摘要
目的观察转染基质金属蛋白酶2抑制剂(tissue inhibitor of matrix metalloproteinase2,TIMP-2)基因的豚鼠巩膜成纤维细胞不同时间增生能力及基质金属蛋白酶2(matrix metalloproteinase2,MMP-2)和TIMP-2的表达,探讨基因治疗近视的可行性。方法随机选取三色健康豚鼠,组织块法体外培养原代巩膜成纤维细胞,免疫组织化学法进行波形蛋白的鉴定,取第3～6代细胞进行实验。分子克隆构建携带TIMP-2基因的真核表达载体,转染豚鼠巩膜成纤维细胞,MTT法观察转染后12h、24h、48h、3d、5d、7d细胞增生能力。提取总RNA,二步法逆转录-聚合酶链反应检测MMP-2mRNA及TIMP-2mRNA的表达水平。结果豚鼠巩膜成纤维细胞原代、传代培养生长良好,波形蛋白鉴定阳性。转染3d、5d、7d TIMP-2基因转染组成纤维细胞的增生速度分别为0.6724±0.0092、0.7963±0.0060、0.8462±0.0064,明显快于正常对照组成纤维细胞(0.5810±0.0120、0.6525±0.0072、0.7129±0.0132),差异均具有显著统计学意义(均为P<0.01)。转染48h MMP-2mRNA表达即开始减少,转染3d时表达明显减少,达到最低,除转染12h与24h之间外,转染组与正常对照组、各转染不同时间组间MMP-2mRNA表达差异均有统计学意义(均为P<0.05)。TIMP-2mRNA表达在转染48h时即开始增加,除12h与24h外,转染组与正常对照组、各转染不同时间组间TIMP-2mRNA表达差异均有统计学意义(均为P<0.05)。结论转染TIMP-2基因对豚鼠巩膜成纤维细胞有促增生作用,TIMP-2基因转染豚鼠巩膜成纤维细胞抑制MMP-2mRNA的表达,可在一定程度上阻止近视发展中巩膜组织的丢失与重塑。
Objective To oberserve the proliferative ability and the expression of matrix metalloproteinase 2（MMP-2） and tissue inhibitor of matrix metalloproteinase 2（TIMP-2） in the sclera fibroblast cell of guinea pig with the transfection of TIMP-2 gene in different time points,and investigate the feasibility of gene therapy in the myopia.Methods The guinea pigs were selected randomly.The sclera fibroblast cells of guinea pig were cultured in vitro with the tissue explant technique and identified by vimentin with the immunohistochemical method.The third to sixth generation of sclera fibroblast cell of guinea pig were used in experiment.The eukaryotic expression vector with carring the TIMP-2 gene was build with molecular cloning.Sclera fibroblast cells of guinea pig were transfected,and their proliferative abilities were observed with MTT method at 12 hours,24 hours,48 hours,3 days,5 days and 7 days after transfection.The total RNA was extracted and the expression levels of MMP-2 and TIMP-2 mRNA were observed by two-step reverse transcription-polymerase chain reaction.Results The primary cultured and subcultured sclear fibroblast cells of guinea pig growed well and the vimentin was positive.The proliferative rates of fibroblast cells were 0.672 4±0.009 2,0.796 3±0.006 0,0.846 2±0.006 4 at 3 days,5 days and 7 days of TIMP-2 tranfection groups,which were obriously faster than 0.581 0±0.012 0,0.652 5±0.007 2,0.712 9±0.013 2 in normal control group,there were significant differences（all P〈0.01）.The expression of MMP-2 mRNA began to decrease at 48 hours of transfection,and reached to the lowest at 3 days of transfection,after which it decreased slightly.It existed statistical different between the groups of transfection except between 12 hours and 48 hours（all P〈0.05）.The expression of TIMP-2 mRNA began to increase at 48 hours of transfection,and increased significantly at 3 days of transfection,after which it increased slightly.It existed statistical different between the groups of transfection excep
出处
《眼科新进展》
CAS
北大核心
2011年第11期1016-1020,1024,共6页
Recent Advances in Ophthalmology
基金
山东省科技厅攻关计划项目资助(编号:2007GG30002025)~~
