羊膜-纤维蛋白胶泥的制备及其对重度眼表碱烧伤治疗作用的实验研究  

Therapeutic effect of amniotic membrane-fibrin sealant cement on severe ocular surface alkali burn in rabbits

作　　者：李楠钰 张文佳[1] 胡竹林[1] Li Nanyu;Zhang Wenjia;Hu Zhulin(The Second People Hospital of Yunnan Province,Yunnan Eye Hospital,Yunnan Institute of Ophthalmology,Yunnan Key Laboratory of Ophthalmic Disease Prevention and Treatment,Yunnan Clinical Medical Center for Ophthalmic Diseases,Yunnan Clinical Medical Research Center for Eye Diseases,Kunming 650021,China)

机构地区：[1]云南省第二人民医院云南省眼科医院云南省眼科研究所云南省眼科疾病防治研究重点实验室云南省眼科疾病临床医学中心云南省眼部疾病临床医学研究中心,昆明650021

出　　处：《中华眼科杂志》2022年第8期606-614,共9页Chinese Journal of Ophthalmology

基　　金：云南省科技计划项目重大科技专项(2018ZF009);云南省卫生健康委员会医学领军人才培养计划(L-2019029);昆明医科大学应用基础研究联合专项(202001AY070001-255)。

摘　　要：目的制备出具备生物学活性的羊膜粉并探索其保存条件,探讨该羊膜粉制成的羊膜-纤维蛋白胶泥(AM-FS)对兔重度眼表碱烧伤模型的疗效。方法实验研究。取新鲜羊膜风干后液氮冷却研磨成颗粒直径<260μm的羊膜粉并进行灭菌。检测该羊膜粉制备后及不同温度[室温(25℃)、4℃、-20℃]下保存10、20、30 d后转化生长因子β(TGF-β)、神经生长因子受体(NGFR)、肝细胞生长因子(HGF)、表皮生长因子(EGF)及碱性成纤维生长因子(bFGF)的表达,并与新鲜羊膜进行比较。倍比稀释制备0.125~65 mg/ml共10种羊膜粉浓度及不含羊膜粉的AM-FS,将兔角膜上皮细胞(CEC)置于其中培养72 h,选取波长450 nm处的吸光度(A)值最大者,以其羊膜粉浓度进行后续动物实验。将32只兔(32只右眼)制作重度眼表碱烧伤模型,采用随机数表法分为AM-FS组、新鲜羊膜移植组、纤维蛋白胶(FS)组及抗生素对照组每组8只兔(8只眼),给予不同眼表干预手段,之后每周观察角膜修复情况,第28 d取材行HE染色观察角膜组织,并行免疫组织化学染色观察单核细胞趋化蛋白1(MCP-1)和血管内皮生长因子(VEGF)的表达。对生长因子或受体的表达差异倍数进行对数处理后得到的logFC值并用BH法校正;采用t检验及方差分析对数据进行统计学分析。结果羊膜粉中TGF-β与NGFR的表达量低于新鲜羊膜,logFC分别为-0.11、-2.07。羊膜粉中HGF、EGF、bFGF的表达量均高于新鲜羊膜,logFC分别为0.72,0.46,2.62(P<0.05);保存10、20 d的羊膜粉中HGF、bFGF、EGF的表达均不低于新鲜羊膜;30 d时羊膜粉除HGF、bFGF外其余生长因子或受体的表达较新鲜羊膜下降,室温保存者HGF、bFGF、EGF的表达均不低于4℃和-20℃保存者。CEC培养72 h后0.25 mg/ml的羊膜粉的A值最大(0.98±0.05)。AM-FS组与新鲜羊膜移植组角膜恢复情况较好,第28 d的角膜混浊程度评分分别为(3.75±0.46)和(3.50±0.46)分,低于抗生素对照组Objective To prepare a biologically active amniotic membrane powder and explore its preservation conditions,and to evaluate the efficacy of the amniotic membrane(AM)-fibrin sealant(FS)cement made from the amniotic powder on the rabbit severe ocular surface alkali burn model.Methods Experimental research.Fresh AM was air-dried,cooled with liquid nitrogen,ground into amniotic powder and sterilized by radiation.The expression of transformed growth factor,nerve growth factor receptor(NGFR),hepatocyte growth factor(HGF),epidermal growth factor(EGF)and basic fibroblast growth factor(bFGF)after preparation and 10,20 and 30 days after storage at room temperature,4℃and-20℃was tested and compared with that in the fresh AM.The AM-FS cement containing different concentrations of amniotic powder and no amniotic powder was diluted.Rabbit corneal epithelial cells were cultured for 72 hours.The effects of different concentrations of amniotic powder on epithelial cell growth were observed by light microscopy,and the amniotic powder concentration with the largest absorbance value at 450 nm was selected for subsequent animal experiments.Thirty-two right eyes of 32 rabbits as the severe ocular surface alkali burn model were divided using the random counting method into the AM-FS cement group,fresh AM transplantation group,FS group and antibiotic control group(8 rabbits each group)and given different interventions.After weekly observation of corneal repair,hematoxylin and eosin staining,immunohistochemical staining of monocyte chemotaxis protein 1(MCP-1)and vascular endothelial growth factor(VEGF)were performed and detected by light microscopy at 28 days.The logFC values of the growth factor or receptor expression difference ratio were corrected by BH;the data were analyzed by t-test and analysis of variance.Results The expression of TGF in the amniotic membrane powder compared with the fresh amniotic membrane group(logFC=-0.11),and the expression of NGFR(HGF,EGF,bFGF)was higher than that of the fresh amniotic membrane group(logF

关 键 词：烧伤 化学 眼烧伤 羊膜 粉剂 纤维蛋白组织黏着剂 疾病模型 动物 

分 类 号：R644[医药卫生—外科学]