本文選題：康柏西普　切入點：兔眼小梁切除術(shù)　出處：《西南醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)的受體能與抗血管內(nèi)皮生長因子藥物康柏西普結(jié)合,使VEGF失去生物活性,從而對新生血管的生成和纖維組織的增生產(chǎn)生抑制作用。本實驗通過研究不同劑量的康柏西普對兔眼小梁切除術(shù)后濾過泡及眼壓的改變,來探討其抑制濾過道纖維瘢痕形成的效果,及其與絲裂霉素C(MMC)作用的比較,探討青光眼術(shù)后結(jié)膜下注射康柏西普能否能成為抗濾過道瘢痕形成的新手段。方法:1.選擇健康成年大耳兔30只(60只眼),體重2.0-2.5kg,雌雄不限,測量基礎(chǔ)眼壓后行小梁切除術(shù),成功制作兔眼抗青光眼手術(shù)動物模型。30只兔(60只眼)隨機分為五組,且每只兔子雙眼所用藥物不同,并于術(shù)中兔耳處用標(biāo)記筆做標(biāo)記:第一組:6只兔右眼術(shù)畢后將0.1mL生理鹽水注射于結(jié)膜下,左眼于術(shù)中鞏膜瓣下放置浸有0.2g/L的絲裂霉素的棉片3分鐘,后用生理鹽水沖洗;第二組:6只兔右眼于術(shù)中鞏膜瓣下放置浸有0.2g/L的絲裂霉素的棉片3分鐘,后用生理鹽水沖洗,左眼術(shù)畢后將0.025mL康柏西普于注射于結(jié)膜下;第三組:6只兔右眼術(shù)畢后將0.025mL康柏西普注射于結(jié)膜下,左眼術(shù)畢后將0.05mL康柏西普注射于結(jié)膜下;第四組:6只兔右眼術(shù)畢后將0.05m L康柏西普注射于結(jié)膜下,左眼術(shù)畢后將0.075mL康柏西普注射于結(jié)膜下;第五組:6只兔右眼術(shù)畢后將0.075ml康柏西普注射于結(jié)膜下,左眼術(shù)畢將0.1ml生理鹽水注射于結(jié)膜下。2.術(shù)后在不同時間點觀察濾過道傷口的愈合及結(jié)膜的充血情況,眼前節(jié)相關(guān)反應(yīng)情況,包括角膜是否有水腫、前房深度及是否有炎性反應(yīng)、晶狀體透明度,觀察濾過泡在不同時間點的形態(tài),照相記錄,于不同時間點監(jiān)測眼壓,并記錄。術(shù)后在不同時間點分批處死兔子(術(shù)后7天、15天、30天),每次每組隨機選取2只,共10只;將處死后的兔子完整取下眼球固定于4%甲醛固定液中24h,切取術(shù)區(qū)組織,包埋、切片后行伊紅、免疫組化及苦味酸-天狼星玫瑰紅染色。通過伊紅染色可以觀察到術(shù)眼濾過通道炎癥細(xì)胞浸潤情況,高倍鏡下(×200倍)進行觀察、照相。經(jīng)過苦味酸-天狼星玫瑰紅染色的切片在偏振光顯微鏡下可以觀察到術(shù)區(qū)Ⅰ型和Ⅲ型膠原纖維(偏振光顯微鏡下Ⅰ型膠原纖維呈現(xiàn)出紅色或黃色雙折光,Ⅲ型呈現(xiàn)出藍(lán)綠色雙折光)。每張切片放置于高倍鏡下(×200倍),于術(shù)區(qū)組織處隨機選擇5個視野,應(yīng)用圖像分析軟件image-proplus7.0,分析兔眼濾過通道術(shù)后不同時間點膠原纖維增生情況,以偏振光顯微鏡高倍鏡(×200)下選取的視野中增生膠原纖維所占視野面積的百分比作為標(biāo)準(zhǔn)。通過免疫組化染色,可以觀察到術(shù)區(qū)不同時間點成纖維細(xì)胞的增生情況(標(biāo)準(zhǔn)為細(xì)胞核內(nèi)有棕黃色顆粒沉著者)。結(jié)果:1.由同一經(jīng)驗豐富的青光眼手術(shù)醫(yī)生制作兔眼抗青光眼手術(shù)模型60只眼。試驗結(jié)果顯示各組在術(shù)后眼前節(jié)變化情況(角膜、前房、晶狀體等相關(guān)情況)在相同時間點比較,各組間變化無明顯差異。術(shù)后7天內(nèi),各試驗組兔眼術(shù)區(qū)結(jié)膜都有不同程度的充血,濾過泡均隆起、彌散(功能性濾泡),各組間比較差異無統(tǒng)計學(xué)意義(p0.05);7天后,生理鹽水組濾泡逐漸變小、扁平,直至術(shù)后30天,生理鹽水組術(shù)區(qū)濾泡逐漸包裹呈囊樣,瘢痕明顯,壁厚,血管豐富(非功能性濾泡);7天后康柏西普0.025ml組濾泡變小、扁平,面積較前有縮小,但大部分為功能性濾泡;而康柏西普0.05ml組、康柏西普0.075ml組及mmc組均呈壁薄、隆起、彌散樣的功能性濾泡,術(shù)后30天康柏西普0.025ml組、康柏西普0.05ml組、康柏西普0.075ml組分別與生理鹽水組比較差異有統(tǒng)計學(xué)意義(p0.01)。術(shù)后第1天各組眼壓比術(shù)前明顯下降,與術(shù)前眼壓比較差異皆有統(tǒng)計學(xué)意義(p0.01),各組間差異無統(tǒng)計學(xué)意義(p0.05);術(shù)后15天后生理鹽水組眼壓逐漸升高,至術(shù)后30天,眼壓接近正常,與術(shù)前眼壓比較差異無統(tǒng)計學(xué)意義(p0.05)。康柏西普0.025ml組在術(shù)后15天后眼壓逐漸升高,但較術(shù)前偏低;而康柏西普0.05ml組、康柏西普0.075ml組及mmc組眼壓仍保持穩(wěn)定、較低水平;術(shù)后30天康柏西普0.05ml組、0.075ml組眼壓分別與康柏西普0.025ml組、生理鹽水組眼壓比較差異有統(tǒng)計學(xué)意義(p0.01),康柏西普0.025ml組眼壓與生理鹽水組眼壓比較差異有統(tǒng)計學(xué)意義(p0.01),康柏西普0.05ml組、康柏西普0.075ml組眼壓及mmc組眼壓比較差異無統(tǒng)計學(xué)意義(p0.05)。2.術(shù)后于不同時間點分批處死兔子(分別于術(shù)后7天、15天、30天),完整取下眼球后固定,切取術(shù)區(qū)組織包埋、切片、染色。(1)組織切片行伊紅染色后結(jié)果顯示:各組術(shù)后術(shù)區(qū)結(jié)膜下均可見有不同程度的炎癥細(xì)胞浸潤,主要包括有淋巴細(xì)胞、漿細(xì)胞(淋巴細(xì)胞小而圓,核漿比很大,核濃染成深藍(lán)紫色;漿細(xì)胞胞漿略嗜堿,核染色深、偏位,大小介于淋巴細(xì)胞和巨噬/多核/巨細(xì)胞之間)。在生理鹽水組術(shù)區(qū)球結(jié)膜下有大量炎癥細(xì)胞浸潤,康柏西普各劑量組及mmc組術(shù)區(qū)球結(jié)膜下組織結(jié)構(gòu)稀疏,可見有少量炎癥細(xì)胞浸潤。(2)組織切片行免疫組化染色后結(jié)果顯示:康柏西普0.05ml組、康柏西普0.075ml組及mmc組濾過道的成纖維細(xì)胞數(shù)量在相同時間點比較要少于康柏西普0.025ml組�？蛋匚髌�0.05ml組、康柏西普0.075ml組及mmc組濾過道合成在相同時間點合成的成纖維細(xì)胞數(shù)基本相同,在相同時間點比較,組間差異無統(tǒng)計學(xué)意義(p0.05)�？蛋匚髌�0.05ml組分別與康柏西普0.025ml組及生理鹽水組濾過道的成纖維細(xì)胞生成數(shù)量,在相同時間點組間比較差異有統(tǒng)計學(xué)意義(p0.01);康柏西普0.025ml組及生理鹽水組濾過道合成的成纖維細(xì)胞相同時間點組間比較差異有統(tǒng)計學(xué)意義(p0.01)。(3)切片組織行苦味酸-天狼星玫瑰紅染色結(jié)果顯示:各組術(shù)區(qū)組織增生的膠原纖維(Ⅰ型、Ⅲ型)隨著時間的延長,膠原纖維的含量逐漸增加,康柏西普0.05ml組、康柏西普0.075ml組及mmc組濾過道合成Ⅰ型、Ⅲ型膠原纖維含量相對稀少、規(guī)則,在相同時間點組間比較差異無統(tǒng)計學(xué)意義(p0.05)。康柏西普0.05ml組分別與康柏西普0.025ml組及生理鹽水組濾過道合成的Ⅰ型、Ⅲ型膠原纖維相同時間點組間比較差異有統(tǒng)計學(xué)意義(p0.01);生理鹽水組濾過道合成的Ⅰ型、Ⅲ型膠原纖維相對致密、紊亂,與康柏西普各劑量組于相同時間點比較差異有統(tǒng)計學(xué)意義(p0.01)。結(jié)論:1.小梁切除術(shù)后采用濾過泡旁結(jié)膜下注射康柏西普的方法有助于減少濾過道新生血管形成,抑制濾過道瘢痕化,維持濾過泡的形態(tài)。2.康柏西普對眼部組織無明顯毒副作用。3.0.05mL、0.075m L劑量的康柏西普抗瘢痕作用強于0.025mL的劑量;而0.05mL康柏西普劑量與0.075mL康柏西普劑量抗瘢痕作用無明顯差異。4.康柏西普(0.05mL,0.075mL)能有效的在兔眼抗青光眼術(shù)后的初始愈合階段抑制新生血管的生成,最終有效的減少成纖維細(xì)胞的增殖和細(xì)胞的膠原蛋白Ⅰ型、Ⅲ型的合成,實驗表明康柏西普可通過抗VEGF從而抑制瘢痕的形成來防止濾過道瘢痕化。
