【作者】 丁擘晓；

【导师】 王春波；

【作者基本信息】 青岛大学 ， 药理学， 2003， 硕士

【摘要】 目的 探讨在中波紫外线（UVB）辐射下，扇贝多肽（PCF）对人真皮成纤维细胞和昆明种无毛小鼠皮肤氧化损伤的保护作用机制。方法 采用海洋酶生物工程技术，从栉孔扇贝中提取PCF，Mr=800-1000。建立UVB(辐射细胞的强度为1.176×10^-4J/cm²；小鼠为5.15×10^-2J/cm²×30天)对体外培养的人真皮成纤维细胞和昆明种无毛小鼠皮肤氧化损伤的病理模型。细胞实验设计分为6组：对照组，UVB模型组，UVB+0.25％PCF组，UVB+0.5％PCF组，UVB+1％PCF组，和UVB+0.1％VitC组。MTT法检测细胞活性；测定胞浆超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-px）、过氧化氢酶（CAT）、黄嘌呤氧化酶（XOD）活性和活性氧（ROS）、丙二醛（MDA）含量以及细胞抗超氧阴离子能力（A-SAC）、总抗氧化能力（T-AOC）；流式细胞仪Annexin V-FITC法测定细胞的凋亡率和死亡率；以Fluo-3-AM为荧光染料，测定细胞内游离[Ca²⁺]_i；rhodamine 123为荧光染料，测定线粒体膜电位（Δψ_M）；透射电镜观察细胞超微结构的改变；彗星电泳法检测DNA链断裂的程度（辐射强度为211.15J/m²）。动物实验设计分为对照组，UVB模型组，UVB+5％PCF组，UVB+20％PCF组，UVB+10％Vit C组。免疫组化法测定皮肤p53基因蛋白表达、表皮生长因子受体（EGFR）表达以及P物质的含量。结果 在UVB辐射情况下1.PCF能显著提高成纤维细胞的活性；提高胞浆中SOD，GSH-px的活性，同时可降低XOD的活性；降低ROS、MDA含量，提高A-SAC及T-AOC，且在0.25-1％的浓度范围内呈量效关系，而不影响CAT的活性；降低成纤维细胞的凋亡率和死亡率；减少成纤维细胞内游离[Ca²⁺]_i；稳定线粒体的膜电位；2．彗星电泳表明PCF组DNA链断裂较UVB模型组明显减轻。3．免疫组化结果表明PCF可以抑制小鼠表皮细胞突变型p53基因和EGFR的表达以及降低胞浆内P物质含量。4．电镜下可见UVB模型组成纤维细胞胞质内可见空泡形成，线粒体受损，嵴断裂，空泡化变性，粗面内质网脱颗粒，呈囊泡状扩张，高尔基复合体减少；而PCF组细胞受损的情况明显减轻，且可使粗面内质网及高尔基复合体增多。结论 扇贝多肽对中波紫外线诱导人真皮成纤维细胞氧化损伤有保护作用，其机制与能清除氧自由基、提高抗氧化酶活性、降低细胞内游离[Ca²⁺]_i、保护细胞膜相结构、减少DNA断裂有关；对UVB连续氧化损伤无毛小鼠皮肤的保护作用机制与其抑制p53基因突变、EGFR的表达及降低、P物质含量作用有关。更多还原

【Abstract】 Background Polypeptide from Chlamys farreri (PCF, Mr=800-1000) is a novel marine polypeptide which was isolated from Chlamys farreri using enzymetically engineering technique. Previous studies from our laboratory indicated that PCF could inhibit the oxidative damage of ultraviolet A on HeLa cells in vitro and the skin of hairless mice in vivo. To further elucidate a possible role for PCF on UVB-damaged normal human cells and the skin of hairless mice, we established the oxidative damage models of normal human dermal fibroblasts (NHDF) and hairless mice exposed to ultraviolet B (cells:1.176 X 10-4J/cm2; mice: 154.5 J/cm2) to study the protective effect of PCF on human dermis fibroblasts and the skin of hairless mice. Methods The cells were randomly divided into six groups: control group, UVB model group, UVB+0.25 %PCF group, UVB+0.5%PCF group, UVB+1 %PCF group and UVB+0.1 %VitC group. MTT method was used to detect the viability of NHDF. The intracellular SOD, GSH-px, CAT, XOD, MDA, ROS, T-AOC, and A-ASC were measured. The effect of PCF on UVB -induced apoptosis were investigated by Annexin V-FITC assay. Intracellur calcium was determined with the calcium-sensitive fluorochrome Fluo-3 , and mitochondrial transmembrane potential with rhodamine 123. Comet assay was employed to detect the UVB -induced DNA damage. The ultrastructure of NHDF was observed under transmissionelectron microscope. The mice were randomly divided into five groups: control group, UVB model group, UVB+5 %PCF group, UVB+20 %PCF group, and UVB+10 % Vit C group. Immunohistochemical methods were used to examined the expression of mutant p53 gene, EGFR, and substance P. Results The results indicated that PCF could greatly enhance the viability of NHDF and markedly promote SOD,GSH-px, T-AOC, and A-ASC, while the amounts of MDA and ROS, the activity of XOD were decreased. PCF could inhibit UVB-induced apoptosis and DNA damage in NHDF. The concentration of cellular free calcium and the mitochondrial transmembrane potential were increased by PCF. In ultrastructure of NHDF, PCF could greatly decrease UVB-induced damage, especially membrane. Immunohistochemistry showed that PCF could also inhibit the expression of p53 gene, EGFR and substance P. Conclusion PCF can inhibit UVB-induced oxidative damage on normal human dermal fibroblasts. Its mechanism is due to its abilities of scavenging oxygen free radical, inhibiting lipid peroxidation, increasing anti-oxidative enzymes, decreasing intracellular calcium and protection of membrane structure. The protective effect of PCF on the hairless mice skin against UVB-induced damage is associated with its ability of decreasing the overexpression of p53 gene, EGFR, and substance P.更多还原