜細(xì)胞凋亡、電鏡下角膜顯微結(jié)構(gòu)改變,說明HIFU輻照角膜基質(zhì)產(chǎn)生熱效應(yīng),在一段時間內(nèi)引起新生膠原修復(fù)重塑,角膜成纖維細(xì)胞活化,角膜細(xì)胞凋亡,均在術(shù)后90天恢復(fù)正常,表明HIFU角膜基質(zhì)術(shù)具有較好的安全性。3.術(shù)后各時間段,晶狀體均保持透明,組織形態(tài)無改變,SOD及MDA含量正常,無晶狀體上皮凋亡細(xì)胞,表面HIFU角膜基質(zhì)術(shù)能精確、安全地改變角膜曲率,不會對晶狀體產(chǎn)生影響。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R77

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