后巩膜加固术治疗高度近视眼机理的力学生物学研究

发布时间：2018-06-04 18:10

  本文选题：高度近视 + 后巩膜加固术　； 参考：《太原理工大学》2011年博士论文

【摘要】：近视是常见的眼部疾病,有屈光性近视与进行性近视之分。其中,进行性近视又称高度近视或病理性近视,在人群中患病率很高,可发生很多严重并发症,是常见的致盲原因之一。后巩膜加固术能够提高后极部巩膜的强度,阻止眼轴进一步延长,最终阻止视功能进一步恶化,是治疗高度近视较为有效的一种方法。 有关后巩膜加固术的疗效说法不一,有必要对后巩膜加固术的治疗机理进行更深入、全面地研究。本文对建立的新西兰大白兔高度近视眼动物模型实施后巩膜加固手术,术后不同时期获取加固区巩膜组织与巩膜成纤维细胞,研究后巩膜加固术后巩膜及巩膜成纤维细胞蛋白的表达情况、巩膜成纤维细胞的增殖活性和粘弹性、巩膜成纤维细胞对力学刺激的响应,从力学-细胞生物学角度解释后巩膜加固术的治疗机理,这将为该手术在临床上的选择实施和推广提供理论指导。 本文的主要研究内容及结论如下： 1.选取3周龄未脱离母乳喂养的新西兰大白兔40只,随机、单眼进行眼睑缝合手术诱导建立高度近视眼模型,另一眼为对照眼。眼睑缝合后60天,打开眼脸,检验屈光度和测量眼轴长度。经过诱导后,实验眼与对照眼屈光度比较,诱导出1.80±0.72D的相对近视,眼轴相对延长了0.45±0.29mm,差异具有统计学意义(P0.05),表明动物模型建立成功。 2.对高度近视眼模型实施后巩膜加固手术,术后3个月和6个月提取加固区巩膜组织和正常巩膜组织,制备组织切片,通过免疫组化方法检测术后巩膜基质金属蛋白酶-2(MMP-2)及其特异性组织抑制剂(TIMP-2)、转化生长因子-β1 (TGF-β1)及碱性成纤维细胞生长因子(bFGF)的表达。检测结果显示：后巩膜加固术后巩膜组织MMP-2表达减少,而TIMP-2、TGF-β1及bFGF的表达增加。这些变化有利于巩膜成纤维细胞的增殖,抑制巩膜基质蛋白降解,促进胞外基质合成,使巩膜组织增厚。 3.组织块培养法提取并培养正常巩膜组织、术后3个月、6个月原巩膜组织与融合区组织巩膜成纤维细胞,用免疫细胞化学法对获取的细胞进行鉴定,结果显示Vimentin染色阳性,Desmin染色阴性,keratin染色阴性,S-100染色阴性,证实所获得的细胞为巩膜成纤维细胞。 采用ATP荧光检测法检测各组细胞的增殖活性；免疫细胞化学及酶联免疫吸附试验(ELISA)的方法联合检测各组巩膜成纤维细胞MMP-2、TIMP-2、TGF-β1及bFGF的表达；微管吸吮技术检测各组巩膜成纤维细胞的粘弹性参数(瞬时模量E0、平衡模量E∞和表观粘性μ)。研究发现： (1)后巩膜加固术后3个月和6个月巩膜成纤维细胞的增殖活性均比正常巩膜组织有明显提高(P0.05),且术后融合区巩膜成纤维细胞的增殖活性明显高于原巩膜组织(P0.05)。 (2)正常巩膜组织、术后3个月和6个月原巩膜组织的巩膜成纤维细胞之间MMP-2的表达无明显差异(P0.05),术后3个月和6个月融合区巩膜成纤维细胞MMP-2的表达明显低于术后原巩膜组织和正常巩膜组织(P0.05)；术后3个月和6个月原巩膜组织与融合区巩膜成纤维细胞TIMP-2、TGF-β1的表达要明显高于正常巩膜组织(P0.05)；术后3个月和6个月原巩膜组织与融合区巩膜成纤维细胞bFGF的表达要明显高于正常巩膜组织(P0.05),术后3个月和6个月融合区巩膜成纤维细胞bFGF的表达明显高于原巩膜组织(P0.05)；术后3个月和6个月原巩膜组织之间、术后3个月和6个月融合区之间巩膜成纤维细胞MMP-2、TIMP-2、TGF-β1及bFGF的表达均无明显差异(P0.05)。 (3)正常巩膜组织与术后3个月、6个月原巩膜组织巩膜成纤维细胞之间的各项粘弹性参数(E0、E∞和μ)无明显差异(P0.05),术后3个月、6个月融合区巩膜成纤维细胞的各项粘弹性参数(E0、E∞和μ)均小于术后原巩膜组织和正常巩膜组织(P0.05)。 实验结果表明：后巩膜加固术后加固区巩膜成纤维细胞的增殖活性提高,MMP-2表达减少,而TIMP-2、TGF-β1及bFGF的表达增加,与免疫组化结果是一致的,这些变化有利于巩膜成纤维细胞外基质的沉积和巩膜组织增厚。后巩膜加固术后原巩膜组织巩膜成纤维细胞的各项粘弹性参数(E0、E∞和μ)没有发生变化,而融合区新生巩膜成纤维细胞的各项粘弹性参数(E0、E∞和μ)较低。 4.取正常巩膜组织,术后6个月原巩膜组织、术后6个月融合区组织的巩膜成纤维细胞,采用FX-4000系统施加0.1Hz、幅度为3%和6%的周期性拉伸,拉伸48h后收集细胞及细胞上清液,检测各组细胞的增殖活性、MMP-2、TIMP-2、TGF-β1及bFGF的表达和粘弹性。研究发现： (1)经过拉伸培养之后正常巩膜组织和术后6个月原巩膜组织巩膜成纤维细胞的增殖活性与各自静态对照组比较有明显提高(P0.05),正常巩膜组织6%拉伸组细胞的增殖活性明显高于3%拉伸组(P0.05),术后6个月融合区巩膜成纤维细胞在经过力学环境培养后增殖活性与静态组比较无统计学差异(P0.05)。 (2)正常巩膜组织和术后6个月巩膜组织的巩膜成纤维细胞在经过力学环境培养后,MMP-2的表达减少,TIMP-2的表达增加(P0.05),而术后6个月融合区巩膜成纤维细胞MMP-2、TIMP-2的表达与静态组比较无明显差异(P0.05)；三组巩膜成纤维细胞在经过力学环境培养后,TGF-β1的表达均增加(P0.05)；正常巩膜组织和术后6个月巩膜组织的巩膜成纤维细胞在经过力学环境培养后,bFGF的表达增加(P0.05),术后6个月融合区巩膜成纤维细胞bFGF的表达与静态组比较无明显差异(P0.05)。 (3)正常巩膜组织与术后6个月原巩膜组织巩膜成纤维细胞在经过力学环境培养后,细胞的粘弹性参数(E0、E∞和μ)与各自静态培养的细胞相比显著降低(p0.05)；术后6个月融合区巩膜成纤维细胞力学环境培养后细胞的各项粘弹性参数(E0、E∞和μ)均增高(P0.05)；各组细胞3%拉伸组与6%拉伸组之间细胞的粘弹性参数(E0、E∞和μ)无明显差异(P0.05)；三组细胞的粘弹性参数(E0、E∞和μ)在经过力学拉伸后趋于相同。 实验结果表明：力学刺激参与调节后巩膜加固术后巩膜成纤维细胞的生物学和生物力学行为,能够提高正常巩膜及后巩膜加固术后原巩膜组织的增殖活性,且能够影响后巩膜加固术后不同区域巩膜成纤维细胞MMP-2、TIMP-2、TGF-β1及bFGF的表达及生物力学特性。力学刺激引起巩膜成纤维细胞的这些变化有利于巩膜胞外基质的沉积、巩膜成纤维细胞的增殖以及巩膜组织与加固条带之间融合,从而使巩膜组织增厚,提高巩膜的生物力学特性,控制近视的发展。
[Abstract]:Myopic myopia is a common eye disease , and has refractive myopia and progressive myopia . Among them , progressive myopia is also known as high myopia or pathological myopia , which is one of the most common causes of blindness . Posterior scleral reinforcement can improve the intensity of posterior sclera , prevent further prolongation of visual function , and prevent visual function from further worsening . It is a more effective method to treat high myopia .

