羟喜树碱对体外培养的人Tenon囊成纤维细胞自噬的诱导作用  

Inducing effect of hydroxycamptothecin on autophagy of human Tenon capsule fibroblasts in vitro

作　　者：徐心雨 童俊[1] 范舒欣 袁志兰[1] 

机构地区：[1]南京医科大学附属江苏省人民医院眼科,210029

出　　处：《中华实验眼科杂志》2015年第3期196-200,共5页Chinese Journal Of Experimental Ophthalmology

摘　　要：背景 滤过道瘢痕化及人Tenon囊成纤维细胞（HTFs）增生是抗青光眼滤过手术失败的主要原因.目前已有羟喜树碱诱导成纤维细胞凋亡及其机制的相关报道,但是羟喜树碱对HTFs自噬的影响鲜见报道. 目的 探讨羟喜树碱对体外培养的HTFs自噬的影响.方法 收集3例接受斜视手术患者的Tenon囊组织,以组织块培养法,用含体积分数10％胎牛血清（FBS）的DMEM培养基原代培养HTFs并传代,取第3～6代细胞进行培养.分别在培养基中加入0.0、0.5、1.0、4.0、10.0 mg/L羟喜树碱,继续培养HTFs 24 h,以细胞计数试剂盒8（CCK-8）检测各组细胞的增生情况.选取4.0 mg/L羟喜树碱作用HTFs 24 h,用Cyto-ID自噬检测试剂盒进行荧光染色,观察各组细胞中自噬小体和自噬溶酶体成分的分布,用荧光显微镜和流式细胞仪分别对Cyto-ID染色阳性细胞数量及其荧光强度进行测定,并与对照组比较.用逆转录定量检测PCR（qRT-PCR）和Western blot法分别检测各组HTFs中自噬相关蛋白Beclin-1、自噬相关基因5（ATG-5）和微管相关蛋白1轻链3（LC-3）蛋白及其mRNA的相对表达水平.结果 随着羟喜树碱质量浓度的增加,HTFs细胞活力逐渐下降,0.0、0.5、1.0、4.0、10.0 mg/L羟喜树碱组HTFs细胞活力的总体差异有统计学意义（F=19.040,P＜0.001）,1.0、4.0、10.0 mg/L羟喜树碱组HTFs的细胞活力均低于对照组,以10.0mg/L组最低,差异均有统计学意义（P＜0.05,P＜0.01,P＜0.01）.qRT-PCR检测显示,4.0 mg/L羟喜树碱组HTFs中Beclin-1 mRNA、ATG-5mRNA和LC-3 mRNA的相对表达量分别是对照组的（3.225±0.346）、（2.839±0.418）和（3.761±0.224）倍,与对照组比较差异均有统计学意义（P=0.020、0.027、0.007）.Western blot检测表明,4.0 mg/L羟喜树碱组HTFs中Beclin-1、ATG-5和LC-3蛋白表达的灰度值较对照组明显上升,LC-3Ⅱ/Ⅰ灰度值比值分别为0.965±0.159和0.275±0.860,差异均有统计学意义（P=0.003）.Cyto-ID染�Background The fibrosis of filtering area caused by proliferation of human Tenon fibroblasts （HTFs） is one of failure causes following glaucoma surgery.Researches revealed that hydroxycamptothecin can induce the apoptosis of HTFs,but its influence on autophagy of HTFs is unclear.Objective This study attempted to investigate whether hydroxycamptothecin can cause an alteration of autophagic activity in HTFs.Methods Human Tenon capsular tissue was obtained from 3 patients during strabismus correction surgery under the informed consent of patients and their parents for the primary culture and passaged of HTFs in DMEM containing 10％ fetal bovine serum.The generation 3 to 6 cells then were incubated with 0.0,0.5,1.0,4.0,10.0 mg/L hydroxycamptothecin for 24 hours,respectively.A cell counting kit-8 （CCK-8） was used to detect the cell viability in different treated groups.The autophagic activity of HTFs was evaluated by a Cyto-ID autophagy detection kit,and then the autophagic flux was evaluated by counting the Cyto-ID positive cells under a fluorescence microscope,and the green fluorescence intensity was determined by flow cytometry.Quantitative reverse transcriptase PCR （qRT-PCR） and Western blot analysis were employed to assay the relative expressions of autophagic-associated genes and their proteins in HTFs,including Beclin-1,autophagy related gene 5 （ATG-5） and light chain 3 （LC-3）.Results The cell viability of HTFs in the 0.0,0.5,1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were （100.00 ± 6.44） ％,（91.70 ± 6.36） ％,（81.47 ± 6.00） ％,（68.43 ± 6.69） ％ and （59.97 ± 6.98） ％ respectively,showing a gradually declining trend with the increase of hydroxycamptothecin doses,with a significant difference among them （F=19.040,P＜0.001）,and the viability of HTFs in the 1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were significantly decreased than the control group （P＜0.05,P＜0.01,P＜0.01）.qRT-PCR analysis revealed that the relative expression levels of Be

关 键 词：自噬 羟喜树碱 人 Tenon囊 成纤维细胞 细胞培养 青光眼 

分 类 号：R96[医药卫生—药理学]