[Abstract]:Objective: vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) receptor can bind with anti VEGF drugs lose VEGF Compaq SEPR, biological activity, and thus the angiogenesis and inhibit the proliferation of fibrous tissue. Changes of filtering bleb and intraocular pressure in experimental trabeculectomy in rabbits through the study of different dose of Compaq SEPR, to investigate the inhibitory effect of filtering the formation of fibrous scar, and mitomycin C (MMC) to investigate the effect of comparison after glaucoma surgery with subconjunctival injection of Kang Baixi could become a universal filter anti scarring. Methods: 1. healthy adult rabbits 30 (60 eyes), weight 2.0-2.5kg, male or female, measured intraocular pressure after trabeculectomy, the successful production of Rabbit anti glaucoma surgery animal model.30 rabbits (60 eyes) were randomly divided into five groups, and Each rabbit eyes with different drugs, and in operation at the rabbit ears with the marker mark: the first group: 6 rabbits right after surgery 0.1mL saline injection in the left eye under the conjunctiva, in operation under the scleral flap placed into a piece of 0.2g/L mitomycin C for 3 minutes after saline water flushing; second group: 6 rabbits in the right eye surgery under the scleral flap is placed into a piece of 0.2g/L mitomycin C for 3 minutes after washing with normal saline, left eye after surgery 0.025mL injection under conjunctiva in Compaq SEPR; the third group: 6 rabbits right eye after surgery 0.025mL Xipu injection on Compaq under the conjunctiva, the left eye after surgery will be injected into subconjunctival 0.05mL Compaq SEPR; fourth group: 6 rabbits right after operation 0.05m L was injected into subconjunctival Compaq SEPR, left after surgery will be injected into subconjunctival 0.075mL Compaq SEPR; the fifth group: 6 rabbits right after operation 0.075ml Compaq SEPR injection In the left eye surgery under conjunctiva, 0.1ml saline injected into subconjunctival.2. after surgery were observed at different time points and wound healing conjunctival filtration of congestion, anterior segment related reactions, including whether corneal edema, anterior chamber depth and inflammatory reaction is, lens transparency, bleb in observation different time points form, recorded at different time points, monitoring and record the postoperative IOP. Rabbits were killed at different time periods (7 days, 15 days after operation, 30 days), each group 2 rats were randomly selected, a total of 10 rabbits were sacrificed; the complete remove eye fixed on the 4% formalin 24h, tissue cutting, surgical embedding, sections were stained by eosin and immunohistochemical staining and picrosirius red staining. Rose by eosin staining can be observed in inflammatory cell infiltration of eye filtering, high magnification (* 200 times) were observed by photography. After Sirius rose red stained sections can be observed in the operation area of type I and type III collagen fibers in polarized light microscope (type I collagen