【作者】 陈海英；

【导师】 于文功； 王春波；

【作者基本信息】 中国海洋大学 ， 药物化学， 2008， 博士

【摘要】 目的建立紫外线B(UVB)对体外培养的小鼠胸腺淋巴细胞凋亡的病理模型,探讨在UVB辐射下,扇贝多肽(PCF)抑制UVB诱导的小鼠胸腺淋巴细胞辐射损伤的作用机制。方法采用正交实验设计,以流式细胞仪PI染色法测定细胞凋亡率,采用45 mJ/cm2的UVB辐射体外培养的小鼠胸腺淋巴细胞,确立凋亡的病理模型,实验分为六组:正常对照组、UVB模型组、UVB + 5.69 mM PCF组,UVB + 2.84 mM PCF组,UVB + 1.42 mM PCF组和UVB + 5.69 mM VitC组。首先研究PCF对UVB辐射小鼠胸腺淋巴细胞后氧化还原状态的影响,UVB辐射后测定细胞抗羟自由基、抗超氧阴离子的水平、黄嘌啉氧化酶(XOD)和NADPH氧化酶活力;以2,7-二氯氢化荧光素二酯(DCFH-DA)为荧光探针,检测PCF对UVB照射后细胞内ROS生成的影响。其次,研究PCF对UVB诱导小鼠胸腺淋巴细胞凋亡的线粒体通路和表面受体通路的影响,分别用Rhodamine 123和Fluo-3-AM荧光染色测定线粒体膜电位和细胞内游离钙的变化;免疫细胞化学检测细胞Bcl-xl和Bid基因蛋白的表达;流式细胞仪检测caspase-3的活性;western-blot检测线粒体细胞色素C(cytochrome c)的释放、FADD和caspase-8基因蛋白的表达;电镜分析PCF和caspase-8抑制剂(z-IETD-fmk)对UVB诱导的胸腺淋巴细胞凋亡的影响;RT-PCR检测Fas(CD95)mRNA的表达。最后,使用基因芯片技术分析PCF对UVB辐射后小鼠胸腺淋巴细胞基因谱的差异表达影响。Western-blot检测硫氧还蛋白(Trx)、蛋白质丝氨酸苏氨酸激酶(Akt/PKB)的活性,预先加入或不加入PI3K/Akt通路特异性抑制剂LY294002检测细胞凋亡信号调节激酶Ⅰ(apoptosis signal regulating kinase-1,ASK1)、JNK的活性、DNA ladder和线粒体膜电位(?ψМ);免疫细胞化学检测细胞p53基因蛋白的表达;原位杂交检测细胞p38 mRNA、p21 mRNA的表达;RT-PCR检测c-fos mRNA和c-jun mRNA的表达;结果正交实验结果表明,45 mJ/cm2的UVB辐射小鼠胸腺淋巴细胞后2 h为最佳凋亡模型;在1.42 mM~5.69 mM浓度范围内,PCF能提高抗羟自由基和抗超氧阴离子的水平,降低胸腺淋巴细胞中黄嘌啉氧化酶、NADPH氧化酶的活力和ROS的含量。PCF可调节线粒体通路和表面受体通路上凋亡相关分子的表达,稳定线粒体的膜电位,降低淋巴细胞内游离钙离子,使Bcl-xl蛋白表达升高而Bid蛋白表达降低;PCF抑制线粒体cytochrome c的释放,使caspase-3的活性降低;PCF可抑制表面受体通路上Fas mRNA、FADD和caspase-8基因蛋白的表达;当预先给予caspase-8抑制剂和PCF能够减轻UVB对细胞超微结构的损害。基因芯片结果显示,与模型组相比,PCF组有功能差异表达的基因共104个,其中上调基因56个,下调基因48个;western-blot结果提示,PCF能够提高小鼠胸腺淋巴细胞硫氧还蛋白的表达,激活AKT的活性,抑制ASK1-JNK/p38凋亡通路的活化。预先使用PI3K/Akt通路特异性抑制剂LY294002,DNA ladder片段增多,线粒体膜电位降低;此外,PCF可提高细胞周期相关基因p53蛋白的表达和p21 mRNA的表达;降低转录因子c-fos mRNA和c-jun mRNA的表达。结论:PCF能够改善由UVB辐射诱导的细胞氧化损伤,从根本上降低活性氧自由基的产生,是较好的海洋类抗氧化剂;PCF能够抑制线粒体凋亡信号通路和膜表面受体凋亡信号通路;提高抗氧化蛋白、细胞生存相关基因,抑制ASK1-JNK/p38凋亡通路的活化,抑制转录因子的表达,PCF通过调节细胞氧化还原、凋亡、信号转导、细胞周期、转录调节等众多基因的表达,起到抗氧化、抗凋亡、抑制UVB对小鼠胸腺淋巴细胞辐射损伤的作用。上述结果表明,PCF是海洋类抑制UVB辐射的优质防护剂。更多还原

