结缔组织生长因子与转化生长因子及Smad信号通路在兔角膜创伤愈合中的作用  

The role of connective tissue growth factor, transforming growth factor and Smad signaling pathway during corneal wound healing

作　　者：杨咏梅[1] 吴欣怡[2] 杜立群[2] 

机构地区：[1]济南市中心医院眼科,山东大学临床医学院250013 [2]山东大学齐鲁医院眼科

出　　处：《中华眼科杂志》2006年第10期918-924,共7页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金(30328026) 山东省自然科学基金(Y2004c27)

摘　　要：目的探讨结缔组织生长因子(CTGF)与转化生长因子β_1(TGF-β_1)在兔角膜创伤愈合过程中的表达和对创伤愈合的作用,并对二者之间的相互作用及与此有关的 Smad 信号传导通路作初步研究。方法选择 Albino 纯种白兔26只,随机分为4组:(1)空白对照组:2只兔(4只眼),未做任何处理。(2)单纯角膜创伤组:又分术后2、6h,1、3、7、21 d 亚组,每个亚组2只兔(4只眼)。(3)TGF 抗体组:颞侧球结膜下注射15.5μg TGF 抗体,又分为3、7、21d 亚组,每个亚组2只兔(4只眼)。(4)Smad4抗体组:颞侧球结膜下注射20μg Smad4抗体,又分为3、7、21d 亚组,每个亚组2只兔(4只眼)。实验眼首先行直径3mm、包含约0.05mm 实质层的板层角膜切除术,然后 TGF 抗体组术眼球结膜下注射 TGF-β_1抗体,Smad4抗体组术眼球结膜下注射 Smad4抗体,用免疫组织化学和mRNA 原位杂交方法分别检测 CTGF、TGF-β_1、纤维粘连蛋白(FN)在术后2、6h、1、3、7、21d 的表达和Ⅰ型胶原、CTGF mRNA 在术后6h、3、7、21d 的表达及角膜纤维细胞活化增生情况。结果 (1)正常兔眼角膜未发现 CTGF 蛋白和 mRNA 的表达。TGF-β_1蛋白在正常角膜上皮中有表达。(2)术后6h角膜纤维细胞开始活化,创伤后 TGF-β_1在上皮和实质中的表达显著增加,同时 CTGF mRNA 表达上调;术后3d TGF-β_1蛋白、CTGF 及Ⅰ型胶原 mRNA 的表达到高峰;术后7d,3者在上皮中表达明显,TGF-β_1蛋白在实质中表达减少;术后21d,绝大多数纤维细胞恢复到伤前状态,CTGF 蛋白和 mRNA在上皮中偶见表达。(3)TGF 抗体使实质中的 TGF-β_1、CTGF、FN 表达减少,同时下调上皮中 CTGFmRNA 的表达。(4)Smad4抗体抑制实质层 TGF 的表达,而对 CTGF 表达无影响。结论创伤能引起TGF-β_1蛋白、CTGF 蛋白及 mRNA 的表达增加,Ⅰ型胶原和 FN 蛋白合成增加。TGF-β_1促进角膜纤维细胞活化,上调 CTGF 的表达;CTGF 主要调节Ⅰ型胶原和 FN 生成,从而影响角膜�Objective To study the expression and location of connective tissue growth factor （CTGF） and transforming growth factor-β1（TG-β1 ） protein and mRNA in rabbit cornea during the wound healing process. To assess the interaction between CTGF and TGF-β1, as well as the Smad signaling pathway involved. Methods Twenty-six Albino white rabbits were used as experimental animals and randomly divided into 4 groups ： （ 1 ） Control group ： two rabbits. （ 2 ） Simple corneal injury group ： a 3 mm diameter and 0.05 mm depth corneal tissue was excised by a trephine at the anterior central cornea as a corneal wound model in 12 rabbits. Two rabbits were randomly sacrificed at 2 h, 6 h, 1 d, 3 d, 7 d and 21 d after the trauma. （3） TGF-β1 antibodies treated group： 6 rabbits were injected with TGF-β1 antibodies （ 15.5 μg） subconjunctivaly after corneal trephine. Two rabbits were randomly sacrificed at 3 d, 7 d and 21 d after the injection. （4） Smad4 antibodies treated group： 6 rabbits were injected with Smad4 antibodies （20 μg） subconjunctivaly after corneal trephine. Two rabbits were randomly sacrificed at 3 d, 7 d and 21 d after the injection. Protein of CTGF, TGF-β1 , and FN was assessed with immunohistoehemistry. CTGF and type one collagen mRNA were measured in by in situ hybridization. Results （1） CTGF protein or mRNA did not exist in normal rabbit corneas, but TGF-β1 protein was expressed in normal rabbit cornea epithelium. （2） Cornea fibroblasts activated 6 h after the operation. Expression of CTGF, TGF-β1, FN protein and mRNA of CTGF and type one collagen were upregulated after cornea injury, and reached the highest level in 3 days. The expression was reduced to the basal level 21 days later. （3） Injection of TGF-β1 antibodies reduced the expression of CTGF, TGF-β1 and FN in the cornea stroma and down-regulated the expression of CTGF in corneal epithelial cells. （4） Injection of Smad4 antibodies inhibited the expression of TGF in the stroma but did not chang

关 键 词：角膜 伤口愈合 即早蛋白质类 胞间信号肽类和蛋白质类 转化生长因子β 信号传递 

分 类 号：R779.1[医药卫生—眼科]