過(guò)氧化物酶體增殖物激活受體δ在大鼠角膜準(zhǔn)分子激光切削術(shù)后的抗纖維化作用
發(fā)布時(shí)間：2018-12-16 07:20
【摘要】：目的:角膜渾濁是造成視力下降的重要原因之一。角膜創(chuàng)傷愈合過(guò)程中,肌成纖維細(xì)胞的出現(xiàn)和異常角膜基質(zhì)的形成是角膜纖維化的重要原因。糖皮質(zhì)激素和絲裂霉素C可以抑制角膜纖維化,但具有明顯副作用。因此,研發(fā)安全有效的抗纖維化藥物極為必要。過(guò)氧化物酶體增殖物激活受體(PPAR)δ在細(xì)胞增殖、分化和組織創(chuàng)傷愈合等方面具有重要作用。研究證實(shí)PPARδ激動(dòng)劑抑制血管平滑肌細(xì)胞、心肌成纖維細(xì)胞的增殖和膠原合成,并通過(guò)抗炎作用減輕肝纖維化。但目前關(guān)于PPARδ是否具有抗角膜基質(zhì)纖維化作用方面尚無(wú)相關(guān)研究。本工作利用準(zhǔn)分子激光切削角膜造成基質(zhì)損傷,應(yīng)用GW501516激活PPARδ,觀察GW501516對(duì)角膜創(chuàng)傷愈合的影響,首次實(shí)驗(yàn)并探討PPARδ的抗角膜基質(zhì)纖維化作用及機(jī)制。方法:1.利用準(zhǔn)分子激光治療性角膜切削術(shù)(PTK)切削SD大鼠的中央角膜,切削直徑4.5 mm,切削深度50μm或70μm。術(shù)后裂隙燈檢查對(duì)比角膜渾濁度,選擇合適的切削深度。角膜共聚焦顯微鏡檢查及HE染色檢查評(píng)估角膜創(chuàng)傷愈合反應(yīng)。2.實(shí)驗(yàn)分4組,分別球結(jié)膜下注射PBS,GW501516(PPARδ激動(dòng)劑),GSK3787(PPARδ拮抗劑)或GW501516聯(lián)合GSK3787。術(shù)后給藥每周2次直到處死大鼠。3.術(shù)后裂隙燈檢查眼表情況:術(shù)后早期利用熒光素染色法觀察上皮愈合情況,術(shù)后每周觀察角膜新生血管情況,進(jìn)行中央角膜渾濁度評(píng)分,共觀察4周。4.術(shù)后1周、2周及4周,共聚焦顯微鏡觀察活體角膜基質(zhì)細(xì)胞及細(xì)胞外基質(zhì)的變化。觀察并對(duì)比各組前部角膜基質(zhì)的相對(duì)反光強(qiáng)度。5.術(shù)后2周及4周,HE染色法觀察角膜組織愈合情況并對(duì)中央角膜的基質(zhì)細(xì)胞進(jìn)行計(jì)數(shù)。6.術(shù)后2周及4周,免疫熒光染色法檢測(cè)角膜組織中α-SMA、III型膠原和纖維連接蛋白的表達(dá)。7.術(shù)后4周,實(shí)時(shí)熒光定量PCR檢測(cè)Ki67抗原、α-SMA、III型膠原、I型膠原和纖維連接蛋白的mRNA相對(duì)表達(dá)量。結(jié)果:1.利用PTK切削SD大鼠的中央角膜,切削直徑4.5 mm,切削深度70μm,該方法安全有效,可獲得理想的角膜渾濁程度并引起角膜損傷修復(fù)反應(yīng)。2.激活PPARδ延遲角膜上皮愈合:術(shù)后24和48小時(shí),GW501516組角膜上皮缺損面積大于其它各組,具有統(tǒng)計(jì)學(xué)差異。3.激活PPARδ促進(jìn)角膜新生血管形成:術(shù)后1周及2周,GW501516組角膜新生血管面積大于其它各組,具有統(tǒng)計(jì)學(xué)差異。4.激活PPARδ改善角膜透明度:術(shù)后4周,GW501516組角膜渾濁度評(píng)分較GSK3787組低,具有統(tǒng)計(jì)學(xué)差異。5.激活PPARδ抑制基質(zhì)細(xì)胞的激活和增殖:術(shù)后4周,共聚焦顯微鏡活體檢查見(jiàn)GW501516組基質(zhì)細(xì)胞形態(tài)安靜,細(xì)胞外基質(zhì)反光較低;GSK3787組基質(zhì)細(xì)胞形態(tài)活躍,細(xì)胞外基質(zhì)反光較強(qiáng)。和其他各組相比,GW501516組前部基質(zhì)相對(duì)反光強(qiáng)度降低,具有統(tǒng)計(jì)學(xué)差異�；|(zhì)細(xì)胞計(jì)數(shù)示GW501516組基質(zhì)細(xì)胞數(shù)明顯低于PBS組和GSK3787組,具有統(tǒng)計(jì)學(xué)差異。同時(shí),GW501516組角膜基質(zhì)的Ki67抗原mRNA表達(dá)較其它各組降低,具有統(tǒng)計(jì)學(xué)差異。6.激活PPARδ抑制基質(zhì)細(xì)胞轉(zhuǎn)化為肌成纖維細(xì)胞:術(shù)后2周,免疫熒光染色證實(shí)前部及中部角膜基質(zhì)內(nèi)存在α-SMA陽(yáng)性細(xì)胞,各組間未見(jiàn)明顯差異。術(shù)后4周,GW501516組的α-SMAmRNA較其它各組降低,具有統(tǒng)計(jì)學(xué)差異。7.激活PPARδ抑制細(xì)胞外基質(zhì)的合成:術(shù)后4周,同PBS組和GSK3787組相比,免疫熒光染色證實(shí)GW501516組的Ⅲ型膠原和纖維連接蛋白表達(dá)降低。同時(shí),GW501516組中Ⅲ型膠原、纖維連接蛋白和Ⅰ型膠原的mRNA表達(dá)均明顯下降,和其他各組相比有統(tǒng)計(jì)學(xué)差異。結(jié)論:PPARδ激動(dòng)劑在角膜組織創(chuàng)傷愈合中有抗纖維化作用,它能抑制基質(zhì)細(xì)胞的激活和增殖,降低基質(zhì)細(xì)胞數(shù)量,緩解基質(zhì)細(xì)胞向肌成纖維細(xì)胞的轉(zhuǎn)化,減少細(xì)胞外基質(zhì)合成。從而在基質(zhì)重塑期改善角膜透明度。但PPARδ激動(dòng)劑延遲角膜上皮愈合并促進(jìn)角膜新生血管形成,因而需謹(jǐn)慎選擇治療對(duì)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，
本文編號(hào)：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，
本文編號(hào)：2381985
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