纳米矢车菊-3-葡萄糖苷干预UVB急性光损伤小鼠皮肤p53线粒体凋亡通路的研究
发布时间：2018-07-31 08:09
【摘要】：【目的】1.建立UVB急性光损伤动物模型;2.探究矢车菊素-3葡萄糖(C3G)苷以及纳米包被的矢车菊素-3葡萄糖苷(Nano-C3G)对UVB急性光损伤动物模型皮肤的影响并对比其效果;3.通过p53参与的线粒体细胞凋亡通路,探究上述影响因素的干预机制。【方法】1.外观:肉眼观察评估;2.皮肤水分及皮肤弹性:多头皮肤检测仪相应探头检测皮肤水分以及皮肤弹性;3.组织病理:皮肤组织苏木素-伊红(Hematoxylin-Eosin,HE)染色法;皮肤组织TUNEL细胞凋亡免疫组织组化染色;4.氧化应激:硫代巴比妥酸(TBA)法检测丙二醛(MDA)、脂质过氧化物(LPO);5.DNA损伤:酶联免疫吸附实验(ELISA)法检测8羟基脱氧鸟苷(8-OHd G);6.p53:免疫印迹法(Western blotting)检测p53表达水平;7.凋亡相关蛋白:a)酶联免疫吸附实验(ELISA)法检测Bcl-2家族蛋白(Bax、Bcl-2);b)免疫印迹法(Western blotting)检测Caspase家族(Caspase-3、Caspase-9);【结果】1.建立以高UVB剂量照射剃毛法除毛的昆明小鼠,并继续饲养24小时的急性光损伤模型。2.Nano-C3G(125-500u M)能改善UVB急性损伤小鼠皮肤外观、皮肤水分;TUNEL细胞凋亡指数下降;且同等高剂量条件下(500 u M),Nano-C3G药效较C3G强;3.Nano-C3G(125-500u M)能够有效降低UVB诱导的氧化应激产物LPO、MDA含量(p0.0001);4.Nano-C3G(125-500u M)能够有效减少UVB诱导的8-OHd G(p0.0001);且同等高剂量条件下(500 u M),Nano-C3G药效较C3G强(p0.05);5.Nano-C3G(125-500u M)能够有效下调UVB诱导的p53表达(p0.0001),平衡Bcl-2家族(抗凋亡蛋白Bcl-2、促凋亡蛋白Bax)(p0.0001),下调Caspase家族(Caspase-3、9)表达(p0.05);且同等高剂量条件下(500 u M),Nano-C3G药效较C3G强(p0.05);【结论】1.UVB照射诱导小鼠皮肤角质形成细胞凋亡,可通过光动力性氧化应激损伤或非光动力性直接损伤DNA,激活p53基因表达,通过调控Bcl-2家族蛋白等一系列细胞因子,最终激活Caspase家族以启动凋亡级联反应;2.Nano-C3G、C3G能对抗UVB急性光损伤导致的小鼠皮肤表皮组织的细胞凋亡,且同等浓度下Nano-C3G的能力更强;3.Nano-C3G、C3G抑制UVB所导致的细胞凋亡,可以通过对抗其中的氧化应激反应、减少DNA损伤,降低p53基因表达,调控Bcl-2家族中抗凋亡蛋白Bcl-2、抑凋亡蛋白Bax的表达,减少凋亡相关细胞因子的释放,最终减少Caspase家族表达实现。
[Abstract]:[objective] 1. The animal model of UVB acute light injury was established. To investigate the effects of cornulin-3 glucose (C3G) glucoside (C3G) and nano-encapsulated cornulin-3 glucoside (Nano-C3G) on the skin of UVB acute light injury animal model. Through p53 involved in mitochondrial apoptosis pathway, the intervention mechanism of the above factors was explored. [methods] 1. Appearance: naked eye observation and evaluation 2. Skin moisture and skin elasticity: multi-head skin detector to detect skin moisture and skin elasticity. 3. Histopathology: Hematoxylin-eosin HE staining and immunohistochemical staining for TUNEL cell apoptosis. Oxidative stress: malondialdehyde (MDA),) lipid peroxide (LPO) damage was detected by thiobarbituric acid (TBA) assay; enzyme linked immunosorbent assay (ELISA) was used to detect 8-hydroxydeoxyguanosine (8-OHd G) 6.p53; Western blot (Western blotting) was used to detect p53 expression. Apoptosis-related protein: a) Enzyme-linked immunosorbent assay (ELISA) was used to detect Bcl-2 family protein (BaxanBcl-2) and (Western blotting) was used to detect Caspase family (Caspase-3 Caspase-9). [results] 1. Kunming mice with shaved hair were irradiated with high UVB dose and kept for 24 hours. 2. Nano-C3G (125-500u M) could improve the skin appearance of mice with acute UVB injury and decrease the apoptosis index of Tunel cells. At the same high dosage (500 u M) Nano-C3G) than C3G, 3.Nano-C3G (125-500u M) can effectively reduce the content of LPO-MDA induced by UVB (p0.0001) 4.Nano-C3G (125-500u M) can effectively reduce the 8-OHd G (p0.0001) induced by UVB, and the drug efficacy of 500u M) Nano-C3G is better than that of C3G (p0.05) 5.Nano-C3G (125-500u). M) can effectively down-regulate p53 expression induced by UVB (p0.0001), balance Bcl-2 family (anti-apoptotic protein Bcl-2, pro-apoptotic protein Bax) (p0.0001), down-regulate the expression of Caspase family (Caspase-3O9) (p0.05), and at the same high dosage (500 u M) + Nano-C3G), the drug efficacy of 1.UVB is better than that of C3G; [conclusion] 1.UVB irradiation is less effective than C3G. [conclusion] Apoptosis of keratinocytes in rat skin The expression of p53 gene can be activated by photodynamic oxidative stress injury or non-photodynamic direct damage of DNA, and a series of cytokines such as Bcl-2 family proteins can be regulated. Finally, the Caspase family was activated to initiate the apoptotic cascade reaction. 2. Nano-C3GN C3G could antagonize the apoptosis of mouse skin epidermis induced by acute light injury of UVB, and the ability of Nano-C3G at the same concentration was stronger. 