血管抑素与丝裂霉素C对原发性翼状胬肉作用的研究

发布时间：2018-01-16 05:17

本文关键词：血管抑素与丝裂霉素C对原发性翼状胬肉作用的研究　出处：《南方医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：研究背景：翼状胬肉是眼科的常见病、多发病,为结膜组织的增生变性疾病。当翼状胬肉不断进展,会牵拉角膜形成散光,影响视力及患者的美观,其次,因为肥厚的体部造成眼部异物感、流泪等不适症状都促使患者就医。翼状胬肉的具体发病机制还不是十分明确,但是随着科学技术的不断发展,对翼状胬肉的发病机制的研究越来越深入,越来越多的研究显示：翼状胬肉是一种增生性疾病。从近几年的病理学和病理生理学研究可发现：新生血管和纤维结缔组织是翼状胬肉上皮下的主要结构。特别是最近国外一些关于翼状胬肉细胞因子的基础学研究结果显示：血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)标记阳性的细胞数量和密度在翼状胬肉组织中含量明显比正常结膜组织中的增多及碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)具有促进微血管内皮细胞VEGF合成和分泌作用,而它存在于成纤维细胞培养上清液中。从这些基础研究我们可以看出新生血管的形成在翼状胬肉的发生发展中具有非常重要的作用,以及新生血管或血管内皮细胞和成纤维细胞之间存在的密切关系,以上这些因素共同参与并促进了翼状胬肉的形成和发展。由以上研究及发现可知,新生血管形成对于翼状胬肉发生、发展具有重要的促进作用。在新生血管的形成过程中,血管新生刺激因素和抑制因素缺一不可。目前发现较多的血管新生刺激因素包括多种生长因子和细胞因子,如VEGF、成纤维生长因子(FGF)等。血管新生抑制因素主要由血管抑素,内皮抑素等组成。血管抑素(Angiostation AS)是被国外学者发现的目前最强有力的最重要的新生血管生成抑制剂之一,可以抑制基质增强的纤溶酶原的活性导致细胞侵犯。多项实验研究均显示血管抑素作用的直接靶点为新生血管的内皮细胞,而对其他细胞及正常静止状态的内皮细胞无抑制作用。从理论上可以认为血管抑素通过抑制新生血管形成达到辅助治疗抑制复发的目的。虽然对于翼状胬肉的主要治疗方案仍是手术治疗,但是近年来根据对翼状胬肉的发病机制研究、动物实验、细胞培养和临床实验研究,并针对发病机制和原理,出现多种治疗方法及治疗药物,以期望尽量降低术后复发率以致避免复发的可能性,主要包括：丝裂霉素C (MMC)应用、激素治疗、冷冻治疗、激光治疗。虽然目前对于翼状胬肉的治疗方法多样,也有一定降低复发率的效果,但仍然未有一种疗效较为确切、实用,复发率低的治疗方法。本课题主要目的是通过配制血管抑素眼用制剂联合应用MMC,以研究血管抑素眼用制剂联合应用MMC对翼状胬肉的术后复发的抑制作用及可能机制。主要研究内容：一、血管抑素与丝裂霉素对离体翼状胬肉的作用研究 (一)目的：血管抑素(AS)联合丝裂霉素C (MMC)对离体培养的翼状胬肉组织的作用研究。 (二)方法：将20例临床手术切除的原发性翼状胬肉标本浸润于DHanks液中,然后利用组织块培养法采用含20%小牛血清的DMEM培养基将细胞常规培养于温箱中。待原代细胞贴壁培养生长至基本铺满培养瓶底时用0.25%胰酶消化,按比例1：2继续传代培养,取第3代或第4代细胞用SABC法进行抗波形蛋白免疫细胞化学染色进行细胞鉴定,鉴定后用于实验。用MTT法测定血管抑素、丝裂霉素C对人翼状胬肉细胞增殖的影响：实验分为四组：分别为A组(空白对照培养组)、B组(50ml/L血管抑素培养组)、C组(0.2mg/mL丝裂霉素培养组)、D组(50ml/L血管抑素与0.2mg/mL丝裂霉素联合培养组),主要观察翼状胬肉细胞在各培养组的生长情(包括数量和形态等情况)及吸光度值情况,计算不同时间、不同质量浓度的血管抑素对人翼状胬肉细胞的抑制率,记录实验结果；以时间为横轴,光吸收值为纵轴绘制细胞生长曲线；通过观察用药后24h、48h、72h三组翼状胬肉细胞中VEGF蛋白及bFGF蛋白的表达含量比较抑制率；通过RT-PCR分析总的mRNA的表达情况,以Folds=2-△△Ct表示VEGF与bFGF mRNA在实验组与对照组中表达的倍比关系；通过Western blot探讨VEGF与bFGF总蛋白的表达情况,根据标准曲线计算出蛋白浓度。采用SPSS13.0统计软件包分析,所得数据以(?)±s表示,MTT实验数据采用析因设计资料方差分析,PCR数据采用两两配对检验；以P0.05表示在统计学上有显著性差异。 (三)结果： 1、MTT法测定血管抑素丝裂霉素对人翼状胬肉细胞增殖的影响不同药物作用于人翼状胬肉细胞24h后,可见A组细胞排列紧密,增生明显,有连成片的趋势；B组与C组细胞呈点片状增生,排列疏密有致；D组细胞呈稀疏状排列。A490吸光度值均低于对照组,对人翼状胬肉增殖的抑制作用随着各实验药物的改变而不同。D组的人翼状胬肉细胞于24,48,72h后较A、B、C三组明显受抑制,实验组吸光度值均低于对照组,各组之间经统计学检验具有统计学差异(F=433.4P0.01)；各实验组的抑制作用较对照组明显增强,B、C组抑制作用相当,D组的抑制作用最强,且各实验组的抑制作用随时间的延长逐渐增强；72h时最大抑制率达到80.00%,且与对照组相比差异均有显著性意义(P0.01)。说明血管抑素联合丝裂霉素对离体培养的翼状胬肉细胞的抑制作用高于前三组。 2、实时荧光定量PCR结果以2-ΔΔct作为统计量,实验组中的VEGF mRNA与bFGF mRNA总和与对照组的表达比较有显著差异(P=0.000)；B组与C组中的VEGF mRNA与bFGF mRNA总和的表达无显著差异(P=0.931)；A与D组比较存在显著差异(P=0.000)；B组、C组与D组比较存在显著差异(t=-6.904,P=0.002)。与A组比较B、C、D组VEGF与bFGF mRNA的表达分别下降了约40%、40%、73%。D组总mRNA明显高于A、B、C三组。 3.各组VEGF与bFGF,总蛋白表达的影响 Western blot结果显示各实验组作用人翼状胬肉细胞后,三组中VEGF蛋白表达含量不同,且随着实验组添加药物不同而蛋白表达不同,D组的蛋白表达量明显低于A、B、C三组。 (四)结论血管抑素与丝裂霉素对翼状胬肉组织均有抑制作用,当两种药物同时使用时,抑制作用明显增强；为在人体翼状胬肉患者中应用奠定了基础。但是对于该两种药物不同浓度梯度的抑制作用及正常结膜组织的损伤等作用未能通过实验中进一步分析。二、血管抑素联合丝裂霉素C防止原发性翼状胬肉术后复发的临床疗效观察 (一)目的：观察血管抑素(Angiostatin AS)联合丝裂霉素C(Mitomycin MMC)防止原发性翼状胬肉术后复发的临床疗效。 (二)方法：根据拟定的纳入标准,选自2008年2月至2011年8月在我院就诊的原发性翼状胬肉患者365例(369眼),其中男性187例(189眼),女性178例(180眼),年龄35-75岁。