【摘要】 目的：探讨重组人色素上皮细胞衍生因子（recombinant human pigment epitheliumderived factor, rhPEDF）的体外生物学活性及其抑制大鼠角膜新生血管的作用及机制。方法：建立鸡胚脲囊膜(chick chorioallantoic membrane, CAM)模型，光镜下观察rhPEDF对正常鸡胚脲囊膜微血管形态及数目的影响。照体外实验，给大鼠角膜碱烧伤模型10mol/L rhPEDF后，进行裂隙灯显微镜观察，并定量分析角膜新生血管面积；采用组织病理学检查，比较对照组与用药组的变化。结果：光镜下可见，0.4μg/L rhPEDF对新生血管有显著抑制作用（P<0.01），鸡胚脲囊膜微血管数减少，并且管径变细。大鼠实验组角膜新生血管较对照组3，7，14d没有显著变化。结论： rhPEDF具有抑制新生血管的生物学活性。由于角膜新生血管形成机制的复杂性，应用于角膜新生血管的治疗还需要进一步深入研究。
【关键词】 角膜新生血管；重组人色素上皮衍生因子；鸡胚脲囊膜
In vitro activity of recombinant human PEDF protein and its inhibition to rat corneal neovascularization
YaNi Wang, XianNing Liu, XiuPing Zhu, Na An, XiaoZhao Yang, Hua Yang, HongBing Zhang
Foundation items: Scientific technical research development plan project in Shaanxi Province, China [No.2005k10G1(9)];Scientific and technological brainstorm project in Xian, China（No.GG06145）
Shaanxi Provincial Ophthalmic Institution, Xian No.1 Hospital, Xian 710002, Shaanxi Province, China
Abstract
AIM: To evaluate the in vitro activity of recombinant human PEDF protein (rhPEDF) and investigate its effect and mechanism in inhibiting rat corneal neovascularization (CNV).
METHODS: The models of angiogenesis of chick chorioallantoic membrane(CAM) were established, the effects of rhPEDF to normal CAM capillary vessel morphology were observed under the light microscope and blood vessels were counted. The models of 24 SD rat CNV were established and randomly divided into two groups: 12 in experimental group(the concentration of rhPEDF according to CAM experiment) and 12 in control group, CNV was observed by the slit lamp biomicroscope examination. The area of CNV was calculated quantitatively. Two groups were compared by histopathological method.
RESULTS: Decrease of blood vessels, slender caliber were observed under the light microscope after treatment with rhPEDF. CAM capillary vessels were significantly inhibited by rhPEDF at the concentration of 0.4μg/L (P<0.01). There was no significant difference after 3,7,14 days between the two groups.
CONCLUSION: rhPEDF has biologic activity in inhibiting neovascularization. As the complexity of mechanism of CNV, the application of rhPEDF in curing CNV still needs to be improved.
KEYWORDS: corneal neovascularization; rhPEDF; chick chorioallantoic membrane
0引言
角膜新生血管（corneal neovascularization,CNV）的形成可引起角膜血管翳、最终可严重导致视力下降，甚至视力丧失，也是角膜移植术后发生排斥反应的高危因素，是最常见的致盲原因之一[13]。目前传统治疗药物均有较强的副作用。色素上皮细胞衍生因子（pigment epitheliumderived factor, PEDF）被证实是生物体内固有的一种重要的新生血管抑制因子，现已能从人和鼠中克隆和纯化[4，5]。我们探讨原核表达的rhPEDF体外生物学活性和对大鼠角膜碱烧伤后所形成新生血管的抑制作用。
1材料和方法
1.1材料 rhPEDF蛋白（本研究所制备），7天龄鸡胚胎蛋（兰州军区后勤养鸡场），SD大鼠（西安交通大学动物中心），其余生化试剂（上海生工）。rhPEDF体外活性测定。20只7天龄鸡胚胎蛋，参照文献[6]制作鸡胚脲囊膜模型，将rhPEDF制备成0.4μg/L的溶液滴加于无菌滤纸片上，覆盖于测试区，37℃继续孵育48h后，滴加预冷的甲醇与丙酮混合液，室温固定30min，将测试区的尿囊膜移至载玻片上，显微镜下统计滤纸片周围6mm范围内的血管数。
图1 鸡胚脲囊膜法检测rhPEDF体外生物学活性组 A：PSB；B：rhPEDF。（略）
图2 大鼠碱烧伤后CNV的生长状况 A：伤后1d；B：伤后3d；C：伤后7d；D：伤后14d。（略）
1.2方法 100g/L水合氯醛3mL/kg ip麻醉，检查所有鼠眼无眼前节病变，盐酸利多卡因滴眼液点眼2次后，棉签吸除结膜囊液体，将直径3mm的滤纸片浸泡于1mol/L NaOH溶液中30s，控干多余溶液，然后置于大鼠角膜中央1min后，生理盐水迅速冲洗角膜及结膜囊1min，前3d涂红霉素软膏，常规喂养。随机数字法将24只大鼠分为2组，每组12只。统一左眼为碱烧伤眼。碱烧伤后即刻，A组滴用生理盐水，4次/d，作为对照组；B组滴用10mol/L rhPEDF蛋白溶液4次/d。大鼠于碱烧伤24h后每日显微镜下观察并拍照记录角膜缘CNV的长度和数量。测量时，以连续弯曲度小、新生血管朝向角膜混浊中心生长的最长血管为准，并计算CNV面积(A)，公式：A=C/12×3.1416[r2(rl)2]。C为新生血管累及角膜的圆周钟点数， l为CNV从角膜缘深入角膜的长度，r为大鼠角膜半径(3mm)。另分别于碱烧伤后1，3，7，14 d处死大鼠，取角膜行石蜡包埋、切片、脱蜡和水化后，行HE染色。
统计学分析：所有检测值均以±s表示，应用SPSS 11.5统计学软件对各组数据进行分析，经方差齐性检验后，采用t检验。以P<0.05为差异有统计学意义。
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