【作者】 谢靖；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 生药学， 2011， 博士

【摘要】 目的建立⁶⁰Coγ辐射对体外培养的小鼠胸腺细胞损伤的病理模型,探讨在⁶⁰Co辐射下,紫薯素抑制⁶⁰Co诱导的小鼠胸腺细胞辐射损伤的作用机制。方法建立紫薯素干预⁶⁰Coγ辐射小鼠胸腺细胞的模型。应用MTT,首先检测⁶⁰Coγ辐射在0～12h范围内对小鼠胸腺细胞活力的影响,⁶⁰Co分为0,2,4,6Gy四个不同剂量进行单次辐射。不同剂量的紫薯素预处理细胞,MTT检测紫薯素对辐射细胞活力的影响、抗辐射的有效剂量以及其有无毒性反应。紫薯素对辐射细胞的作用检测中,通过DNA ladder检测细胞晚期凋亡;以流式细胞仪Annexin V-FITC/PI染色检测细胞早期凋亡;流式细胞仪PI染色检测细胞周期。探讨紫薯素辐射防护作用的机制。测定紫薯素对氧化应激的影响:2,7-二氯氢化荧光素二酯（DCFH-DA）为荧光探针,检测紫薯素对⁶⁰Co照射后细胞内活性氧（ROS）的影响;酶生化法测定抗氧化酶过氧化物歧化酶（SOD）和谷胱甘肽过氧物酶（GSH-px）的活力。研究紫薯素对⁶⁰Co诱导小鼠胸腺细胞凋亡的线粒体通路的影响:JC-1荧光染色测定线粒体膜电位;western-blot检测细胞色素C、caspase3和PARP的蛋白表达以及Bcl-2和Bax的蛋白表达;对caspase 3和caspase 9进行酶活性检测。研究紫薯素对⁶⁰Co诱导小鼠胸腺细胞凋亡的死亡受体通路的影响:RT-PCR检测Fas mRNA;western-blot测定FADD的蛋白表达;caspase8酶活性的检测。检测紫薯素对⁶⁰Co诱导小鼠胸腺细胞的MAPK途径的影响:western-blot测定磷酸化的ERK、JNK、p38MAPK的蛋白表达。探讨紫薯素对⁶⁰Co诱导小鼠胸腺细胞p53的影响:RT-PCR检测p53 mRNA;Western-blot检测p53、p21Cip1和p27Kip1的蛋白表达。结果MTT结果显示: ⁶⁰Coγ辐射作用于细胞,降低了细胞活力;辐射各组细胞活力从4h开始急剧衰减。4Gy辐射后4h的细胞活力接近50%。本研究选择了4Gy辐射后4h建立细胞损伤模型。辐射后,在0.625g/L～2.500g/L浓度范围内,紫薯素能够提升辐射小鼠胸腺细胞的活力,其提升细胞活力的能力与剂量呈量效依赖关系,而且在此剂量范围内,紫薯素对细胞无毒性。DNA ladder显示:辐射后小鼠胸腺细胞产生明显的ladder,紫薯素干预使DNA ladder减弱。流式检测显示:紫薯素降低了辐射导致的小鼠胸腺细胞早期凋亡,同时缓解了辐射导致的G2/M期细胞周期阻滞。紫薯素的防辐射机制试验结果显示:紫薯素降低了细胞内ROS的含量,提高了抗氧化酶SOD与GSH-px的活性;稳定了线粒体膜电位,减少细胞色素C的释放,降低caspase 3和PARP的蛋白表达,抑制caspase 9和caspase 3的酶活性,并使Bcl-2蛋白表达升高而Bax蛋白表达降低;对Fas mRNA和FADD蛋白表达无显著作用,但中、高剂量的紫薯素对caspase 8的酶活性有一定的提升;MAPK通路中,紫薯素对ERK活性的影响不明显,对磷酸化的JNK和p38MAPK表达有显著的抑制作用;紫薯素提高p53 mRNA与p53的蛋白表达,抑制p27Kip1的蛋白表达,对p21Cip1蛋白表达的作用不明显。结论:紫薯素能够有效地抑制⁶⁰Co辐射引起的小鼠胸腺细胞损伤,提高细胞活力,抑制细胞凋亡,缓解细胞周期阻滞。紫薯素的辐射防护机制包括:抑制了由⁶⁰Co辐射诱导的细胞氧化损伤,降低ROS,提高抗氧化酶的活性,通过改善氧化还原状态抑制辐射诱导的凋亡;抑制线粒体凋亡信号通路,同时调节Bcl-2、Bax蛋白达到抑制凋亡的目的;对死亡受体凋亡信号通路无作用;MAPK途径中,抑制了JNK与p38MAPK磷酸化,对ERK活性没有影响;通过抑制p53的转录激活和蛋白表达,抑制辐射导致的细胞凋亡;通过抑制p53以及其下游的p27Kip1,缓解细胞周期阻滞。紫薯素通过调节多种基因与蛋白的表达,达到其抗氧化、抗凋亡、缓解细胞周期阻滞的辐射防护作用,有望成为应用广泛的⁶⁰Co辐射防护剂。更多还原

【Abstract】 Objective: Establish the ⁶⁰Coγray-induced irradiation damage model in murine thymocytes. Investigate the protective effects of purple sweet potato（PSP）pigments on ⁶⁰Co-induced damage.Methods: After ⁶⁰Coγray-single dose-irradiation with various doses of 0, 2, 4, 6Gy, the murine thymocytes viabilities are detected by MTT at the post-irradiated time point ranged from 0 to 12h. Treated the thymocytes with indicated dose of irradiation in the present of different concentrations of PSP pigments, the cell viabilities are detected by MTT to investigate the effective and safe concentrations of PSP pigments against irradiation. After the model of PSP pigments against irradiaion is established, DNA ladder is preformed to test whether PSP pigments have the protective effect on late apoptosis. The early apoptosis and cell cycle of thymocytes are analyzed by flow cytometry with annexin V-FITC/PI staining and PI staining.The mechanisms of the radioprotective effects of PSP pigments are explored. The antioxidative effects of PSP pigments are detected. DCFH-DA fluorescent probe is applied to investigate the intra-cellular ROS. The activities of antioxidative enzymes SOD and GSH-px are detected by biochemistry methods. The effects of PSP pigments on mitochondrion-mediated apoptosis are analyzed. Mitochondrial membrane potential is detected by JC-1 fluorescent probe. The released cytochrome C, caspase 3, PARP, Bcl-2 and Bax proteins are investigated by western-blot. The enzyme activities of caspase 3 and caspase 