体外转染P27抑癌基因抑制人眼球筋膜囊成纤维细胞增生的研究  

Inhibition effect of P27 tumor suppressor gene transfection on human Tenon's fibroblast cells

作　　者：孙璐[1] 骆玮[1] 李静敏[1] 

机构地区：[1]大连医科大学附属二院眼科,116027

出　　处：《眼科研究》2010年第12期1119-1123,共5页Chinese Ophthalmic Research

摘　　要：目的探讨阳离子脂质体介导的P27抑癌基因对体外培养的人眼球筋膜囊成纤维细胞增生的抑制作用。方法收集斜视患者矫正手术过程中获取的球筋膜囊组织制备成组织块,采用贴壁法在DMEM+质量分数10%胎牛血清培养液中进行培养和传代。采用脂质体介导的方法,将构建的增强型绿色荧光蛋白(pEGFP)-N1P27真核表达质粒转染体外培养的人眼球筋膜囊成纤维细胞为pEGFP-N1P27转染组,同时以重组质粒pEGFP-N1的空质粒转染组为空载体组,另设部分未转染的成纤维细胞为未转染组。Westernblot法检测P27蛋白在人眼球筋膜囊成纤维细胞中的表达,用流式细胞技术进行细胞周期分析,MTT法检测P27基因对培养的人眼球筋膜囊成纤维细胞活性的影响,检测值以细胞的吸光度(A490)值表示。结果荧光倒置显微镜下见转染成功的成纤维细胞呈绿色荧光。Westernblot检测显示,pEGFP-N1P27转染组中可见宽大的蛋白条带,证实人眼球筋膜囊成纤维细胞在蛋白水平表达P27基因。细胞周期分析结果表明,pEGFP-N1P27转染组73.16%的人眼球筋膜囊成纤维细胞处于G0-G1期,26.84%处于S期,而空载体组的人眼球筋膜囊成纤维细胞在G0-G1期、S期者分别为63.29%和58.16%,未转染组分别为36.71%和41.84%。MTT法检测表明,pEGFP-N1P27转染组人眼球筋膜囊成纤维细胞的A490值为0.079±0.054,明显低于空载体组的0.127±0.106和未转染组的0.180±0.007,差异均有统计学意义(P=0.000,P=0.011)。结论转染的P27基因对体外培养的人眼球筋膜囊成纤维细胞的增生具有抑制作用,并能够抑制成纤维细胞的活性。Background The failure rate of glaucoma filtrating surgery is 15%-30% during 2-year duration and the main reason is proliferation of fibroblast and scarring of filtering tunnel.Clinical researches found some adverse effects after administration of anti-scarring drugs.ObjectivePresent study was to discuss the cationic liposome-mediated P27 tumor suppressor gene in cultured human Tenon＇s fibroblast inhibition in vitro.MethodsHuman Tenon＇s capsule tissue was collected during the strabismus correct surgery,and adherent explants culture method was used to cultivate the fibroblast in DMEM＋10%FCS.With liposome-mediated method,the constructed pEGFP-N1 P27 eukaryotic expression plasmid was transfected in vitro into cultured human Tenon＇s fibroblasts,and recombinant plasmid pEGFP-N1 was transfected in the same way as the empty vector transfection group.Only cultured fibroblasts were used as non-transfection group.Expression of P27 in human Tenon＇s fibroblasts was detected by Western blot.The cell cycle after transfection was observed by flow cytometry.The proliferation activity of the fibroblasts following the pEGFP-N1 P27 transfection was assessed by methylthiazlyl tetrazolium（MTT）assay.ResultsThe transfected fibroblasts cells showed the green fluorescence under the inverted fluorescence microscope.The fibroblasts transfected by pEGFP-N1 P27 gene presented with the green fluorescence.Western blot assay showed that P27 gene was expressed in the fibroblasts at the protein level.The percentage of the fibroblasts in G0-G1 phase and S phase was 73.16% and 26.84% respectively in pEGFP-N1 P27 transfection group,63.29% and 58.16% in pEGFP-N1 transfection group,36.71% and 41.84% in non-transfection group.After transfection of pEGFP-N1 P27,the A490 value of the fibroblasts was significantly declined in comparison with pEGFP-N1 group（0.079±0.054 vs 0.127±0.106）and non-transfection group（0.079±0.054 vs 0.180±0.007）（P=0.000;P=0.011）.ConclusionThe P27 gene transfection may inhibit the proliferation of huma

关 键 词：抑癌基因P27 人眼球筋膜囊 成纤维细胞 基因转染 脂质体 

分 类 号：R775[医药卫生—眼科]