【Abstract】 AIM The study establishs the apoptotic models of thymocytes caused by UVB radiation in vitro to investigate the inhibit effects and mechanism of PCF (polypeptides from Chlamys farreri) on thymocytes from the mouse damaged by UVB radiation. MATHODS Using 45mJ/cm2 UVB to radiate mouse thymocytes, UVB-induced apoptotic model of thymocytes is established by orthogonal design and apoptotic rate is determined by flow cytometry PI staining. Cells are divided into six groups: control group, UVB model group, UVB+5.69 mM PCF group, UVB+2.84 mM PCF group, UVB+1.42 mM PCF group, UVB+5.69 mM Vitamine C group. First, the study investigates the effect of PCF on redox state in mouse thymocytes after UVB radiation. The anti-hydroxy radical, anti-superoxide anion level, activity of xanthine oxidase(XOD)and NADPH oxidase in thymocytes is detected after UVB radiation. Effect of PCF on UVB-induced production of ROS is detected by 2’, 7’-Dichlorofluorescin diacetate (DCFH-DA). Second, the study investigates the effect of PCF on apoptosis signal transduction pathway from mitochondria and cell surface receptor in mouse thymocytes induced by UVB radiation.The mitochondria membrane potential and the free calcium variation on thymocytes are tested using Rhodamine 123 and Fluo-3-AM fluorescein stain respectively. Bcl-xl and Bid proteins are examined by immunocytochemical technica. The caspase-3 activity is measured by Flow Cytometry (FCM). Western blot analysis is performed to determine the release of cytochrome c, expressing levels of FADD and caspase-8. The effects of PCF and caspase-8 inhibitor z-IETD-fmk on UVB-induced apoptosis of thymocytes are investigated by electron microscope. Expressing levels of Fas mRNA are detected by RT-PCR. And last, the effect of PCF on the different of gene expression profile in thymocytes after UVB radiation are studied by gene chip technique. The activation of thioredoxin (Trx) and protein kinase B (Akt/PKB) is investigated by western-blot. ASK1, JNK activation, DNA ladder and Mitochondria memberine potential are also investigated after pretreatment with or without PI3K/Akt pathway inhibitor LY294002. P53 protein is examined by immunocytochemical technica. The p38mRNA and P21 mRNA expressions are detected through the technique of in situ hybridiation. The expression of c-fos and c-jun is observed by RT-PCR. RESULTS Results of orthogonal experiment suggest that 45mJ/cm2 UVB and 2 h incubation after radiation make up the best apoptotic model. 1.42~5.69 mM PCF increases the anti-hydroxy radical and anti-superoxide anion level, decreases the activity of xanthine oxidase(XOD), NADPH oxidase and the ROS accumulation in thymocytes. PCF can modulate the expression of molecular that related to apoptosis signal transduction pathway from mitochondria and cell surface receptor. PCF can stabilize the mitochondria membrane potential, decrease free calcium, increase the expression of Bcl-xl, reduces the expression of Bid and the releasing of cytochrome c and caspase-3 activity. PCF can inhibit the expression levels of FADD and caspase-8 that related to apoptosis signal tranduction pathway from cell surface receptor. The ultrastructural of thymocytes is less damaged by UVB when pretreatment with the caspase-8 inhibitor z-IETD-fmk and PCF. The ruslts of genechip indicate that 104 genes which have identified functions show a differential expression in PCF group, with 56 genes up-regulated and 48 genes down-regulated when compared with model group. Western-blot shows that PCF can active Trx and Akt, inhibit the activation of the ASK1-JNK/p38 apoptosis pathway. When pretreatment with PI3K/Akt pathway inhibitor LY294002 with PCF, the fragment of DNA ladder is increased while the mitochondria memberine potential is decreased. PCF can elevate the expression of p53 protein, p21 mRNA which related to cell cycle, cut down the expression of transcription factors c-fos and c-jun mRNA. CONCLUSION PCF can reverse the cell damage radiated by UVB. PCF fundamentally depresses active oxygen free radical product, function as a good antioxidant reagent from ocean. PCF can inhibit apoptosis signal transduction pathway from mitochondria and cell surface receptor, increase antioxygen protein and genes which related to survival, inhibit the activation of the ASK1-JNK/p38 apoptosis pathway, cut down the expression of transcription factors. PCF exerts its antioxidation, anti-apoptosis and anti-UVB radiative damage by modulate the expression of numerous genes that related to cellular oxidoreduction, apoptosis, signal transduction, cell cycle and transcription regulation. These results show that PCF is a good preventive regimen from ocean against UVB radiation.更多还原