9 are also detected. The effects of PSP pigments on death receptor-mediated apoptosis are determined by Fas mRNA RT-PCR analysis, FADD protein western-blot analysis and caspase 8 enzyme activity detection. The MAPK pathways including ERK, JNK and p38MAPK are analyzed by western-blot. The effects of PSP pigments on p53 are determined by p53 mRNA RT-PCR detection and p53, p21Cip1 and p27Kip1 protein western-blot analysis.Results: MTT showes that ⁶⁰Coγray decreases the cell viabilities and a sharp decline of cell viability starts from 4h post-irradiation. 4Gy irradiation causes approximate 50% cell viability loss at 4h after ⁶⁰Coγray irradiation. So the ⁶⁰Co-irradiating model should be established at 4h post-4Gy-irradiation. With 4Gy irradiation, the cell viablities are increased in the present of 0.625g/L～2.500g/L PSP pigments in a concentration-dependent manner and no cytotoxcity is observed in these PSP pigments concentrations. DNA ladders are smeared and weak in PSP pigments pretreated groups compared to that of the irradiated group. Flow cytometry results show that PSP pigments can decrease the early apoptosis and attenuate the cell cycle arrest induced by irradiation.The mechanisms of radioprotective effects of PSP pigments are explored and the results demonstrate that PSP pigments can decrease the intra-cellular ROS and increase the activities of SOD and GSH-px as well. PSP pigments stabilize the mitochondrial membrane potential, decrease the released cytochrome C, caspase 3 and PARP proteins. The activities of caspase 3 and caspase 9 are inhibited by PSP pigments. Bcl-2 protein is increased by PSP pigments while Bax is decreased to draw the cells towards antiapoptosis. PSP pigments show no significant effect on Fas mRNA and FADD protein but the activity of caspase 8 is increased in the present of medium and large dose of PSP pigments. MAPK pathway including JNK and p38MAPK is inhibited by PSP pigments but no changes happen to ERK. P53 mRNA and p53 protein are both depressed by PSP pigments and p27Kip1 is decreased consequently to attenuate the cell cycle arrest. PSP pigments have no obvious effects on p21Cip1 protein.Conclusion: PSP pigments can inhibit the ⁶⁰Coγray-induced damage in murine thymocytes. The cell viability is increased, apoptosis is decreased and cell cycle arrest induced by irradiation is attenuated in the present of PSP pigments. The radioprotective effects are related to the anti-oxidative effects of PSP pigments, which scavenging the intra-cellular ROS and enhancing the activities of anti-oxidative enymes to rebanlance the redox. The mitochondrial-mediated apoptosis pathway but not the death receptor-mediated apoptosis pathway is inhibited by PSP pigments. Bcl-2 and Bax are involved in the anti-apoptosis effects of PSP pigments. PSP pigments depress JNK and p38MAPK pathways but keep no influence on ERK. P53 mRNA is inactivated and p53 protein is decreased by PSP pigments pretreatment. Cell cycle arrest related p53 flare-up is cooling down by PSP pigments and p27Kip1 but not p21Cip1 is consequently decreased. Thus, the radio-protective effects of PSP pigments are via modulation of multiple genes and proteins to yield the anti-oxidation, anti-apoptosis and anti-cell cycle arrest. The PSP pigments might become a promising candidate for radio-protector.更多还原