【作者】 朱海莲；

【导师】 王莒生；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2012， 博士

【摘要】 白癜风作为一种以皮肤局限性色素代谢障碍为表现的后天性皮肤病,是皮肤科常见病。该病临床表现比较单纯,诊断简单,属于慢性难治之症,近年来该病的发病率呈上升的趋势。目前该病的病因病机尚未明了,认为可能与免疫、内分泌、遗传、精神神经、微量元素、自由基清除等诸多因素有关。白癜风的病理机制目前尚无定论,但越来越多的证据提示,机体的氧化-抗氧化平衡失调,导致局部微环境中的活性自由基大量地聚集,引起了氧化应激,诱导凋亡,成为引起白癜风发病的一个重要因素。线粒体在细胞凋亡中起着主开关的作用。目前西医治疗白癜风多采用光敏药联合紫外线照射、激素和免疫抑制剂、外科疗法等,但疗效不理想、且长期应用易长期应用易产生副作用,而中医治疗本病具有复发率低、副作用小、远期疗效好等优势,有较广阔的应用前景。中医认为本病多属风搏皮肤、气血失和、肝肾不足,而滋补肝肾方补益肝肾、养血祛风,是临床治疗白癜风的有效方剂。本研究通过体外观察滋补肝肾方含药血清及其主要成分芍药苷对黑素细胞氧化致黑素细胞凋亡的保护作用,探索中药治疗白癜风的作用靶点,为临床应用提供理论依据。本研究共分两部分：1滋补肝肾方及拆方含药血清对氧化诱导的黑素细胞凋亡的作用以过氧化氢诱导小鼠B16黑素细胞凋亡为体外细胞模型,观察该方及补肝肾、养血及祛风拆方的含药血清对黑素细胞氧化致黑素细胞凋亡及脂质过氧化状态的影响,并进一步探讨其抗氧化应激的机制。1.1 B16黑素细胞氧化损伤模型的建立及滋补肝肾方的作用采用MTT法观察细胞活性。2mmol/l、1mmol/l、0.5mmol/l、0.25mmol/l的H2O2作用0.5h对黑素细胞增殖的抑制呈浓度依赖性(分别P<0.01)；1mmol/l的H2O2作用不同时间对黑素细胞增殖的抑制呈时间依赖性(分别P<0.05)。1mmol/l的H2O2作用0.5h对B16细胞活性的抑制率为47.3%-54.1%,接近IC50,为本实验的药物观察浓度和时间。滋补肝肾全方及拆方组细胞活性不同程度地升高氧化损伤后B16细胞活性,以全方组作用最为明显,比模型组高约31.2%(P<0.05)；其次为祛风组,比模型组高约26.7%(P<0.05)。1.2滋补肝肾方对黑素细胞脂质过氧化作用的影响采用生化法观察滋补肝肾含药血清对于氧化损伤B16细胞中SOD、CAT、GSH-Px的活性,抑制(OH-)的能力及MDA含量的影响。结果如下：与正常对照组相比,模型组抗氧化酶SOD(25.78±6.69)、CAT(1.32±0.06)、GSH-Px(305.72±13.82)和抑制OH-(385.13±57.32)的能力都有所下降而脂质过氧化最终产物MDA(1.69±0.04)含量增多,且均有显著性差异(分别P<0.05,P<0.01)。说明H2O2刺激细胞后,降低了细胞的抗氧化能力,导致细胞内自由基增多,从而抑制细胞活性。与模型组相比,滋补肝肾各中药组能显著提高H2O2损伤细胞中抗氧化酶SOD、CAT和GSH-Px的活性,抑制脂质过氧化产物MDA和氧自由基(OH-)在细胞中的累积浓度,且全方组的保护作用最好,体现了中药综合作用的优越性。在各拆方中,SOD、CAT及抑制(OH-)都是补肝肾组最好,体现了滋补肝肾方在这些方面以补肝肾法则为主、养血及祛风法则起辅助作用。