IGF-1在糖尿病大鼠角膜创伤愈合过程中的机制研究

发布时间：2018-06-12 10:24

本文选题：胰岛素样生长因子-1 + 糖尿病　；参考：《山东大学》2010年硕士论文

【摘要】： 目的糖尿病角膜创伤愈合延迟是目前眼科临床最常见问题之一,治疗棘手,发病机制尚不明确。IGF-1可促进细胞迁移、增殖,抑制细胞凋亡,在创伤愈合中发挥重要作用。本实验通过建立糖尿病大鼠角膜创伤模型,检测创伤愈合过程中IGF-1的表达,从分子水平探求糖尿病角膜创伤愈合延迟的可能机制。方法1.建立糖尿病大鼠模型：65只雄性Wistar大鼠,体重170-200g,随机分为糖尿病组和正常对照组。糖尿病组腹腔注射链脲霉素(STZ)制作糖尿病大鼠模型(n=35),正常对照组腹腔注射等量柠檬酸盐缓冲液(n=30)。造模后饲养8周,每周测血糖和体重。2.建立大鼠角膜创伤模型：用直径5.0毫米的环钻在角膜中央标记创伤区,刮除标记区内的角膜上皮。3.分别在角膜上皮移除术后不同时间点(0h,12h,24h,36h,48h,60h,72h,4d-16d),在裂隙灯下观察角膜创伤愈合及感染情况；进行荧光素钠染色实验,拍照记录并分析角膜上皮创伤愈合率。4.分别在角膜上皮移除术后不同时间点(0h,12h,1d,2d,3d,4d,5d,7d)处死正常大鼠(n=24)及糖尿病大鼠(n=24),取角膜组织,HE染色分析角膜创伤愈合的病理组织学变化；real time RT-PCR检测IGF-1mRNA在不同时间点的表达；免疫荧光法检测IGF-1的表达位置,分析累积光密度(integrated optical density, IOD)检测IGF-1蛋白表达量。结果1.糖尿病大鼠出现典型的“三多一少”症状：多饮、多食、多尿、消瘦,体重显著低于正常大鼠(P0.01),血糖浓度持续显著高于正常大鼠(P0.01)。到第8周时,糖尿病大鼠体重比正常大鼠体重低25.8%(262.11±12.11g,353.44±10.85g,P0.01),血糖浓度比正常大鼠血糖浓度高4.63倍(18.43±1.04 mmol／L,3.98±0.78mmol／L,P0.01)。2.与正常大鼠相比,糖尿病大鼠角膜上皮创伤愈合速率显著延迟。创伤后60h,正常大鼠角膜上皮创伤完全愈合,糖尿病大鼠角膜上皮创伤愈合率为80.7%,两组的愈合率在24,36,48及60h均有显著性差异(P0.01)。3.HE染色显示,炎症细胞在糖尿病大鼠角膜创伤区的出现时间延迟,停留时间延长,伴基质水肿、大量角膜缘新生血管生成。4.未创伤时,正常大鼠角膜中未检测到IGF-1mRNA表达,糖尿病大鼠角膜中可见少量表达。正常大鼠角膜中IGF-1 mRNA在创伤后1d表达上调,2d达高峰,高峰持续至5d时表达下调。糖尿病大鼠角膜中IGF-1mRNA在创伤后2d表达上调,3d达高峰,持续高表达。创伤后2d-4d,正常大鼠角膜中IGF-1mRNA的表达水平显著高于糖尿病大鼠(P0.01),分别高4.8,1.6及2.2倍。5.未创伤时,正常大鼠角膜中几乎无IGF-1表达,糖尿病大鼠角膜上皮及基质中可见少量IGF-1表达。创伤后,正常大鼠创伤区角膜基质及新覆盖的角膜上皮中,IGF-1在伤后1d开始表达,3d达高峰,5d表达量降低,但仍可见有表达。糖尿病大鼠角膜上皮及创伤区基质中IGF-1在伤后1d上升,3d达高峰,5天时表达显著下降。分析IOD结果显示,正常大鼠角膜创伤愈合过程中IGF-1的表达量显著高于糖尿病大鼠(P0.01),在1d,3d,5d时,分别高4.6倍,2.2倍及2.4倍。结论IGF-1表达延迟和表达不足可能是导致糖尿病角膜创伤愈合延迟的重要机制之一。
[Abstract]:Objective diabetic corneal wound healing delay is one of the most common clinical problems in Ophthalmology, the treatment is difficult. The pathogenesis is not clear.IGF-1 can promote cell migration, proliferation, inhibit cell apoptosis and play an important role in wound healing. This experiment was established by establishing a diabetic rat corneal wound model to detect IGF-1 in the healing process. To explore the possible mechanism of delayed healing of diabetic corneal injury from molecular level.
Method 1. the diabetic rat model was established: 65 male Wistar rats and weight 170-200g were randomly divided into diabetes group and normal control group. Diabetic rats were injected with streptozotocin (STZ) to make diabetic rat model (n=35). The normal control group was injected with equal amount of citrate buffer (n=30). After making the model for 8 weeks, the blood glucose and body were measured weekly. A rat model of corneal trauma was established with a 5 millimeter diameter ring with a diameter of 5 millimeters in the cornea. Corneal epithelium.3. was removed at different time points after removal of the corneal epithelium (0h, 12h, 24h, 36h, 48h, 60H, 72h, 4d-16d) in the corneal epithelium. The corneal wound healing and infection were observed under the slit lamp. Fluorescein sodium staining was performed under the slit lamp. Corneal epithelial wound healing rate (0h, 12h, 1D, 2D, 3D, 4D, 5D, 7D) were sacrificed to the normal rats (n=24) and diabetic rats (n=24) after the corneal epithelial removal, and the corneal tissue was taken to analyze the histopathological changes of corneal wound healing. The expression of IGF-1 was detected by immunofluorescence, and the expression of IGF-1 protein was analyzed by integrated optical density (IOD).