3. Nano-C3GN C3G inhibited the apoptosis induced by UVB. It can reduce DNA damage, reduce p53 gene expression, regulate the expression of anti-apoptotic protein Bcl-2, inhibit the expression of apoptotic protein Bax, and reduce the release of apoptosis-related cytokines by antagonizing the oxidative stress reaction, reducing the expression of p53 gene, regulating the expression of anti-apoptotic protein Bcl-2 in the Bcl-2 family. Finally, the realization of Caspase family expression is reduced.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R275.9
[Abstract]:[objective] 1. The animal model of UVB acute light injury was established. To investigate the effects of cornulin-3 glucose (C3G) glucoside (C3G) and nano-encapsulated cornulin-3 glucoside (Nano-C3G) on the skin of UVB acute light injury animal model. Through p53 involved in mitochondrial apoptosis pathway, the intervention mechanism of the above factors was explored. [methods] 1. Appearance: naked eye observation and evaluation 2. Skin moisture and skin elasticity: multi-head skin detector to detect skin moisture and skin elasticity. 3. Histopathology: Hematoxylin-eosin HE staining and immunohistochemical staining for TUNEL cell apoptosis. Oxidative stress: malondialdehyde (MDA),) lipid peroxide (LPO) damage was detected by thiobarbituric acid (TBA) assay; enzyme linked immunosorbent assay (ELISA) was used to detect 8-hydroxydeoxyguanosine (8-OHd G) 6.p53; Western blot (Western blotting) was used to detect p53 expression. Apoptosis-related protein: a) Enzyme-linked immunosorbent assay (ELISA) was used to detect Bcl-2 family protein (BaxanBcl-2) and (Western blotting) was used to detect Caspase family (Caspase-3 Caspase-9). [results] 1. Kunming mice with shaved hair were irradiated with high UVB dose and kept for 24 hours. 2. Nano-C3G (125-500u M) could improve the skin appearance of mice with acute UVB injury and decrease the apoptosis index of Tunel cells. At the same high dosage (500 u M) Nano-C3G) than C3G, 3.Nano-C3G (125-500u M) can effectively reduce the content of LPO-MDA induced by UVB (p0.0001) 4.Nano-C3G (125-500u M) can effectively reduce the 8-OHd G (p0.0001) induced by UVB, and the drug efficacy of 500u M) Nano-C3G is better than that of C3G (p0.05) 5.Nano-C3G (125-500u). M) can effectively down-regulate p53 expression induced by UVB (p0.0001), balance Bcl-2 family (anti-apoptotic protein Bcl-2, pro-apoptotic protein Bax) (p0.0001), down-regulate the expression of Caspase family (Caspase-3O9) (p0.05), and at the same high dosage (500 u M) + Nano-C3G), the drug efficacy of 1.UVB is better than that of C3G; [conclusion] 1.UVB irradiation is less effective than C3G. [conclusion] Apoptosis of keratinocytes in rat skin The expression of p53 gene can be activated by photodynamic oxidative stress injury or non-photodynamic direct damage of DNA, and a series of cytokines such as Bcl-2 family proteins can be regulated. Finally, the Caspase family was activated to initiate the apoptotic cascade reaction. 2. Nano-C3GN C3G could antagonize the apoptosis of mouse skin epidermis induced by acute light injury of UVB, and the ability of Nano-C3G at the same concentration was stronger. 3. Nano-C3GN C3G inhibited the apoptosis induced by UVB. It can reduce DNA damage, reduce p53 gene expression, regulate the expression of anti-apoptotic protein Bcl-2, inhibit the expression of apoptotic protein Bax, and reduce the release of apoptosis-related cytokines by antagonizing the oxidative stress reaction, reducing the expression of p53 gene, regulating the expression of anti-apoptotic protein Bcl-2 in the Bcl-2 family. Finally, the realization of Caspase family expression is reduced.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R275.9
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