α1抗胰蛋白酶抑制角膜碱烧伤的实验机制研究

目的研究α1抗胰蛋白酶(alpha-1 antitrypsin,AAT)抑制角膜碱烧伤新生血管的效应和可能机制。方法2018年1月~2018年6月应用NaOH构建小鼠碱烧伤模型,应用AAT制备的滴眼液干预碱烧伤模型。通过眼前段照相、CD31角膜铺片免疫荧光观察AAT对角膜碱烧伤模型新生血管的抑制作用。H&E切片观察角膜组织中炎症细胞浸润的情况。RT-PCR检测炎症血管因子的mRNA表达情况。结果裂隙灯眼前段照相发现AAT明显抑制小鼠碱烧伤角膜新生血管的生长,造模后7d、14d新生血管增生的面积较对照组减少24.6%、32.3%,差异具有统计学意义(P<0.05),CD31角膜组织免疫铺片检测取得相近的实验结果,7d、14d新生血管密度较对照组降低8.2%、15.1%。HE切片也提示AAT明显抑制了角膜组织中炎症细胞的浸润,造模14d对照组、实验组炎症细胞浸润个数为231.4±80.7、113.5±56.1,二者之间具有统计学差异。RT-PCR检测发现AAT下调角膜组织β-FGF,CCL2,IL-6,MMP9等血管炎症因子的mRNA表达水平。结论AAT可能通过抑制角膜组织的免疫炎症发挥抑制新生血管的作用,AAT应用治疗角膜化学性烧伤疾病具有广阔的临床使用前景。

Investigation of alpha-1 antitrypsin in inhibiting corneal neovascularization by alkali burn

Objetive To identify whether AAT could inhibit corneal neovascularization in mice and to investigate its underlying molecular mechanism.Methods The model of corneal neovascularization induced by alkali burn was treated with AAT and then the area and density of the new vessels were calculated with the slit lamp photography and CD31 immunofluorescence staining on wholemount of cornea.to evaluation of the infiltration of inflammatory cells by H&E staining after AAT treated to alkali burned corneas.qPCR was used to confirm the AAT’s inhibitive effect to the inflammatory factors,vascular growth factors and metalloproteinase in alkali-burned cornea to explore the mechanism involved in this treatment.Results AAT could reduce the area and density of neovascularization in retina of alkali-burned cornea,indicating that AAT can treat alkali burn induced corneal neovascularization.The area of neovascularization was reduced 24.6%,32.3%which were statistically significantly different after 7 d and 14 d AAT treatment compared to control group.Aslo the density of neovascularization was 8.2%,15.1%less after 7 d and 14 d in AAT group than control group.The infiltration of inflammatory cells remarkably reduced in the alkali-burned cornea which were 231.4±80.7,113.5±56.1 in control group and AAT group.In the meantime,the mRNA expression of cytokines such as b-FGF,CCL2,IL-6,MMP9 were reduced,indicating that AAT could inhibit corneal neovascularization by alleviating the inflammation of alkali-burned cornea.Conclusion AAT inhibited the infiltration of inflammatory cells,as well as the release of the inflammatory and vascular factors,and significantly suppressed the corneal neovascularization.Further study need to explore the implicated mechanism that AAT may exlert its effect by regulating PKA system of macrophages which were the main infiltrated inflammatory cells.