基于LCS探讨重组人TGF-β2对HTF细胞增殖及转分化的作用

Effects of recombinant human TGF-β2 on proliferation and transdifferentiation of HTF based on live cell station

目的基于活细胞工作站(LCS)探讨不同剂量重组人转化生长因子-β(transforming growth factor-beta,TGF-β2)对人Tenon’s囊成纤维细胞(HTF)增殖及转分化的作用及机制,为确定建立青光眼滤过术后HTF纤维化瘢痕细胞模型的最佳实验条件提供理论基础。方法培养原代HTF,分别用不同浓度TGF-β2(0,1,5,10,20,100 ng/ml)培养。CCK-8检测细胞活性。LCS分析不同浓度TGF-β2处理0,12,24,48,72 h时细胞增殖率。流式细胞术碘化丙啶染色检测细胞周期。Western blot检测细胞增殖指标增殖细胞核抗原(PCNA)蛋白,细胞转分化指标α-平滑肌肌动蛋白(α-SMA)、纤连蛋白(FN)蛋白的表达。结果1~100 ng/ml的TGF-β2对HTF无明显细胞毒性作用。相对于TGF-β2干预0,12,24,72 h时,TGF-β2干预48 h时促HTF增殖作用最为显著(P<0.05),因此采用TGF-β2干预HTF 48 h进行后续研究。与0 ng/ml的TGF-β2相比,5,10,20 ng/ml的TGF-β2干预促进HTF细胞转分化指标α-SMA、FN的表达(P<0.05)。与0 ng/ml的TGF-β2比较,5,10 ng/ml的TGF-β2干预促进HTF增殖(P<0.05),提高HTF增殖指标PCNA的表达(P<0.05),降低G0/G1期细胞的比例(P<0.05),增加S期细胞的比例(P<0.05)。结论LCS系统是研究TGF-β2对HTF细胞作用的有效方法。5~10 ng/ml的TGF-β2能有效促进HTF细胞转分化,通过促进细胞进入S期增加增殖期细胞的比例,进而促进HTF细胞增殖。推荐5~10 ng/ml的TGF-β2干预48 h建立HTF纤维化瘢痕细胞模型。

Objective To study the effect and mechanism of different doses of recombinant human transforming growth factor-beta(TGF-β2)on the proliferation and the transdifferentiation of human Tenon’s capsule fibroblasts(HTFs)based on live cell station(LCS),and lay a theoretical basis for determining the optimal experimental conditions for HTF fibrosis model after glaucoma filtration surgery.Methods Primary HTFs were cultured and treated with 0,1,5,10,20,100 ng/ml TGF-β2,respectively.Cell viability was detected by CCK-8.Cell proliferation rate was measured after TGF-β2 treatment for 0,12,24,48,72 h using the LCS analysis system.Cell cycle was detected by flow cytometry with propidium iodide staining.The expression levels of cell proliferation index proliferating cell nuclear antigen(PCNA)protein and cell transdifferentiation indexα-smooth muscle actin(α-SMA)and fibronectin(FN)proteins were detected by Western blot.Results TGF-β2 treatment at 1-100 ng/ml had no obvious cytotoxic effect on HTF.After treatment with TGF-β2 for 0,12,24,48,72 h,the effect of TGF-β2 on HTF proliferation was the most significant at 48 h.Therefore,the follow-up study was carried out in HTFs by TGF-β2 for 48 h.Compared with 0 ng/ml TGF-β2,5,10,20 ng/ml TGF-β2 promoted the transdifferentiation of HTF cells(P<0.05).Compared with 0 ng/ml TGF-β2,5,10 ng/ml TGF-β2 promoted the cell proliferation(P<0.05),increased the expression of proliferation index PCNA(P<0.05),decreased the proportion of G0/G1-stage cells(P<0.05),and increased the proportion of S stage cells(P<0.05).Conclusion LCS system is an effective method to study the effects of TGF-β2 on HTF.TGF-β2 at 5-10 ng/ml can effectively promote the transdifferentiation of HTF and the proliferation of HTF by promoting the transition of the cell cycle to the S phase.The experimental conditions of 5-10 ng/ml TGF-β2 intervention for 48 h are recommended to establish HTF fibrosis model.