青光安颗粒剂对TGF-β1诱导的HTFs增殖影响的实验研究

Effect of Qingguang’ an granule on the proliferation of HTFs induced by TGF-beta 1

目的探讨青光安颗粒剂抑制转化生长因子β1 (TGF-β1)诱导的人类Tenon 成纤维细胞(HTFs)增殖的可能机制。方法从接受青光眼滤过手术(GFS)的个体获得人结膜下Tenon 胶囊样品。实验设计分为3 组:对照组(HTFs 未经处理,n = 10);TGF-β1 诱导组(100 ng/ ml TGF-β1 诱导HTFs,构建GFS 术后细胞模型,n = 10);TGF-β1+青光安治疗组(TGF-β1 诱导+青光安颗粒剂血清处理HTFs,n = 30)。TGF-β1+青光安治疗组按照青光安血清的剂量进一步分为3 组:TGF-β1+青光安高剂量组(TGF-β1 诱导+青光安颗粒剂血清5 ml处理HTFs);TGF-β1+青光安中剂量组(TGF-β1 诱导+青光安颗粒剂血清2.5 ml 处理HTFs);TGF-β1+青光安低剂量组(TGF-β1 诱导+青光安颗粒剂血清处理1 ml HTFs);每组设10 个培养皿。通过CCK-8 检测青光安对TGF-β1 诱导HTFs 增殖的影响;通过流式细胞术评估青光安对细胞周期的影响;通过Cyto-ID 免疫细胞化学染色测定青光安颗粒剂对HTFs 细胞自噬的影响;采用RT-PCR 和Western Blot 法测定自噬体形成的必需蛋白质Beclin-1,ATG-5 和LC3-Ⅲ基因和蛋白的表达水平。多组间比较采用单因素方差分析,组间两两比较采用LSD-t 检验。结果青光安能够抑制TGF-β1 诱导HTFs 的增殖能力,TGF-β1+青光安高剂量组细胞增殖水平(13.52±1.24)较TGF-β1 诱导组(23.42±1.25)降低,差异有统计学意义(F = 12.347,P < 0.01);TGF-β1 诱导G0/ G1 期的比例(88.29±0.35)降低,而S 期的比例(9.04±0.25)升高,而青光安治疗能够导致G0/ G1 期的比例(91.18±1.04)增加,而S 期的比例(5.41±0.59)降低,差异有统计学意义(F = 13.857,P < 0.01);与TGF-β1 诱导组相比,用TGF-β1+青光安治疗组导致荧光染色强度(1.84±0.14)降低,HTFs 阳性细胞数目(112.46±12.11)减少,差异有统计学意义(F =12.347,P = 18.472);TGF-β1+青光安治疗组能抑制TGF-β1 诱导对自噬基因Beclin-1、ATG-5 和LC- 3 Ⅲ mRNA 和蛋白质表达增加(P < 0.05)。结论青光安颗粒剂抑制TGF-β1 诱导的HTFs增殖,且可能机制为青光安诱导HTFs 细胞周期停滞于G0/G1 期,而且青光安可减少TGF-β1 诱导的HTFs 自噬。

Objective To explore the possible mechanism of Qingguang' an granule inhibiting the proliferation of HTFs induced by TGF-β1. Methods Samples of human subconjunctival Tenon capsules were obtained from individuals undergoing GFS surgery. The experimental design was divided into three groups: a control group(HTFs untreated, n = 10);TGF-β1 induction group(100 ng/ml TGF-β1 induced HTFs, constructed cell model after GFS, n = 10);TGF-β1 + Qingguang' an treatment group(TGF-β1 induced + Qingguang' an granule serum treated HTFs, n = 30). The treatment group was further divided into three groups according to the dose of Qingguang' an serum: the high dose group of TGF-β1 + Qingguang' an (TGF-β1 induction +Qingguang' an granule serum 5 ml for HTFs), the medium dose group of TGF-β1 + Qingguang' an granule serum 2.5 ml for HTFs, and the low dose group of TGF-β1 + Qingguang' an granule serum 1 ml for HTFs;There were 10 Petri dishes in each group. The effect of glaucoma on the proliferation of TGF-β1-induced HTFs was detected by CCK-8;the effect of glaucoma on cell cycle was evaluated by flow cytometry;the Cyans granules were determined by Cyto-ID immunocytochemical staining. The effect of autophagic cells;the expression levels of the essential proteins Beclin-1, ATG-5 and LC3-Ⅲ genes and proteins formed by autophagosomes were determined by RT-PCR and Western Blot. Results Qingguang' an inhibited the proliferation of HTFs cells induced by TGF-beta1. The proliferation level of HTFs cells in the high dose group of TGF-β1+Qingguang' an(13.52±1.24) was lower than that in the high dose group of TGF-β1(23.42±1.25), with a statistically significant difference(F = 12.347, P < 0.001);the proportion of G0/G1 phase induced by TGF-β1(88.29±0.35) was lower, while the proportion of S phase (9.04±0.25)was higher. The proportion of G0/G1 phase was increased(91.18±1.04), while that of S phase was decreased(5.41+0.59), with a significant difference(F = 13.857, P = 0.007);compared with TGF-β1 induced group, the intensity of fluorescence staining decreased(1.84+0.14) and the number of HTFs-positive cells was decreased (112.46+12.11), with a significant difference (F = 12.347, P = 18.472). TGF-β1 + Qingguang' an treatment group inhibited the increase of expression of autophagy gene Beclin-1, ATG-5 and LC-3 Ⅲ mRNA and protein induced by TGF-β1(P < 0.05). Conclusion Qingguang' an granule inhibits the proliferation of HTFs induced by TGF-β1, and the possible mechanism is that Qingguang' an induces HTFs cell cycle to stagnate at G0/G1 phase, and Qingguang' an can reduce the autophagy of HTFs induced by TGF-β1.