【作者】 窦梅；

【导师】 王春波；

【作者基本信息】 青岛大学 ， 药理学， 2004， 硕士

【摘要】 目的 探讨在单次长波紫外线（UVA）辐射下，扇贝多肽（PCF）对人HaCaT角质形成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 采用海洋生物工程技术，从栉孔扇贝中提取PCF,Mr＝879。建立UVA氧化损伤细胞和无毛小鼠的病理模型，辐射强度分别为：细胞5J／cm²；无毛小鼠4.13J／cm²。细胞实验设计分为7组：对照组，UVA模型组，UVA+0.125％PCF组，UVA+0.25％PCF组，UVA+0.5％PCF组，UVA+1％PCF组，和UVA+0.1％VitC组。MTT法检测细胞活性；酶生化法测定胞浆超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-px）、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PI染色法测定细胞的凋亡率；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（△ψM）；透射电镜观察细胞超微结构的改变；彗星电泳法检测DNA的损伤程度；原位杂交技术检测细胞内p21mRNA的变化。动物实验设计分为5组：对照组，UVA模型组，UVA+5％PCF组，UVA+20％PCF组，UVA+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、P物质、Ⅰ型胶原及Ⅳ型胶原的含量。 结果 在5J／cm²UVA辐射下，在0.125％～1％的浓度范围内PCF能剂量依赖性的提高角质形成细胞活性；减轻对细胞DNA的损伤；降低胞浆中ROS、MDA含量；提高SOD，GSH-px的活力；提高A-SAC及T-AOC的含量；降低细胞凋亡率；稳定线粒体的膜电位；减少细胞内游离Ca²⁺（以上各指标加药组与模型组比较，P＜0.05，差别均有显著性意义）；电镜下可见UVA模型组角质形成细胞绒毛消失，细胞核固缩，胞质内空泡形成，线粒体受损，嵴断裂，空泡化变性，大量髓样小体产生；而PCF组细胞受损情况较模型组明显减轻；p21mRNA原位杂交发现PCF可明显抑制其表达，并呈量效关系。免疫组化结果表明PCF可以抑制无毛小鼠表皮细胞突变型p53基因蛋白和EGFR蛋白的表达（P＜0.05）；降低胞浆内P物质含量（P＜0.05）；Ⅰ型胶原含量减少（P＜0.05）；但对Ⅳ型胶原没有影响（P＞0.05）。 中文摘要 结论扇贝多肤对单次UVA（SJ/cm，）诱导的人HaCaT角质形成细胞氧化损伤有保护作用。其机制与清除氧自由基、提高抗氧化酶如SOD，GSH一px等活性、提高A一SAC及T一AOC的含量、降低细胞内游离「CaZ+〕;、保护线粒体膜结构、减少DNA链断裂、抑制p21 mRNA表达及细胞凋亡有关。对单次UVA（4 .13）/cmZ)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、降低P物质含量、及减少I型胶原的表达作用有关。更多还原

【Abstract】 Objective To investigate the protective effect of PCF on the oxidative damages in HaCaT keratinocytes and the skin of hairless mice induced by single UVA.Methods Polypeptide from Chlamys farreri (PCF,Mr=879) was isolated from Chla-mys farreri using enzymetically engineering technique. The cells were randomly divided into seven groups: control group, UVA model group, UVA+0. 125%PCF group, UVA+ 0. 25%PCF group, UVA + 0. 5%PCF group, UVA + 1%PCF group and UVB + 0. 1 % VitC group. MTT method was used to detect the viability of the cells. The intracellular SOD, GSH-px, MDA, ROS, T-AOC, and A-ASC were measured by biochemical methods. The effects of PCF on UVA-induced apoptosis, intracellur calcium, and mitochondrial membrane potential were investigated by flow cytometer. UVA-induced DNA damage was detected by comet assay. The ultrastructures of HaCaT keratinocytes were observed under transmission electron microscope. In situ hybridization was used to examine the changes of p21 mRNA expression. The mice were randomly divided into five groups: control group, UVA model group, UVA+ 5% PCF group, UVA+ 20 % PCF group, and UVA+10 % Vit C group. The expression of mutant p53 protein, EGFR, substance P, collegen type I and type IV were examined by immunohistochemical methods.Results PCF greatly enhanced the viability of HaCaT keratinocytes and markedly increased the activities of SOD and GSH-px, the contents of T-AOC and A-ASC, while decreased the amounts of MDA and ROS (all P<0.05 compared PCF groups with model group). UVA-induced apoptosis and DNA damage in HaCaT keratinocytes were inhibited by PCF. The concentration of cellular free calcium decreased and the mitochondrial membrane potential were increased by PCF (P<0. 05). In ultrastructure, PCF greatly decreased UVA-induced damage, especially membrane injuries. p21mRNA expression was inhibited in PCF groups. All results were in a dose-dependent manner. Immunohistochem-istry showed that PCF also inhibited the expression of p53 protein, EGFR, substance P and collegen type I (P<0. 05), but there was no statistical significance in the expression of collegen type W(P>0. 05).Conclusion PCF can inhibit UVA-induced oxidative damage on HaCaT keratino-cytes. The mechanism were due to its abilities to scavenge oxygen free radical, inhibit lipid peroxidation, increase anti-oxidarvt enzymes, decrease intracellular calcium, protect the membrane structure, and block the expression of p21mRNA. The protective effect of PCF on the hairless mice skin against UVA-induced damage is associated with its ability of decreasing the overexpression of p53 protein, EGFR, substance P and collegen type I.更多还原