摘要
背景青光眼滤过性手术后手术区域瘢痕化是导致抗青光眼手术失败的主要因素，因此越来越多的研究关注瘢痕形成的原因和过程。目的比较青光眼滤过术后滤过区人瘢痕组织来源成纤维细胞（HSFs）与人正常Tenon囊来源成纤维细胞（HTFs）增生迁移能力的差异以及微小RNA200a（miR-200a）对结膜成纤维细胞生物学行为的抑制作用，并探讨产生这种差异的可能机制。方法收集斜视手术过程中切取的正常Tenon囊组织和青光眼滤过术后手术区瘢痕组织，对成纤维细胞进行原代培养，采用细胞计数试剂盒8（CCK8）法检测不同来源成纤维细胞的增生能力；采用细胞划痕试验检测细胞的相对迁移距离；采用实时荧光定量PCR法检测不同来源成纤维细胞中转化生长因子-β1（TGF-β1）mRNA和miR-200a mRNA的表达。于HTFs培养基中分别加入0、1、2和5ng／ml TGF-β1拟似物，于HSFs培养基中分别加入0．00、0．25、0．50、1．00μg／ml TGF-β1抑制剂，细胞处理24h，采用CCK8法检测细胞增生能力；用2ng／ml TGF-β1拟似物处理HTFs，用1．00μg／ml TGF-β1抑制剂处理HSFs，分别采用划痕法检测不同处理组细胞的相对迁移距离，采用实时荧光定量PCR法检测不同处理组细胞中miR-200a mRNA的相对表达量。结果原代培养的细胞胞体较大，呈纺锤形或星形，细胞核呈卵圆形，对角蛋白抗体呈弱阳性反应，对波形蛋白抗体呈阳性反应。HSFs的增生值（A值）为1．476±0．110，明显高于HTFs的0．958±0．074，差异有统计学意义（t=24．900，P=0．016）；HSFs中TGF-β1 mRNA相对表达量高于HTFs，是HTFs的（1．739±0．205）倍，差异有统计学意义（t=6．358，P=0．024）；HSFs中miR-200a mRNA的相对表达量明显低于HTFs，是HTFs的（46．23±10．78）％，差异有统计学意义（t=7．394，P=0．018）。与添加2ng／ml TGF-β1拟似物的HTFs相比，添加TGF-β1拟似�
Background Scarring of surgical area,the most important factor,leads to the failure of glaucoma filtering surgeries. Therefore, more and more attentions are paid to the causes and process of scar formation. Objective This study was to compare the differences of proliferation and migrating abilities of fibroblasts between filtering bleb scar tissue and normal Tenon capsular tissue, and to investigate the inhibitory effects of miRNA-200a （miR-200a） on biological behavior of conjunctival fibroblasts. Methods Normal Tenon capsular tissue and filtering bleb scar tissue were collected during the strabismus surgery and glaucoma filtering surgery,respectively for the primarily culture of fibroblasts. The proliferation （absorbency,A） of the ceils was assayed by cell counting kit-8 （CCK8） method; the relative migrating distance of the ceils was measured by cell scratch test; and the relative expressions of transforming growth factor-β1 （TGF-β1）mRNA and miR-200a mRNA in the cells were detected by real- time fluorescence quantitative PCR. TGF-β1 mimic of 0,1,2 and 5 ng/ml was added in the medium of human normal Tenon capsular-derived fibroblasts （HTFs） , and 0. 00,0.25,0.50,1.00 μg/ml TGF-β1 inhibitor was added in the medium of human scarring-derived fibroblasts （HSFs） for 24 hours,respectively,and CCK8 was used to evaluate the proliferation of the cells. The relative migrating distance as well as the relative expressions of miR-200a mRNA were analyzed in the 2 ng/ml TGF-β1 mimic-or 1.00 μg/ml TGF-β1 inhibitor-treated cells. Results The primary conjunctival presented the spindle and star-like in shape with large body and oval nuclei. The cells showed the positive response for keratin and vimentin antibodies. The A values were 1. 476±0. 110 in the HSFs and 0. 958±0.074 in the HTFs,with a significant difference between them （t = 24. 900,P = 0. 016）. The relative expressions of TGF-β1 mRNA were significantly higher in the HSFs than those in the HTFs, and the relative expressions of miR-200
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2016年第12期1087-1091,共5页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目（81271001）
