康柏西普对兔眼小梁切除术后滤过通道瘢痕形成的影响

发布时间：2018-03-02 23:29

本文选题：康柏西普　切入点：兔眼小梁切除术　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:血管内皮生长因子(vascular endothelial growth factor,VEGF)的受体能与抗血管内皮生长因子药物康柏西普结合,使VEGF失去生物活性,从而对新生血管的生成和纤维组织的增生产生抑制作用。本实验通过研究不同剂量的康柏西普对兔眼小梁切除术后滤过泡及眼压的改变,来探讨其抑制滤过道纤维瘢痕形成的效果,及其与丝裂霉素C(MMC)作用的比较,探讨青光眼术后结膜下注射康柏西普能否能成为抗滤过道瘢痕形成的新手段。方法:1.选择健康成年大耳兔30只(60只眼),体重2.0-2.5kg,雌雄不限,测量基础眼压后行小梁切除术,成功制作兔眼抗青光眼手术动物模型。30只兔(60只眼)随机分为五组,且每只兔子双眼所用药物不同,并于术中兔耳处用标记笔做标记:第一组:6只兔右眼术毕后将0.1mL生理盐水注射于结膜下,左眼于术中巩膜瓣下放置浸有0.2g/L的丝裂霉素的棉片3分钟,后用生理盐水冲洗;第二组:6只兔右眼于术中巩膜瓣下放置浸有0.2g/L的丝裂霉素的棉片3分钟,后用生理盐水冲洗,左眼术毕后将0.025mL康柏西普于注射于结膜下;第三组:6只兔右眼术毕后将0.025mL康柏西普注射于结膜下,左眼术毕后将0.05mL康柏西普注射于结膜下;第四组:6只兔右眼术毕后将0.05m L康柏西普注射于结膜下,左眼术毕后将0.075mL康柏西普注射于结膜下;第五组:6只兔右眼术毕后将0.075ml康柏西普注射于结膜下,左眼术毕将0.1ml生理盐水注射于结膜下。2.术后在不同时间点观察滤过道伤口的愈合及结膜的充血情况,眼前节相关反应情况,包括角膜是否有水肿、前房深度及是否有炎性反应、晶状体透明度,观察滤过泡在不同时间点的形态,照相记录,于不同时间点监测眼压,并记录。术后在不同时间点分批处死兔子(术后7天、15天、30天),每次每组随机选取2只,共10只;将处死后的兔子完整取下眼球固定于4%甲醛固定液中24h,切取术区组织,包埋、切片后行伊红、免疫组化及苦味酸-天狼星玫瑰红染色。通过伊红染色可以观察到术眼滤过通道炎症细胞浸润情况,高倍镜下(×200倍)进行观察、照相。经过苦味酸-天狼星玫瑰红染色的切片在偏振光显微镜下可以观察到术区Ⅰ型和Ⅲ型胶原纤维(偏振光显微镜下Ⅰ型胶原纤维呈现出红色或黄色双折光,Ⅲ型呈现出蓝绿色双折光)。每张切片放置于高倍镜下(×200倍),于术区组织处随机选择5个视野,应用图像分析软件image-proplus7.0,分析兔眼滤过通道术后不同时间点胶原纤维增生情况,以偏振光显微镜高倍镜(×200)下选取的视野中增生胶原纤维所占视野面积的百分比作为标准。通过免疫组化染色,可以观察到术区不同时间点成纤维细胞的增生情况(标准为细胞核内有棕黄色颗粒沉着者)。结果:1.由同一经验丰富的青光眼手术医生制作兔眼抗青光眼手术模型60只眼。试验结果显示各组在术后眼前节变化情况(角膜、前房、晶状体等相关情况)在相同时间点比较,各组间变化无明显差异。术后7天内,各试验组兔眼术区结膜都有不同程度的充血,滤过泡均隆起、弥散(功能性滤泡),各组间比较差异无统计学意义(p0.05);7天后,生理盐水组滤泡逐渐变小、扁平,直至术后30天,生理盐水组术区滤泡逐渐包裹呈囊样,瘢痕明显,壁厚,血管丰富(非功能性滤泡);7天后康柏西普0.025ml组滤泡变小、扁平,面积较前有缩小,但大部分为功能性滤泡;而康柏西普0.05ml组、康柏西普0.075ml组及mmc组均呈壁薄、隆起、弥散样的功能性滤泡,术后30天康柏西普0.025ml组、康柏西普0.05ml组、康柏西普0.075ml组分别与生理盐水组比较差异有统计学意义(p0.01)。