fibers under polarized light microscopy showed a red or yellow double refraction type blue-green double refraction). Each section is placed in a high magnification (* 200 times in the operation area), group 5 randomly selected vision, using image analysis software image-proplus7.0, analysis of filtering channels in rabbits at different time points after implantation of collagen fiber hyperplasia, with polarized light microscopy at high magnification (* 200) proliferation of collagen fibers selected in the view of the percentage of the area in the visual field as the standard. Through the immune group staining can be observed in the operation area, different time points into fiber cell hyperplasia (standard cell nucleus brown yellow granules were). Results: 1. by the same experienced doctor glaucoma surgery in rabbits Eye anti glaucoma surgery in 60 eyes. The experimental results show that the model was in front of the postoperative section changes (cornea, anterior chamber, lens and other relevant circumstances) comparison points at the same time, there was no significant difference between the changes. 7 days after operation, the experimental group of rabbit conjunctival area has a different degree of congestion, filtration bubbles uplift, diffusion (follicular function), no statistically significant differences between the two groups (P0.05); 7 days later, the saline group follicles became smaller, flat, until 30 days after surgery, saline group operation area gradually showed cystic follicular package, obvious scar thickness, rich blood vessels (non functional follicular); 7 days later, Compaq SIPP 0.025ml group follicular becomes small, flat, narrow area than ever before, but for the majority of functional follicles; while Compaq SEPR group 0.05ml, Compaq SEPR 0.075ml group and MMC group showed a thin wall, uplift, functional follicular dispersive samples, after 30 days of Compaq SEPR 0 Group 25ml, Compaq SEPR 0.05ml group, the difference was statistically significant compared with saline group group 0.075ml Xipu Compaq (P0.01). First days after operation were decreased significantly compared with the preoperative IOP, and preoperative intraocular pressure difference has statistical significance (P0.01), each group showed no statistically significant difference (P0.05); after 15 days of saline group IOP gradually increased to 30 days after surgery, intraocular pressure close to normal, no statistically significant differences compared with the preoperative intraocular pressure (P0.05). 0.025ml Xipu Compaq group after 15 days after the intraocular pressure increased gradually, but compared with the preoperative low; while Compaq Compaq SEPR Xipu 0.05ml group, 0.075ml group and intraocular pressure of group MMC remained stable, low level; after 30 days of Xipu Compaq 0.05ml group, 0.075ml group and 0.025ml group respectively Compaq SEPR intraocular pressure, intraocular pressure of saline group was statistically significant difference (P0.01), general Kang Baixi IOP of 0.025ml group and saline group There are statistically significant differences between intraocular pressure (P0.01), Compaq SEPR 0.05ml group, no significant Compaq intraocular pressure of group 0.075ml and group MMC Xipu intraocular pressure difference (P0.05) after.2. in rabbits were sacrificed at different time points (respectively after 7 days, 15 days, 30 days), remove the eye after complete fixed. The operation area