狭鳕鱼皮胶原蛋白肽对人皮肤成纤维细胞生物学特性的影响

发布时间：2018-03-29 02:23

本文选题：胶原　切入点：皮肤　出处：《青岛大学》2017年硕士论文

【摘要】：目的:通过体外培养人皮肤成纤维细胞,观察狭鳕鱼皮胶原蛋白肽对其生物学性状的影响。方法:人皮肤成纤维细胞通过体外培养,培养液中加入不同浓度的胶原蛋白肽,分为实验对照组和空白对照组,使用酶标法检测狭鳕鱼皮胶原蛋白肽对成纤维细胞增殖的影响、使用Annexin V/PI双染法检测狭鳕鱼皮胶原蛋白肽对成纤维细胞凋亡的影响;使用Elisa试剂盒检测狭鳕鱼皮胶原蛋白肽对成纤维细胞分泌TGF-β1、SOD等指标的表达变化;使用Western blot法检测I型胶原蛋白的表达;使用划痕实验法检测胶原蛋白肽对人皮肤成纤维细胞迁移率的影响。结果:1.细胞增殖:不同浓度的胶原蛋白肽处理人皮肤成纤维细胞48h后,通过CCK-8试剂盒检测胶原蛋白肽对人皮肤成纤维细胞增殖的作用。结果显示经胶原蛋白肽刺激后,成纤维细胞增殖速度呈现剂量依赖效应,随着胶原蛋白肽浓度的增高,细胞的增殖速度也加快,与空白对照组相比具有显著性差异(P0.05)。在胶原蛋白肽浓度为25mg/ml的条件下,成纤维细胞增殖速度最快。2.细胞凋亡:25mg/ml胶原蛋白肽处理24h后,我们通过Annexin-v-FITC/PI试剂盒检测了胶原蛋白肽对人皮肤成纤维细胞凋亡的影响。经胶原蛋白肽处理后,相比空白对照组,成纤维细胞的凋亡比率显著下降(P0.05)胶原蛋白肽可有效促进人皮肤成纤维细胞的增殖,抑制细胞凋亡,并可显著提高成纤维细胞分泌TGF-β1、SOD等活性因子的含量(t=4.07,F=774.23、2 100.63,P0.05)。与空白对照组相比,胶原蛋白肽组细胞迁移能力增强(P0.05)3.Elisa实验结果:经不同浓度胶原蛋白肽刺激后,结果显示TGF-β1和SOD表达均升高,并呈剂量依赖性,25mg/ml及50mg/ml的浓度较空白对照组差异均具有极其显著性差异(P0.01),其中,以50mg/ml作用最为显著。4.划痕实验:结果显示,经过胶原蛋白肽作用后的细胞迁移率显著提高(P0.01)。5.Western blot实验:结果显示,经过胶原蛋白肽作用后的人皮肤成纤维细胞I型胶原蛋白的表达量提高(P0.05)。结论:狭鳕鱼皮胶原蛋白肽能促进成纤维细胞的增殖,提高人皮肤成纤维细胞存活率,以胶原蛋白浓度为25mg/ml时作用最为显著,并能抑制细胞的凋亡。狭鳕鱼皮胶原蛋白肽对TGF-βI、SOD的分泌表达具有显著的促进作用,并呈剂量依赖性。狭鳕鱼皮胶原蛋白肽能促进人皮肤成纤维细胞的迁移,促进I型胶原蛋白合成可能在创伤愈合中发挥重要作用。
[Abstract]:Objective: to observe the effect of collagen peptide on the biological characteristics of human skin fibroblasts cultured in vitro. Methods: human skin fibroblasts were cultured in vitro, and different concentrations of collagen peptides were added into the culture medium. The fibroblasts were divided into experimental control group and blank control group. The effect of collagen peptide on fibroblast proliferation was detected by enzyme labeling method, and the effect of collagen peptide on fibroblast apoptosis was detected by Annexin V/PI double staining. Elisa kit was used to detect the expression of TGF- 尾 1 SOD in fibroblasts, and Western blot method was used to detect the expression of type I collagen. The effect of collagen peptide on the migration of human skin fibroblasts was detected by scratch test. Results: 1. Cell proliferation: human skin fibroblasts were treated with different concentrations of collagen peptides for 48 hours. The effect of collagen peptide on the proliferation of human skin fibroblasts was detected by CCK-8 kit. The results showed that the proliferation rate of fibroblasts induced by collagen peptide was dose-dependent, with the increase of collagen peptide concentration. When the concentration of collagen peptide was 25mg/ml, the proliferation rate of fibroblasts was the fastest. 2. Apoptosis: 25 mg / ml collagen peptide was treated for 24 h. We tested the effect of collagen peptide on apoptosis of human skin fibroblasts by Annexin-v-FITC/PI kit. The apoptotic ratio of fibroblasts was significantly decreased (P 0.05). Collagen peptide could effectively promote the proliferation of human skin fibroblasts, inhibit cell apoptosis, and significantly increase the content of active factors such as TGF- 尾 1 SOD in fibroblasts. The cell migration ability of collagen peptide group was enhanced. The results of Elisa showed that the expression of TGF- 尾 1 and SOD were increased after different concentrations of collagen peptide were stimulated, and the expression of TGF- 尾 1 and SOD increased. In a dose-dependent manner, the concentrations of 25mg / ml and 50mg/ml were significantly higher than those of the control group (P 0.01). Among them, the effect of 50mg/ml was the most significant. 4. Scratch test: the results showed that, After treatment with collagen peptide, the cell migration rate was significantly increased by P0.01U. 5. Western blot assay: the results showed that, The expression of type I collagen in human skin fibroblasts was increased after the treatment of collagen peptide. Conclusion: the collagen peptide of cod skin can promote the proliferation of fibroblasts and increase the survival rate of human skin fibroblasts. When the concentration of collagen was 25mg/ml, the effect was the most significant and the apoptosis was inhibited. The collagen peptide of cod skin could significantly promote the secretion and expression of TGF- 尾 Ion SOD. The collagen peptide of cod skin can promote the migration of human skin fibroblasts and the synthesis of type I collagen may play an important role in wound healing.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R641

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