随机分成3组,分别采用A组行逆行性翼状胬肉切除术联合丝裂霉素(121眼),B组行逆行性翼状胬肉切除术联合血管抑素(122眼),C组行逆行性翼状胬肉切除术联合丝裂霉素及血管抑素(126眼)等方法治疗,按Prabhasawat等标准进行裂隙灯下疗效评定。365例患者均为初次手术,手术均在显微镜下由同一医师完成：常规消毒、铺巾、麻醉后,利用弯剪充分游离翼状胬肉与巩膜面后,用有齿镊夹住翼状胬肉的颈部,将胬肉头部从角膜面旋撕分离。残余组织用显微无齿镊夹起撕掉,分离并剪除胬肉体部结膜下的纤维组织,烧灼止血暴露术区巩膜3mm×4mm。其中A组MMC的应用：将浸有新配制的0.2mg/mL MMC的棉片放于胬肉体、根部球结膜下巩膜暴露区范围,5min后去除棉片,用100m1生理盐水彻底冲洗术区,将其游离缘间断缝合两针固定于浅层巩膜上；B组：结膜缝合方法同A组,术后第1d开放点眼,30μg/mL浓度的血管抑素溶液,用药1周；C组：MMC应用同A组,血管抑素应用同B组。术后给予常规抗感染、营养促修复等治疗,随访12～18个月,比较其复发率。药品：日本株式会社生产丝裂霉素C2mg/瓶,制备0.2mg/mL MMC溶液。血管抑素原液由中山大学生命科学院提供,制备成30μg/mL浓度的溶液。所制备的药品置于4℃冰箱保存(最长时间不超过1wk)备用。使用SPSS13.0统计软件处理,采用X2检验,P0.05为差异有统计学意义。 (三)、结果观察12-18月,A组治愈121眼,复发9眼,复发率为7.4%,其中2眼复发于术后前3个月,其余7眼复发于第9-12个月,术后轻度不适12只眼；B组治愈122眼,复发7眼,复发率为5.7%,其中2眼复发于术后前3个月,其余5眼复发于第9-12个月,术后轻度不适10只眼；C组治愈126眼,复发1眼,复发率为：0.8%,术后12个月复发,术后轻度不适11只眼；三组均未见严重术后并发症发生。统计学结果示：经多个独立样本率x2检验得出X2=6.731P=0.035(P0.05),3组间差异具有显著性意义,可认为不同的治疗方法的复发率有显著差异,根据平均秩次进一步得出：逆行性切除法联合AS及MMC治疗方法复发率最低,其次是逆行性切除法联合AS,最后是逆行性切除法联合MMC。 (四)结论逆行性翼状胬肉手术联合丝裂霉素C及血管抑制素治疗原发性翼状胬肉,其疗效显著,复发率低,值得推广。
[Abstract]:Background of Study : Pterygium is a common disease in ophthalmology , and is a disease of hyperplasia and degeneration of conjunctival tissue . When Pterygium is continuously progressing , the cornea can be pulled to form astigmatism , which affects the vision and the beauty of the patient , and secondly , because the hypertrophy body part causes discomfort symptoms such as eye discomfort , tears , etc . , the patient is prompted to see a doctor . The specific pathogenesis of Pterygium is not very clear , but with the development of science and technology , the research on the pathogenesis of Pterygium is more and more deeply , and more and more studies have shown that the quantity and density of vascular endothelial growth factor ( VEGF ) - positive cells are the main structures in the formation of Pterygium . The results of these studies show that the formation of vascular endothelial growth factor ( VEGF ) is very important in the development of Pterygium . It is known from the above studies and findings that the formation of angiogenesis plays an important role in the development of Pterygium . In the course of the formation of neovascularization , the factors of angiogenesis and the inhibition of angiogenesis are not indispensable . At present , it is found that many angiogenic factors include various growth factors and cytokines , such as VEGF , fibroblast growth factor ( FGF ) , etc . Although the main treatment plan for Pterygium is still surgical treatment , in recent years , according to the research on the pathogenesis , animal experiment , cell culture and clinical experiment of Pterygium , and aiming at the pathogenesis and principle , there are a variety of treatment methods and therapeutic drugs , so as to reduce the recurrence rate as far as possible , including : mitomycin C ( MMC ) application , hormone therapy , cryotherapy and laser therapy . The purpose of this study was to study the inhibitory effect and possible mechanism of MMC on recurrence of Pterygium with MMC in combination with MMC . Main research content : Study on the