In this paper , the mechanism of scleral reinforcement after posterior scleral reinforcement was studied in this paper . The mechanism of scleral reinforcement after scleral reinforcement after scleral reinforcement was studied . The mechanism of scleral reinforcement was explained from the perspective of mechanical - cell biology , which would provide theoretical guidance for the clinical selection and popularization of the operation .

The main research contents and conclusions are as follows :

1 . Forty - four New Zealand white rabbits who were not separated from breast - feeding were randomly and single - eye to establish a high myopia model , and the other one was the control eye . After induction , the eyes were opened , the dioptric power was measured and the length of the eye axis was measured . After induction , the relative myopia of 0.80 卤 0.72D was induced in the experimental eye and the control eye . The difference was statistically significant ( P0.05 ) , indicating that the animal model was established successfully .

The expressions of MMP - 2 and TIMP - 2 , TGF - 尾1 and bFGF in scleral tissues were detected by immunohistochemistry . The results showed that the expression of MMP - 2 and TIMP - 2 , TGF - 尾1 and bFGF increased . These changes were beneficial to the proliferation of scleral fibroblasts , inhibited the degradation of scleral matrix protein , promoted the synthesis of extracellular matrix , and thickened scleral tissue .

3 . Tissue culture method was used to extract and culture the normal scleral tissue . After 3 months of operation , the scleral fibroblasts were cultured in the scleral tissue and the fusion area . The results showed that Vimentin was positive , Desmin staining was negative , keratin staining was negative , S - 100 was negative , and it was confirmed that the obtained cells were scleral fibroblasts .

ATP fluorescence assay was used to detect the proliferative activity of each group of cells .
The expressions of MMP - 2 , TIMP - 2 , TGF - 尾1 and bFGF were detected by ELISA .
The viscoelastic parameters ( instantaneous modulus E0 , equilibrium modulus E 鈭，

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