术后第1天各组眼压比术前明显下降,与术前眼压比较差异皆有统计学意义(p0.01),各组间差异无统计学意义(p0.05);术后15天后生理盐水组眼压逐渐升高,至术后30天,眼压接近正常,与术前眼压比较差异无统计学意义(p0.05)。康柏西普0.025ml组在术后15天后眼压逐渐升高,但较术前偏低;而康柏西普0.05ml组、康柏西普0.075ml组及mmc组眼压仍保持稳定、较低水平;术后30天康柏西普0.05ml组、0.075ml组眼压分别与康柏西普0.025ml组、生理盐水组眼压比较差异有统计学意义(p0.01),康柏西普0.025ml组眼压与生理盐水组眼压比较差异有统计学意义(p0.01),康柏西普0.05ml组、康柏西普0.075ml组眼压及mmc组眼压比较差异无统计学意义(p0.05)。2.术后于不同时间点分批处死兔子(分别于术后7天、15天、30天),完整取下眼球后固定,切取术区组织包埋、切片、染色。(1)组织切片行伊红染色后结果显示:各组术后术区结膜下均可见有不同程度的炎症细胞浸润,主要包括有淋巴细胞、浆细胞(淋巴细胞小而圆,核浆比很大,核浓染成深蓝紫色;浆细胞胞浆略嗜碱,核染色深、偏位,大小介于淋巴细胞和巨噬/多核/巨细胞之间)。在生理盐水组术区球结膜下有大量炎症细胞浸润,康柏西普各剂量组及mmc组术区球结膜下组织结构稀疏,可见有少量炎症细胞浸润。(2)组织切片行免疫组化染色后结果显示:康柏西普0.05ml组、康柏西普0.075ml组及mmc组滤过道的成纤维细胞数量在相同时间点比较要少于康柏西普0.025ml组。康柏西普0.05ml组、康柏西普0.075ml组及mmc组滤过道合成在相同时间点合成的成纤维细胞数基本相同,在相同时间点比较,组间差异无统计学意义(p0.05)。康柏西普0.05ml组分别与康柏西普0.025ml组及生理盐水组滤过道的成纤维细胞生成数量,在相同时间点组间比较差异有统计学意义(p0.01);康柏西普0.025ml组及生理盐水组滤过道合成的成纤维细胞相同时间点组间比较差异有统计学意义(p0.01)。(3)切片组织行苦味酸-天狼星玫瑰红染色结果显示:各组术区组织增生的胶原纤维(Ⅰ型、Ⅲ型)随着时间的延长,胶原纤维的含量逐渐增加,康柏西普0.05ml组、康柏西普0.075ml组及mmc组滤过道合成Ⅰ型、Ⅲ型胶原纤维含量相对稀少、规则,在相同时间点组间比较差异无统计学意义(p0.05)。康柏西普0.05ml组分别与康柏西普0.025ml组及生理盐水组滤过道合成的Ⅰ型、Ⅲ型胶原纤维相同时间点组间比较差异有统计学意义(p0.01);生理盐水组滤过道合成的Ⅰ型、Ⅲ型胶原纤维相对致密、紊乱,与康柏西普各剂量组于相同时间点比较差异有统计学意义(p0.01)。结论:1.小梁切除术后采用滤过泡旁结膜下注射康柏西普的方法有助于减少滤过道新生血管形成,抑制滤过道瘢痕化,维持滤过泡的形态。2.康柏西普对眼部组织无明显毒副作用。3.0.05mL、0.075m L剂量的康柏西普抗瘢痕作用强于0.025mL的剂量;而0.05mL康柏西普剂量与0.075mL康柏西普剂量抗瘢痕作用无明显差异。4.康柏西普(0.05mL,0.075mL)能有效的在兔眼抗青光眼术后的初始愈合阶段抑制新生血管的生成,最终有效的减少成纤维细胞的增殖和细胞的胶原蛋白Ⅰ型、Ⅲ型的合成,实验表明康柏西普可通过抗VEGF从而抑制瘢痕的形成来防止滤过道瘢痕化。
[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.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R779.6

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