过氧化物酶体增殖物激活受体δ在大鼠角膜准分子激光切削术后的抗纤维化作用
发布时间：2018-12-16 07:20
【摘要】：目的:角膜浑浊是造成视力下降的重要原因之一。角膜创伤愈合过程中,肌成纤维细胞的出现和异常角膜基质的形成是角膜纤维化的重要原因。糖皮质激素和丝裂霉素C可以抑制角膜纤维化,但具有明显副作用。因此,研发安全有效的抗纤维化药物极为必要。过氧化物酶体增殖物激活受体(PPAR)δ在细胞增殖、分化和组织创伤愈合等方面具有重要作用。研究证实PPARδ激动剂抑制血管平滑肌细胞、心肌成纤维细胞的增殖和胶原合成,并通过抗炎作用减轻肝纤维化。但目前关于PPARδ是否具有抗角膜基质纤维化作用方面尚无相关研究。本工作利用准分子激光切削角膜造成基质损伤,应用GW501516激活PPARδ,观察GW501516对角膜创伤愈合的影响,首次实验并探讨PPARδ的抗角膜基质纤维化作用及机制。方法:1.利用准分子激光治疗性角膜切削术(PTK)切削SD大鼠的中央角膜,切削直径4.5 mm,切削深度50μm或70μm。术后裂隙灯检查对比角膜浑浊度,选择合适的切削深度。角膜共聚焦显微镜检查及HE染色检查评估角膜创伤愈合反应。2.实验分4组,分别球结膜下注射PBS,GW501516(PPARδ激动剂),GSK3787(PPARδ拮抗剂)或GW501516联合GSK3787。术后给药每周2次直到处死大鼠。3.术后裂隙灯检查眼表情况:术后早期利用荧光素染色法观察上皮愈合情况,术后每周观察角膜新生血管情况,进行中央角膜浑浊度评分,共观察4周。4.术后1周、2周及4周,共聚焦显微镜观察活体角膜基质细胞及细胞外基质的变化。观察并对比各组前部角膜基质的相对反光强度。5.术后2周及4周,HE染色法观察角膜组织愈合情况并对中央角膜的基质细胞进行计数。6.术后2周及4周,免疫荧光染色法检测角膜组织中α-SMA、III型胶原和纤维连接蛋白的表达。7.术后4周,实时荧光定量PCR检测Ki67抗原、α-SMA、III型胶原、I型胶原和纤维连接蛋白的mRNA相对表达量。结果:1.利用PTK切削SD大鼠的中央角膜,切削直径4.5 mm,切削深度70μm,该方法安全有效,可获得理想的角膜浑浊程度并引起角膜损伤修复反应。2.激活PPARδ延迟角膜上皮愈合:术后24和48小时,GW501516组角膜上皮缺损面积大于其它各组,具有统计学差异。3.激活PPARδ促进角膜新生血管形成:术后1周及2周,GW501516组角膜新生血管面积大于其它各组,具有统计学差异。4.激活PPARδ改善角膜透明度:术后4周,GW501516组角膜浑浊度评分较GSK3787组低,具有统计学差异。5.激活PPARδ抑制基质细胞的激活和增殖:术后4周,共聚焦显微镜活体检查见GW501516组基质细胞形态安静,细胞外基质反光较低;GSK3787组基质细胞形态活跃,细胞外基质反光较强。和其他各组相比,GW501516组前部基质相对反光强度降低,具有统计学差异。基质细胞计数示GW501516组基质细胞数明显低于PBS组和GSK3787组,具有统计学差异。同时,GW501516组角膜基质的Ki67抗原mRNA表达较其它各组降低,具有统计学差异。6.激活PPARδ抑制基质细胞转化为肌成纤维细胞:术后2周,免疫荧光染色证实前部及中部角膜基质内存在α-SMA阳性细胞,各组间未见明显差异。术后4周,GW501516组的α-SMAmRNA较其它各组降低,具有统计学差异。7.激活PPARδ抑制细胞外基质的合成:术后4周,同PBS组和GSK3787组相比,免疫荧光染色证实GW501516组的Ⅲ型胶原和纤维连接蛋白表达降低。同时,GW501516组中Ⅲ型胶原、纤维连接蛋白和Ⅰ型胶原的mRNA表达均明显下降,和其他各组相比有统计学差异。结论:PPARδ激动剂在角膜组织创伤愈合中有抗纤维化作用,它能抑制基质细胞的激活和增殖,降低基质细胞数量,缓解基质细胞向肌成纤维细胞的转化,减少细胞外基质合成。从而在基质重塑期改善角膜透明度。但PPARδ激动剂延迟角膜上皮愈合并促进角膜新生血管形成,因而需谨慎选择治疗对象。
[Abstract]:Objective: The opacity of the cornea is one of the important reasons for the loss of vision. In the course of corneal wound healing, the appearance of myofibroblast and the formation of abnormal corneal stroma are the important causes of corneal fibrosis. Glucocorticoid and mitomycin C can inhibit corneal fibrosis, but have obvious side effects. Therefore, it is necessary to develop a safe and effective anti-fibrosis drug. Peroxisome proliferator-activated receptor (PPAR) antigen plays an important role in cell proliferation, differentiation and tissue wound healing. The results show that the PPAR agonist can inhibit the proliferation and collagen synthesis of vascular smooth muscle cells and myocardial fibroblasts, and reduce the hepatic fibrosis by anti-inflammatory action. However, there is no relevant research about whether the PPAR antigen has an anti-corneal stroma fibrosis effect. Objective To study the effect of GW501516 on corneal wound healing by using an excimer laser cutting cornea, and to observe the effect of GW501516 on corneal wound healing. Method: 1. The central cornea of SD rats was cut with excimer laser (PTK), the cutting diameter was 4.5mm, the cutting depth was 50. m u.m or 70. m The corneal wound healing was assessed by a confocal microscope and a HE staining. In 4 groups, PBS, GW501516 (PPAR agonist), GSK3787 (PPAR antagonist) or GW501516 in combination with GSK3787 were injected under the bulbar conjunctiva, respectively. The administration was administered twice a week until the rats were sacrificed. After the operation, the condition of the eye was examined by the slit lamp. In the early postoperative period, the condition of the epithelial healing was observed by the fluorescein staining method, and the corneal neovascularization was observed on a weekly basis, and the central corneal haze score was measured. 