Results 1. diabetic rats had typical symptoms of "more than three more and one less": polydipsia, polyuria, emaciation, weight loss significantly lower than normal rats (P0.01), and the blood glucose concentration was significantly higher than that of normal rats (P0.01). By eighth weeks, the weight of diabetic rats was 25.8% (262.11 + 12.11g, 353.44 + 10.85g, P0.01), and the blood glucose concentration ratio was higher than that of normal rats. In normal rats, the blood glucose concentration was 4.63 times higher (18.43 + 1.04 mmol / L, 3.98 + 0.78mmol / L, P0.01).2., compared with normal rats, the healing rate of corneal epithelial wound in diabetic rats was significantly delayed. After trauma, the corneal epithelial wound healing was completely healed in 60H, and the healing rate of corneal epithelial wound in diabetic rats was 80.7%. The healing rate of the two groups was 24,36,4 The significant difference between 8 and 60H (P0.01).3.HE staining showed that the occurrence time of the inflammatory cells in the corneal wound area of the diabetic rats was delayed, the stay time was prolonged, the matrix edema and a large number of corneal limbus neovascularization were not wound, and the expression of IGF-1mRNA was not detected in the normal rat cornea, and a small amount of expression was seen in the cornea of diabetic rats. The expression of IGF-1 mRNA in the cornea of normal rats was up-regulated, 2D reached its peak, and the expression of IGF-1mRNA was down regulated when the peak was continued to 5D. The 2D expression of IGF-1mRNA in the cornea of diabetic rats was up to up, the peak of 3D reached its peak, and the expression of IGF-1mRNA in the cornea of normal rats was significantly higher than that of the diabetic rats (P0.01), and the high 4.8,1.6 was higher than that of the diabetic rats (P0.01). The high 4.8,1.6 in the cornea of normal rats was higher than that of the diabetic rats (P0.01). The 4.8,1.6 was higher than that of the diabetic rats. There was almost no IGF-1 expression in normal rats' cornea and a small amount of IGF-1 expression in the corneal epithelium and matrix of diabetic rats. After trauma, IGF-1 was expressed in 1D after injury in normal rats and in the newly covered corneal epithelium. The expression of 3D reached the peak and the expression of 5D decreased, but the expression of IGF-1 was still visible. The expression of diabetes was still visible. Diabetes was still expressed. The expression of diabetes was still obvious. The 1D increased after injury in the corneal epithelium and wound area of the rat, and the expression of 3D reached a peak at 5 days. The analysis of IOD showed that the expression of IGF-1 in the healing process of normal rats was significantly higher than that of diabetic rats (P0.01), which was 4.6 times, 2.2 times and 2.4 times higher in 1D, 3D, and 5D respectively.
Conclusion delayed expression of IGF-1 and insufficient expression may be one of the important mechanisms leading to delayed wound healing in diabetic patients.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R587.1;R772.2

【参考文献】