地龙蛋白对人增生性瘢痕细胞增殖抑制作用机制研究

Earthworm Protein of Hyperplastic Scar Cell Proliferation Inhibition Mechanism Research

目的:研究地龙蛋白对人增生性瘢痕细胞增殖抑制作用机制。方法:培养人增生性瘢痕成纤维细胞(HSF),将传代好的HSF细胞分为四组,分别加入地龙样品浓度为0 mg/m L(对照组)、0.3125 mg/m L(低剂量组)、0.625 mg/m L(中剂量组)、1.25 mg/m L(高剂量组)。应用MTS法检测细胞活力,流式细胞仪检测细胞凋亡率,RT-PCR法和Western-blot法测定Cyclin D1、Caspase-3、TGF-β1的表达,Western-blot法测定Bcl-2、Mcl-2的表达。结果:地龙蛋白对HSF细胞的增殖活力有抑制作用。浓度0.625mg/m L、1.25 mg/m L样品组作用细胞48 h后凋亡率为分别为31.5%和69.2%,(P<0.01);RT-PCR显示,浓度0.625 mg/m L、1.25mg/m L样品组能够增加Caspase-3的基因表达,降低Cyclin D1的基因表达,降低TGF-β1的基因表达,(P<0.01);浓度0.3125mg/m L样品组与空白对照组比较有显著性差异(P<0.05)。W-B方法显示,各样品组能够增加Caspase-3的蛋白表达量,降低Cyclin D 1的蛋白表达量,降低TGF-β1的蛋白表达量,(P<0.01);浓度0.625 mg/m L、1.25 mg/m L样品组能够降低Bcl-2蛋白表达量,(P<0.01);浓度0.3125 mg/m L样品组Bcl-2的蛋白表达量与空白对照组比较有显著性差异(P<0.05)。结论:地龙蛋白诱导细胞死亡,作用机制与下调Cyclin D1的基因和蛋白表达,上调Caspase-3的基因和蛋白表达,下调TGF-β1的基因和蛋白表达,下调Bcl-2蛋白表达量有关。

Objective： The mechanism of the proliferation inhibition of hypertrophic scar cells was studied. Methods： Cultured hy- perplastic scar fibroblasts （HSF）, HSF cells were divided into four groups after generation, add earthworm sample separately, the concentration is 0 mg/mL（The control group）, 0.3125 mg/mL（Low dose group）, 0.625 mg/mL （Middle dose group）, 1.25 mg/mL （High dose group）. Cell vitality was detected by MTS method. Cell apoptosis rate was detected by flow cytometry. The expression of Cyclin D1, Caspase - 3 and TGF-β1 was determined by Rt-pcr methodand Western blot method. The expression of bcl-2 and McL-2 was determined by Western - blot method. Results： Earthworm proteinhas inhibitory effect on the proliferation activity of HSF cells. Concentration of 0.625 mg/mL, 1.25 mg/mL sample group effect on the cellseparately after 48 hours, the apoptosis rate of the cell was 31.5% and 69.2%. method showed that concentration of 0.625 mg/mL, 1.25 mg/mL sample group was able to increase gene expression of Caspase-3, reduce gene expression of CyclinD1 and TGF -β1（P〈0.01）. There was a significant difference between the 0.3125 mg/mL sample group and the blank control group （P〈0.05）, Western - blot method showed that Each sample group could increase the protein expression of Caspase-3, reduce the protein expression of CyclinD1, and reduce the protein expression of TGF-beta 1 （P〈0.01）. The concentration of 0.625 mg/mL and 1.25 mg/mL samples was able to reduce the expression ofbcl-2 protein （P〈0.01）. The protein expression of bcl-2 in the concentration of 0.3125 mg/mL sample group was significantly different than that in the blank control group （P〈0.05）. Conclusion： The mechanism of earthworm protein inducing cell death was related to that earthworm protein can cut Cyclin D1 gene and protein expression, raise Caspase 3 gene and protein expression, cut TGF-β1 gene and protein expression, cut the Bcl-2 protein expression quantity.