【作者】 王艳；

【导师】 李建基； 王亨；

【作者基本信息】 扬州大学 ， 临床兽医学， 2015， 硕士

【摘要】 犬角膜损伤是临床常见的眼科疾病,且致盲率较高。羊膜免疫源性低,以其优越的理化特性而成为理想的生物移植材料。因此,为了探索犬角膜损伤的高效疗法,本试验首先建立了犬角膜碱烧伤疾病模型,然后采用羊膜移植术对其进行治疗,并观察病犬眼睛的临床症状、角膜组织学变化及相关细胞因子TNF-α、VEGF和TGF-β1的基因相对表达量情况。选取健康成年比格犬6只,将直径6 mm的圆形滤纸片用1 mol/L NaOH溶液浸湿,置于角膜中央1 min,然后立即用生理盐水冲洗眼球表面,制得犬角膜碱烧伤疾病模型。取剖腹产犬胎儿羊膜,制备犬羊膜片,置于DMEM甘油液中,-80℃保存。本试验用同一条犬的左眼作为对照组,仅进行碱烧伤；右眼为试验组,碱烧伤后进行羊膜移植。术后每日观察犬角膜水肿、角膜新生血管和眼分泌物等情况,于术后3、7、14、21、28和35 d测量眼内压和泪液量,并对角膜混浊、新生血管及上皮修复情况进行评分；术后35 d采集犬角膜组织,将其分成2份,一份用于病理组织学观察,组织切片用HE染色及Masson三色染色；另一份用于荧光定量PCR实验,检测TNF-α、VEGF和TGF-β1的基因相对表达量。试验结果如下：(1)术后两组犬的角膜都出现不同程度的水肿和眼分泌物增多症状,试验组犬眼球表面的羊膜植片较平整,羊膜于1周内溶解,术后5 d角膜缘出现新生血管芽,至第16 d时眼分泌物减少并恢复到正常水平；而对照组犬眼术后5 d角膜缘出现刷状的新生血管,至第28 d时仍有2只眼睛有较多的分泌物,在第6 d时有一只眼的角膜出现穿孔。至术后35 d时,试验组犬眼在角膜混浊程度及血管新生方面明显优于对照组(P<0.05),第21 d时两组犬角膜荧光素染色出现显著差异(P<0.05),试验组犬角膜着色明显轻于对照组,至术后35 d时试验组犬角膜荧光素着色不明显,对照组犬角膜呈浅着色。术后14 d,试验组犬眼的眼内压及泪液量指标升高,但处于正常生理范围之内；对照组犬眼的眼内压极显著降低(P<0.01),泪液量显著减少(P<0.05)；至第35 d时两组犬眼的眼内压及泪液量均恢复至正常水平,但对照组处于正常生理范围的下限。(2)术后35 d,犬角膜病理组织学切片显示：对照组犬角膜有较多的炎性细胞和新生血管,血管腔内有成熟红细胞,成纤维细胞肥大,基底细胞肿胀且间隙增大,胶原纤维排列紊乱；试验组犬角膜炎性细胞和新生血管相对较少,成纤维细胞扁平,胶原纤维排列相对较整齐。(3)术后35 d,试验组犬角膜组织中TNF-α、VEGF和TGF-β1的基因相对表达量极显著低于对照组(P<0.01)。由上述试验结果可知,羊膜具有抑制炎症反应、抑制血管新生和瘢痕的形成、促进角膜损伤愈合的重要作用,对犬角膜碱烧伤有一定的临床疗效。更多还原

【Abstract】 Corneal injury often occurs in canine clinical ophthalmology, which frequently induces blindness. Amniotic membrane has been confirmed to be an ideal biomaterial for transplantation due to its superior physical and chemical properties as well as its low immunogenicity. In order to explore the effective treatment of corneal injury, canine corneal alkali burn was induced in this study as a pathological model, and the amniotic membrane transplant (AMT) was used for the treatment. The clinical symptoms, histological changes and the relative expression of the TNF-α, VEGF and TGF-β1 genes in the corneas were observed.6 healthy adult Beagles were selected during the course of the experiment. The corneal injury model was made using circular filter paper of 6mm diameter with 1mol/L NaOH, which was placed onto the central cornea for 1 min, then rinsed with saline. The amniotic membrane was collected from fetal canine via caesarean section. The pieces of amniotic membrane were prepared and placed in DMEM glycerol solution, then stored at -80℃. The left eyes which received the alkali burn but no treatment were used as the control group, whereas the right eyes of the same canine were the experimental group, which received alkali burn and then treated using AMT. Corneal edema, neovascularization and eye secretions were observed daily post-surgery. The intraocular pressure (IOP) and tear volume were measured on days 3,7,14,21, 28 and 35 post-surgery and the corneal opacity, neovascularization and epithelial healing were scored on days 7,14,21,28 and 35. The corneal tissue was extracted on day 35 post-surgery and divided into 2 halves, one prepared for histopathology observation, including HE staining and Masson trichrome staining, and the other was used for qRT-PCR, to detect the relative expression levels of TNF-α, VEGF and TGF-β1 genes. The results are as follows:(1) Varying degrees of corneal edema and increased eye secretion were observed in the canine corneas of both groups. In the experimental group, the amniotic membrane was smooth and dissolved within one week. Neovascular buds appeared on day 5 post-surgery. Secretion volume returned to normal after 16 days. In the control group, brush-like gross neovascularization could be seen on day 5, one case of corneal perforation was detected on day 6, and excess secretions were observed in the eyes of two canines on day 28. The degree of corneal opacity and neovascularization was less in the experimental group compared to that of the control group on day 35 (P<0.05). Fluorescent staining on day 21 revealed a significant difference between the two groups (P<0.05), with the experimental group displaying a lesser degree of staining than that of the control group. On day 35, the experimental group showed no obvious staining unlike the control group, which still retained traces of dye. The IOP and tear volume increased in the experimental group on day 14, but remained within the normal range, whereas the control group had decreased IOP (P<0.01) and tear volume (P<0.05). By day 35, the indicators returned within normal parameters in both groups, however in the control group they remained in the lower limit.(2) On day 35 post-surgery, histopathological examination of the canine cornea of the control group presented a large number of inflammatory cells and new blood vessels containing mature red blood cells. Hypertrophic fibroblasts, swollen basal cells with increased intercellular gap and disorganized collagen fibers were also noted. The experimental group presented fewer inflammatory cells and new blood vessels as well as flat fibroblasts and relatively organized collagen fibers.(3) The relative expression level of TNF-a, VEGF and TGF-β1 genes was significantly lower in the corneas of the experimental group than those of the control group on day 35 (P< 0.01).The results suggested that amniotic membrane inhibited the inflammation and formation of new blood vessels and scar tissues, promoting the healing of the injured cornea. AMT is a clinically effective treatment for canine corneal alkali burn.更多还原