【摘要】：小鼠糖尿病性角膜病變模型的建立及上皮病變的初步研究 目的：研究鏈脲佐菌素(Streptozocin, STZ)腹腔注射誘導的糖尿病小鼠模型中角膜病變的病理變化及其機制。 方法：建立STZ腹腔注射誘導C57BL/6小鼠的糖尿病模型,在成模2-16周分別行1%虎紅染色檢查角膜上皮完整性；角膜上皮刮除后,熒光素鈉染色觀察比較角膜上皮的愈合速度；病理檢測角膜的組織形態(tài)和結構的變化；免疫組化檢測刮除上皮和未刮除上皮DNA損傷標記8-OHdG的表達情況；透射電子顯微鏡檢查角膜在超微結構水平的變化。 結果：STZ腹腔注射后,C57BL/6小鼠逐漸表現(xiàn)出糖尿病多飲、多食、多尿、體重下降等典型癥狀,其血糖穩(wěn)定維持在較高水平(18mmol/L),體重較正常小鼠明顯偏低�；⒓t染色發(fā)現(xiàn),成模2周的小鼠角膜即已出現(xiàn)點狀著色,且隨著病程的延長,染色面積和程度逐漸加重,到成模16周幾乎呈全角膜著色。在上皮愈合速度方面,刮除角膜上皮后,成模2周的小鼠角膜上皮在40小時即完全愈合(與正常小鼠相似),成模4周小鼠上皮愈合時間為120小時,成模8周、12周、16周上皮完全愈合的時間為144小時左右,其中成模16周的小鼠在96小時至120小時上皮缺損的范圍再次擴大,后逐漸縮小愈合,呈現(xiàn)反復現(xiàn)象。病理檢測顯示,STZ誘導的糖尿病小鼠角膜上皮細胞排列疏松極性逐漸消失,細胞水腫,細胞層數(shù)較正常對照小鼠明顯減少,基質(zhì)層纖維增粗排列紊亂。8-OHdG免疫組化檢測顯示,STZ誘導的小鼠在未刮除角膜上皮和刮除角膜上皮的角膜上皮層和基質(zhì)層可見其陽性染色,尤其在中央角膜上皮刮除后殘存的角膜上皮中表達水平較高,且隨著糖尿病病程延長,表達量逐漸升高。透射電鏡檢測發(fā)現(xiàn),STZ誘導的糖尿病小鼠角膜上皮表面的微絨毛減少,上皮細胞間以及上皮與基底膜間的緊密連接數(shù)目明顯減少。 結論：STZ腹腔注射誘導的糖尿病小鼠角膜,表現(xiàn)出角膜上皮損害,角膜上皮損傷后延遲愈合等癥狀,說明其可以作為研究糖尿病角膜病變的動物模型。此外,糖尿病小鼠角膜上皮在超微結構和DNA氧化應激損傷方面的異常可能是造成上皮損傷愈合延遲的部分原因。
[Abstract]:Establishment of Diabetic Corneal lesion Model and preliminary study on epithelial lesions in mice objective: to study the pathological changes and mechanism of corneal lesions in diabetic mice induced by intraperitoneal injection of streptozotocin (Streptozocin, STZ). Methods: the diabetic model of C57BL/6 mice induced by intraperitoneal injection of STZ was established and the corneal epithelial integrity was examined by 1% tiger red staining at 2-16 weeks. The healing rate of corneal epithelium was observed by fluorescein sodium staining, the changes of corneal tissue morphology and structure were detected by pathology, and the expression of 8-OHdG was detected by immunohistochemistry. The ultrastructural changes of cornea were examined by transmission electron microscope (TEM). Results: after intraperitoneal injection of STZ, C57BL/6 mice gradually showed typical symptoms of diabetes mellitus, polydipsia, polyuria and weight loss, and their blood glucose remained stable at a higher level (18mmol/L), and their body weight was significantly lower than that of normal mice. It was found by tiger red staining that the cornea of mice in the second week of model formation had been stained with dots, and with the prolongation of the course of disease, the staining area and degree became more and more serious, and almost the whole cornea was stained at the 16th week of model formation. In terms of the speed of epithelial healing, the corneal epithelium healed completely in 40 hours (similar to normal mice) in 2 weeks after scraping corneal epithelium, 120 hours in 4 weeks, 8 and 12 weeks in model. The time of complete epithelial healing at 16 weeks was about 144 hours, and the range of epithelial defect in mice with model 16 weeks was enlarged again from 96 hours to 120 hours, and then gradually reduced and healed, showing repeated phenomena. Pathological examination showed that the loose polarity of corneal epithelial cells in diabetic mice induced by STZ disappeared gradually, the cell edema and the number of cell layers decreased significantly compared with the normal control mice. 8-OHdG immunohistochemical examination showed that STZ induced mice had positive staining in corneal epithelium and stroma of unscraped and erased corneal epithelium. Especially in the corneal epithelium remaining after central corneal epithelial curettage, the expression level was higher, and with the prolongation of diabetes course, the expression level gradually increased. Transmission electron microscopy (TEM) showed that the number of tight junctions between epithelial cells and basement membrane decreased significantly in diabetic mice induced by STZ. Conclusion: the cornea of diabetic mice induced by intraperitoneal injection of STZ showed corneal epithelial damage and delayed healing after corneal epithelial injury, which indicated that it could be used as an animal model for the study of diabetic keratopathy. In addition, the abnormal ultrastructure of corneal epithelium and oxidative stress injury of DNA in diabetic mice may be part of the cause of delayed healing of epithelial injury.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R772.2

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