4 weeks were observed. The changes of matrix and extracellular matrix were observed at 1, 2 and 4 weeks after operation. The relative reflection intensity of the anterior corneal stroma was observed and compared. The corneal tissue healing was observed at 2 and 4 weeks after operation and the stromal cells of the central cornea were counted. The expression of C-SMA, type III collagen and fibronectin in the corneal tissue was detected by immunofluorescence staining at 2 and 4 weeks. The relative expression of Ki67 antigen, antigen-SMA, type III collagen, type I collagen and fibronectin was detected by real-time fluorescence quantitative PCR at 4 weeks after operation. Results: 1. The central cornea of SD rats was cut with PTK, the cutting diameter was 4.5 mm and the cutting depth was 70. m PPAR was activated to delay corneal epithelial healing: 24 and 48 hours post-operation, and the area of the corneal epithelial defect in the GW501516 group was greater than that of the other groups, with a statistically significant difference. PPAR was activated to promote the formation of corneal neovascularization: 1 and 2 weeks after operation, and the corneal neovascularization in the GW501516 group was greater than that of the other groups. PPAR was activated to improve the transparency of the cornea: 4 weeks after operation, the corneal haze score in the GW501516 group was lower than that of the GSK3787 group, with a statistical difference of 5. The activation and proliferation of the matrix cells were inhibited by the activation of PPAR. In the 4-week post-operation, the morphology of the stromal cells in the GSK3787 group was quiet and the extracellular matrix was low; the matrix cells of the GSK3787 group were active and the extracellular matrix was reflective. Compared with the other groups, the anterior matrix of the GW501516 group was decreased relative to the light-reflecting intensity, and there was a statistical difference. The matrix cell count showed that the number of matrix cells in the GW501516 group was significantly lower than that of the PBS group and the GSK3787 group, with a statistical difference. At the same time, the expression of Ki67 antigen of the corneal stroma in the GW501516 group was lower than that of the other groups. PPAR was activated to inhibit the transformation of the matrix cells into the myofibroblast: 2 weeks after the operation, the presence of the antigen-SMA positive cells in the anterior and middle corneal stroma was confirmed by immunofluorescence staining, and no significant difference was found among the groups. In the 4-week post-operation, the serum-SMAMmRNA in the GW501516 group was lower in other groups, with a statistical difference of 7. PPAR was activated to inhibit the synthesis of extracellular matrix: 4 weeks after operation, the expression of type 鈪，
本文编号：2381985
[Abstract]:Objective: The opacity of the cornea is one of the important reasons for the loss of vision. In the course of corneal wound healing, the appearance of myofibroblast and the formation of abnormal corneal stroma are the important causes of corneal fibrosis. Glucocorticoid and mitomycin C can inhibit corneal fibrosis, but have obvious side effects. Therefore, it is necessary to develop a safe and effective anti-fibrosis drug. Peroxisome proliferator-activated receptor (PPAR) antigen plays an important role in cell proliferation, differentiation and tissue wound healing. The results show that the PPAR agonist can inhibit the proliferation and collagen synthesis of vascular smooth muscle cells and myocardial fibroblasts, and reduce the hepatic fibrosis by anti-inflammatory action. However, there is no relevant research about whether the PPAR antigen has an anti-corneal stroma fibrosis effect. Objective To study the effect of GW501516 on corneal wound healing by using an excimer laser cutting cornea, and to observe the