of tissue cutting and embedding, slicing and staining. (1) tissue section stained by eosin staining results showed that: the conjunctival operation area of each group showed different degrees of inflammatory cell infiltration, including lymphocytes, plasma cells and small lymphocytes (round, karyoplasmic ratio, nuclear trachychromatic blue purple pulp; cytoplasm slightly basophilic, nuclear staining deep, deviation, size between lymphocytes and macrophages / nuclear / between giant cells in the saline group). Postoperative conjunctival inflammatory cell infiltration, the sparse structure of West Cape Compaq group and MMC group were under conjunctiva, There are few infiltration of inflammatory cells. (2) tissue sections for immunohistochemical staining showed that 0.05ml group Xipu Compaq Compaq Xipu, 0.075ml group and MMC group of filtering passageway the number of fibroblasts at the same time point compared to less than 0.025ml group. Compaq Compaq SEPR Xipu 0.05ml group, 0.075ml group and MMC group Compaq SEPR at the same time the synthesis of filtering bleb in synthesis of fibroblasts is basically the same number of points of comparison, at the same time, there were no significant differences between groups (P0.05). 0.05ml group and the number of Compaq SEPR fiber cells into 0.025ml Compaq SEPR group and the saline group of filtering passageway, at the same time there are differences among the groups statistical significance (P0.01); Compaq SEPR 0.025ml group and saline group filtering bleb fibroblasts synthesis at the same time the difference between groups was statistically significant (P0.01). (3) slice tissue by picric acid Sirius red staining showed that the hyperplasia of area of each collagen (type I, III) with the extension of time, the content of collagen fibers increased gradually, Compaq SEPR 0.05ml group, 0.075ml group and MMC group Compaq SEPR filter corridor is relatively scarce, the synthesis of type I, type III collagen fiber content dimensional rules. At the same time there was no significant difference between groups (P0.05). Group 0.05ml synthesis and Compaq Compaq SEPR Xipu 0.025ml group and saline group respectively filtering bleb type 1, type III collagen fibers at the same time the difference between groups was statistically significant (P0.01); saline group of synthesis filter aisle type III collagen fiber is relatively dense, disorder, and Compaq west each dose group P at the same time the difference was statistically significant (P0.01). Conclusion: 1. after trabeculectomy using the method of bleb beside the subconjunctival injection of Xipu Compaq will help To reduce filtration angiogenesis, inhibit scarring, maintain the morphology of.2. bleb Compaq SEPR no obvious side effect of.3.0.05mL on ocular tissue, anti cicatricial effect 0.075m L dose in the dose of 0.025mL Compaq SEPR; 0.05mL Compaq SEPR 0.075mL Compaq SEPR dose dose and the antiproliferative effects of.4. had no significant difference. Compaq SEPR (0.05mL, 0.075mL) is effective in anti glaucoma surgery in rabbit eyes after the initial healing stage to inhibit tumor angiogenesis, and ultimately reduce the collagen type I cell proliferation and fiber cells, the synthesis of type III, Compaq SEPR experiments show that to prevent scarring by forming and anti VEGF inhibition of scar.

【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R779.6

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