靶向Bcl-2小分子化合物纳米悬混液对碱烧伤诱导的大鼠角膜新生血管的抑制作用及其作用机制研究

发布时间：2018-04-25 11:21

本文选题：角膜新生血管 + 碱烧伤　；参考：《中国人民解放军医学院》2013年博士论文

【摘要】：目的:检测靶向Bcl-2小分子化合物纳米悬混液（Z24-NS）在眼部应用安全性基础上，观察Z24-NS对大鼠角膜碱烧伤后角膜新生血管（cornealneovascularization，CNV）增殖的影响，并初步探讨其抑制效应及其可能的机制，为将来Z24-NS滴眼液应用于临床奠定理论基础。方法： 1．8只SD大鼠随机分为Z24组和生理盐水组两组，，每组4只(4眼)。Z24组左眼滴2％的Z24-NS，生理盐水组左眼滴0.9％的生理盐水。连续滴眼14天，通过行为学观察、裂隙灯显微镜检查、制作病理切片，观察光镜下角膜病理学改变，检测2％Z24-NS滴眼液有无明显毒副作用。 2．36只SD大鼠，采用浸润1mol/L NaOH的3×3mm滤纸贴附左眼角膜中央40s的方法制作大鼠角膜碱烧伤诱导CNV模型，并随机分为Z24-NS滴眼液治疗组、地塞米松磷酸钠滴眼液阳性对照组以及空白组三组，每组12只(12眼)。Z24组左眼滴2％的Z24-NS，地米组左眼滴0.1％地塞米松磷酸钠滴眼液，每日4次，空白组不做任何处理。实验周期为14天。在碱烧伤后3天、7天、14天通过裂隙灯显微镜检查及照相定性、定量观察各组的CNV生长情况并计算CNV面积、角膜混浊程度、炎症反应和前房反应。 3．碱烧伤后14天在第二部分的基础上通过病理组织学观察及计算机图像分析系统分析各组角膜情况，检测各组角膜组织中炎症细胞的数量、微血管密度，通过免疫组织化学方法、RT-PCR和Western Blot检测大鼠角膜VEGF及其受体Flk-1和Bcl-2的mRNA和蛋白的表达情况。结果: 1．短期局部应用2％的Z24-NS滴眼后大鼠无明显甩头、挠抓眼部现象，大鼠无角膜混浊、水肿，角膜上皮无明显缺损，组织学检查显示结构正常，未见炎症细胞浸润。 2．碱烧伤后3、7、14天Z24组和地塞米松组CNV面积、炎症指数及角膜混浊程度均小于空白组（P0.01或P0.05）。与地塞米松组比较，2％的Z24-NS抑制CNV程度、炎症指数及角膜混浊程度差异无统计学意义(P0.05)。 3．碱烧伤后14天Z24组和地塞米松组角膜组织中炎症细胞的数量、微血管密度均小于空白组（P0.01或P0.05），VEGF、 Flk-1及Bcl-2蛋白及mRNA表达明显低于空白组（P0.05），而2％的Z24-NS与地塞米松组比较差异无统计学意义(P0.05)。各实验组VEGF和Flk-1的表达与MVD的数量呈正相关(r1=0.9997，P1=0.015；r2=0.9999，P2=0.011)。结论： 1．2％的Z24-NS滴眼液短期局部应用对大鼠的角膜结构无影响，在眼部应用是安全的。 2．2％的Z24-NS滴眼液对大鼠角膜碱烧伤后CNV的生长具有明显抑制作用，同时相应减轻了角膜混浊程度，其抑制CRNV效果与0.1％地塞米松滴眼液相同。 3．2％的Z24-NS滴眼液有一定的抗炎作用，它可以抑制大鼠角膜新生血管模型中角膜VEGF、Flk-1及Bcl-2的表达，提示Z24-NS可能通过降低角膜VEGF、Flk-1及Bcl-2的产生，从而抑制了CNV的生长。
[Abstract]:Objective : To investigate the effect of Z24 - NS on the proliferation of corneal neovascularization ( CNV ) after corneal alkali burn in rats and to investigate the inhibitory effect and possible mechanism of Z24 - NS on corneal neovascularization after corneal alkali burn in rats .

Method :

1.8 SD rats were randomly divided into two groups : Z24 - NS group and normal saline group .

2 . Thirty - six SD rats were randomly divided into two groups : Z24 - NS eye drops 2 % Z24 - NS , dexamethasone sodium phosphate eye drops 0.1 % sodium phosphate eye drops , 4 times a day .

3 . The expression of VEGF and its receptor Flk - 1 and Bcl - 2 mRNA and protein in corneal tissue were measured by pathological and histological observation and computer image analysis on the second part 14 days after alkali burn . The expression of VEGF and its receptor Flk - 1 and Bcl - 2 mRNA and protein were detected by immunohistochemistry , RT - PCR and Western Blot .

Results :

1 . After short - term topical application of 2 % Z24 - NS , no obvious spin - off was found in the rats , scratching the eyes , the rats had no corneal opacity , edema , no obvious defect of corneal epithelium , histological examination showed normal structure , and no infiltration of inflammatory cells was seen .

Compared with the dexamethasone group , 2 % of Z24 - NS inhibited the degree of CNV , the inflammatory index and the degree of corneal opacity ( P0.05 ) .

The expression of VEGF , Flk - 1 and Bcl - 2 protein and mRNA in the experimental group were significantly lower than those in the blank group ( P0.05 ) . The expression of VEGF and Flk - 1 was positively correlated with MVD ( r1 = 0.9997 , P1 = 0.015 ) .
r2=0.9999,P2=0.011).

Conclusion :

Short - term topical application of 1.2 % Z24 - NS eye drops has no effect on the corneal structure of rats and is safe in eye application .

2 . 2 % Z24 - NS eye drops significantly inhibited the growth of CNV after corneal alkali burn in rats , and the degree of corneal opacity was decreased accordingly , which inhibited the effects of CRNV and 0.1 % dexamethasone eye drops .

3.2 % Z24 - NS eye drops have anti - inflammatory effects , which can inhibit the expression of VEGF , Flk - 1 and Bcl - 2 in corneal neovascularization model in rats , suggesting that Z24 - NS may inhibit the growth of CNV by lowering the production of corneal VEGF , Flk - 1 and Bcl - 2 .

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R772.2

【参考文献】