本文選題：膠原　切入點：皮膚　出處：《青島大學(xué)》2017年碩士論文

【摘要】：目的:通過體外培養(yǎng)人皮膚成纖維細(xì)胞,觀察狹鱈魚皮膠原蛋白肽對其生物學(xué)性狀的影響。方法:人皮膚成纖維細(xì)胞通過體外培養(yǎng),培養(yǎng)液中加入不同濃度的膠原蛋白肽,分為實驗對照組和空白對照組,使用酶標(biāo)法檢測狹鱈魚皮膠原蛋白肽對成纖維細(xì)胞增殖的影響、使用Annexin V/PI雙染法檢測狹鱈魚皮膠原蛋白肽對成纖維細(xì)胞凋亡的影響;使用Elisa試劑盒檢測狹鱈魚皮膠原蛋白肽對成纖維細(xì)胞分泌TGF-β1、SOD等指標(biāo)的表達(dá)變化;使用Western blot法檢測I型膠原蛋白的表達(dá);使用劃痕實驗法檢測膠原蛋白肽對人皮膚成纖維細(xì)胞遷移率的影響。結(jié)果:1.細(xì)胞增殖:不同濃度的膠原蛋白肽處理人皮膚成纖維細(xì)胞48h后,通過CCK-8試劑盒檢測膠原蛋白肽對人皮膚成纖維細(xì)胞增殖的作用。結(jié)果顯示經(jīng)膠原蛋白肽刺激后,成纖維細(xì)胞增殖速度呈現(xiàn)劑量依賴效應(yīng),隨著膠原蛋白肽濃度的增高,細(xì)胞的增殖速度也加快,與空白對照組相比具有顯著性差異(P0.05)。在膠原蛋白肽濃度為25mg/ml的條件下,成纖維細(xì)胞增殖速度最快。2.細(xì)胞凋亡:25mg/ml膠原蛋白肽處理24h后,我們通過Annexin-v-FITC/PI試劑盒檢測了膠原蛋白肽對人皮膚成纖維細(xì)胞凋亡的影響。經(jīng)膠原蛋白肽處理后,相比空白對照組,成纖維細(xì)胞的凋亡比率顯著下降(P0.05)膠原蛋白肽可有效促進(jìn)人皮膚成纖維細(xì)胞的增殖,抑制細(xì)胞凋亡,并可顯著提高成纖維細(xì)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)。與空白對照組相比,膠原蛋白肽組細(xì)胞遷移能力增強(P0.05)3.Elisa實驗結(jié)果:經(jīng)不同濃度膠原蛋白肽刺激后,結(jié)果顯示TGF-β1和SOD表達(dá)均升高,并呈劑量依賴性,25mg/ml及50mg/ml的濃度較空白對照組差異均具有極其顯著性差異(P0.01),其中,以50mg/ml作用最為顯著。4.劃痕實驗:結(jié)果顯示,經(jīng)過膠原蛋白肽作用后的細(xì)胞遷移率顯著提高(P0.01)。5.Western blot實驗:結(jié)果顯示,經(jīng)過膠原蛋白肽作用后的人皮膚成纖維細(xì)胞I型膠原蛋白的表達(dá)量提高(P0.05)。結(jié)論:狹鱈魚皮膠原蛋白肽能促進(jìn)成纖維細(xì)胞的增殖,提高人皮膚成纖維細(xì)胞存活率,以膠原蛋白濃度為25mg/ml時作用最為顯著,并能抑制細(xì)胞的凋亡。狹鱈魚皮膠原蛋白肽對TGF-βI、SOD的分泌表達(dá)具有顯著的促進(jìn)作用,并呈劑量依賴性。狹鱈魚皮膠原蛋白肽能促進(jìn)人皮膚成纖維細(xì)胞的遷移,促進(jìn)I型膠原蛋白合成可能在創(chuàng)傷愈合中發(fā)揮重要作用。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R641

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