摘要
背景研究表明转化生长因子-β2（TGF-β2）能够促进瘢痕形成，但其在青光眼滤过术后局部瘢痕形成中的作用机制值得研究。目的探讨TGF-β2对人Tenon囊成纤维细胞（HTFs）转化以及在滤过术后瘢痕形成中的作用。方法在斜视手术中获取3例患者的Tenon囊组织，并用组织块培养法在含质量分数10％胎牛血清的DMEM中培养，培养的细胞贴壁后达到70％～80％亚融合状态时更换为无血清培养基饥饿培养24h，然后分别加入质量浓度1、2、5、10、20Ixg／LTGF-β2诱导HTFs24h，另用2tzg／L或5μg／LTGF-β2 诱导HTFs6、24、48、72h，阴性对照组培养基中不加TGF-β2 用Westernblot法检测HTFs中α-平滑肌肌动蛋白（n—SMA）和pSmad2蛋白的表达，采用细胞免疫荧光技术检测“-SMA及纤维状肌动蛋白（F—actin）表达的定位，采用凝胶收缩实验检测培养细胞收缩力的变化。结果不同质量浓度TGF—B，组HTFs中0I—SMA表达的强度均明显强于阴性对照组，2tzg／L、5μg／LTGF—B，作用24～48h后HTFs中d—SMA表达强度达峰值，10μg／L、20μg／LTGF-β2组较2μg／L、5μg／LTGF-β2组α—SMA蛋白表达强度减弱。对照组HTFs中有少量α—SMA及F—actin表达，1、2、5μg／LTGF-β2组d—SMA及F—actin荧光表达明显增强，阳性细胞数明显增加，10μg／LTGF-β2组α—SMA及F-actin荧光表达较低质量浓度组减弱。2μg／LTGF-β2作用后pSmad2表达强度明显强于PBS组和FBS组，在作用后30rain～2h表达较强，2h后开始出现下降。对照组HTFs收缩凝胶面积所占原面积的百分数为（78．00-3．13）％，1、2、5、10tzg／LTGF-β2组分别为（63．88±1．78）％、（20．69±O．65）％、（19．49±O．54）％、（16．24~0．84）％，HTFs收缩凝胶面积占原面积的百分数随着TGF-β2质量浓度的增加而明显增加（F组别=859．400，P=0．000）。结论TGF-β2可诱导成纤维细胞转分化，一定程度上其作用呈质量浓度依赖性和时间依赖性，且细胞收缩力随质量浓度的增加而增强，可能是青光眼滤过术后滤过通道建立失败的重要机制。
Background Research showed that transforming growth factorq32 （TGF-β2） promotes scar formation. But its mechanism in scarring after glaucoma filtration surgery is worthy of studying. Objective This study was to investigate the effect of TGF-β2 on myofibroblast transition of human Tenon fibroblasts （HTFs） and scarring after glaucoma filtration surgery. Methods Tenon capsular tissue was obtained from 3 patients with strabismus during the surgery and was incubated in DMEM with 10% fetal bovine serum （FBS）. The cells were collected and passaged in the free-serum medium for 24 hours,and then 1,2,5,10,20 μ/L TGF-β2 was added into the medium respectively,to induce the transformation of HTFs,and 2 μ/L or 5 μg/L TGF-β2was used to treat the HTFs for 6,24,g8 and 72 hours. The control group was not treated with TGFq32 . The expressions of ct-smooth muscle actin （c^-SMA） and phosphorylation of the signaling proteins （pSmad2） in HTFs were detected by Western blot assay. The expressions of oL-SMA and F-actin were located by cell immunofluorescine technique under the confocal immunofluorescenee microscopy. Cell contractility was determined by collagen gel contraction assays. This study was approved by Ethic Committee of Institute of Surgery Research of Daping Hospital,and informed consent was obtained from each patient or custodian initial of the study. Results The expression of a-SMA protein in the HTFs was increased significantly after the treatment of TGF-~2 in comparison with the control group and reached a peak at 24- 48 hours. The ct-SMA expression was gradually weakened in the 10 p,g/L TGF-β2groups. Little of ~^-SMA and F-actin were expressed in the control group. However, strong staining for c^-SMA and F-actin were observed in the 1,2 and 5 ixg/L TGF-β2groups and then the staining weakened at the concentration of 10 p4/L. In addition,pSmad2 showed astronger expression in the 2 p^g/L TGF-β2 group than that in the PBS group and FBS group, with the strongest expression in 30 minutes through 2 hours. The untreated gel contracted （78.00-＋3. 13 ）% fro,n its initial size, and contraction in the 1,2,5,10 p^g/L TGFq3~ group were （63.88＋-1.78）% ,（20.69_＋0.65）%, （19.49＋0.54）%, （ 16.24＋_0.84 ） % , respectively, TGF-[~2 increased HTFs contraction significantly （ Fgroup = 859,400, P = 0. 000 ）. Conclusions TGF-[32 can induce transdifferentiation of Tenon fibroblast into myofibroblast and increase cell contractility,with a concentration-dependent and time-dependent pattern to an extent. It may be the mechanism of scar formation after glaucoma filter surgery.
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2013年第3期215-219,共5页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目（81170852）
重庆市自然科学基金项目（CQCSTC2010BB5199）