Effect of One , Angiostatin and Mitomycin on Pterygium ( i ) Purpose : Effects of angiostatin ( AS ) and mitomycin C ( MMC ) on the tissue of Pterygium in vitro . ( II ) Method : The expression of VEGF and bFGF was determined by means of MTT assay . The results were as follows : A group ( blank control group ) , group B ( 50 ml / L angiostatin culture group ) , group C ( 0.2 mg / mL mitomycin C culture group ) , group D ( 50 ml / L angiostatin and 0.2 mg / mL mitomycin C ) were used . ( 3 ) Result : 1 . Effect of angiostatin mitomycin C on proliferation of human Pterygium root cells by MTT assay The results showed that the inhibitory effect of angiostatin combined with mitomycin C was higher than that of the control group , and the inhibitory effect of group B and C was significantly higher than that of the control group . The inhibitory effect of the experimental group was significantly higher than that of the control group . 2 . Real - time Fluorescence Quantitative PCR Results The expression of VEGF mRNA and bFGF mRNA in the experimental group was significantly different from that of the control group ( P = 0.000 ) . There was a significant difference between the expression of VEGF mRNA and bFGF mRNA in group B and C ( P = 0.931 ) . There was a significant difference between group A and D ( t = - 6.904 , P = 0.002 ) . Compared with group A , the expression of VEGF and bFGF mRNA in group B , C and D decreased by about 40 % , 40 % and 73 % respectively . 3 . Effects of VEGF and bFGF and total protein expression in each group Western blot showed that the expression of VEGF protein was different in three groups , and the expression of VEGF in group D was significantly lower than that of group A , B and C . ( IV ) Conclusion The inhibitory effect of angiostatin and mitomycin C on the tissue of Pterygium is obviously enhanced when the two drugs are simultaneously used . However , the inhibition of different concentration gradients and the damage of normal conjunctival tissue in the patients with Pterygium in the human body fail to be further analyzed in the experiment . Clinical observation of angiostatin combined with mitomycin C in preventing recurrence of primary Pterygium ( i ) Purpose : To observe the clinical effect of angiostatin combined with mitomycin C ( Mitomycin MMC ) on the prevention of recurrence of primary Pterygium . ( II ) Method : A total of 365 cases ( 369 eyes ) of Pterygium incited from February 2008 to August 2011 were divided into three groups . ( iii ) Results The recurrence rate was 7 . 4 % . The recurrence rate was 7 . 4 % in group A . The recurrence rate was 7 . 4 % . The recurrence rate was 0 . 8 % in group B . The recurrence rate was 0 . 035 ( P 0 . 05 ) . ( IV ) Conclusion The treatment of Pterygium combined with mitomycin C and angiostatin in the treatment of primary Pterygium with obvious curative effect and low recurrence rate is worthy of popularization .

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R777.33

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