effect of GW501516 on corneal wound healing. Method: 1. The central cornea of SD rats was cut with excimer laser (PTK), the cutting diameter was 4.5mm, the cutting depth was 50. m u.m or 70. m The corneal wound healing was assessed by a confocal microscope and a HE staining. In 4 groups, PBS, GW501516 (PPAR agonist), GSK3787 (PPAR antagonist) or GW501516 in combination with GSK3787 were injected under the bulbar conjunctiva, respectively. The administration was administered twice a week until the rats were sacrificed. After the operation, the condition of the eye was examined by the slit lamp. In the early postoperative period, the condition of the epithelial healing was observed by the fluorescein staining method, and the corneal neovascularization was observed on a weekly basis, and the central corneal haze score was measured. 4 weeks were observed. The changes of matrix and extracellular matrix were observed at 1, 2 and 4 weeks after operation. The relative reflection intensity of the anterior corneal stroma was observed and compared. The corneal tissue healing was observed at 2 and 4 weeks after operation and the stromal cells of the central cornea were counted. The expression of C-SMA, type III collagen and fibronectin in the corneal tissue was detected by immunofluorescence staining at 2 and 4 weeks. The relative expression of Ki67 antigen, antigen-SMA, type III collagen, type I collagen and fibronectin was detected by real-time fluorescence quantitative PCR at 4 weeks after operation. Results: 1. The central cornea of SD rats was cut with PTK, the cutting diameter was 4.5 mm and the cutting depth was 70. m PPAR was activated to delay corneal epithelial healing: 24 and 48 hours post-operation, and the area of the corneal epithelial defect in the GW501516 group was greater than that of the other groups, with a statistically significant difference. PPAR was activated to promote the formation of corneal neovascularization: 1 and 2 weeks after operation, and the corneal neovascularization in the GW501516 group was greater than that of the other groups. PPAR was activated to improve the transparency of the cornea: 4 weeks after operation, the corneal haze score in the GW501516 group was lower than that of the GSK3787 group, with a statistical difference of 5. The activation and proliferation of the matrix cells were inhibited by the activation of PPAR. In the 4-week post-operation, the morphology of the stromal cells in the GSK3787 group was quiet and the extracellular matrix was low; the matrix cells of the GSK3787 group were active and the extracellular matrix was reflective. Compared with the other groups, the anterior matrix of the GW501516 group was decreased relative to the light-reflecting intensity, and there was a statistical difference. The matrix cell count showed that the number of matrix cells in the GW501516 group was significantly lower than that of the PBS group and the GSK3787 group, with a statistical difference. At the same time, the expression of Ki67 antigen of the corneal stroma in the GW501516 group was lower than that of the other groups. PPAR was activated to inhibit the transformation of the matrix cells into the myofibroblast: 2 weeks after the operation, the presence of the antigen-SMA positive cells in the anterior and middle corneal stroma was confirmed by immunofluorescence staining, and no significant difference was found among the groups. In the 4-week post-operation, the serum-SMAMmRNA in the GW501516 group was lower in other groups, with a statistical difference of 7. PPAR was activated to inhibit the synthesis of extracellular matrix: 4 weeks after operation, the expression of type 鈪，
本文编号：2381985
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