GSH-Px、MDA都是养血组最好,体现了滋补肝肾方在这些方面以养血法则为主、补肝肾及祛风法则起辅助作用。1.3滋补肝肾方对氧化诱导的黑素细胞凋亡及分子机制的作用采用流式细胞仪以AnnexinV-PE/7-AAD染色检测细胞凋亡率,Hoechst33342观察凋亡细胞的核变化,激光共聚焦显微镜以JC-1染色观察B16细胞线粒体膜电位,底物荧光法检测Caspase-3/9的活性,Western blot法测定B16细胞凋亡Bcl-2、Cyt-c蛋白的表达。结果如下：①与正常对照组相比,模型组凋亡率(29.50±3.49)明显增高(P<0.01)；细胞核浓集呈亮蓝色,出现凋亡小体；线粒体膜电位降低,线粒体呈圆点状、粒状,并向细胞核聚集；B16细胞Caspase-3/9的活性显著增高；Cyt-c蛋白表达量升高；Bcl-2蛋白表达量降低。②与模型组相比,滋补肝肾含药血清各组凋亡率明显降低(P<0.01)；仅部分细胞出现核亮蓝呈分叶、碎片状,边集等；滋补肝肾含药血清治疗后,线粒体仍呈线管状、网状,并且弥漫分布在胞浆中,线粒体膜电位降低得到改善；其Caspase-3/9的活性降低,与模型组有显著性的意义(分别P<0.01)；滋补肝肾含药血清各组作用于氧化损伤的B16细胞24h后,可使Cyt-c蛋白表达量降低,Bcl-2蛋白表达量升高,与模型组有明显差异(P<0.01)。滋补肝肾各拆方组均以补肝肾组最好。2芍药苷对黑素细胞氧化损伤分子机制的研究本实验以小鼠B16黑素细胞为研究对象,以不同浓度芍药苷预保护24h继用1mmol/1的H2O2最后作用4h造成细胞凋亡模型,观察其对B16黑素细胞的抗凋亡效果并探讨其抗氧化损伤的作用机制。通过观察细胞形态学的变化,测定细胞凋亡率、线粒体跨膜电位、Caspase-3/9的活性、Bcl-2、Bax、Cyt-c蛋白的表达而深入研究芍药苷抗凋亡作用的分子调控机制,方法同上。结果如下：①与正常对照组相比,模型组凋亡率(28.13±3.31)明显增高(P<0.01)；核体积变小、固缩和片断化,呈亮蓝色,出现凋亡小体；线粒体膜电位降低,线粒体呈圆点状、粒状,并向细胞核聚集；B16细胞Caspase-3/9的活性显著增高；Cyt-c蛋白表达量升高；Bcl-2蛋白表达量降低。②与模型组相比,200μg/ml、100μg/ml、50μg/ml芍药苷的凋亡率明显低于模型组(P<0.01)且呈剂量依赖性；经各组芍药苷治疗后,仅部分细胞出现核亮蓝呈分叶、碎片状,边集等；线粒体膜电位降低得到改善,线粒体仍呈线管状、网状,并且弥漫分布在胞浆中；芍药苷各组Caspase-3/9的活性升高,与模型组有显著性的差异(分别P<0.01)且呈剂量依赖性；芍药苷各组作用于氧化损伤的B16细胞24h后,可使Cyt-c蛋白表达量降低,与模型组均具有显著性差异(P<0.01),Bcl-2蛋白表达量升高,与模型组有明显差异(P<0.01)。芍药苷对黑素细胞的保护作用呈剂量依赖性。综上所述,本论文从H2O2诱导小鼠B16黑素细胞凋亡模型研究,滋补肝肾含药血清能够抑制B16黑素细胞的凋亡和脂质过氧化状态,具有抗氧化应激损伤的功效。滋补肝肾含药血清和芍药苷抑制氧化应激凋亡的机制可能是通过提高细胞凋亡相关基因Bcl-2蛋白的表达,抑制线粒体膜通透性,维持跨膜电位,进而阻止线粒体内细胞色素C的释放和Caspase蛋白的级联激活反应,抑制细胞凋亡。本论文揭示了线粒体信号转导通路可能是滋补肝肾含药血清和芍药苷干预氧化应激诱导B16黑素细胞凋亡的调控机制之一。更多还原

【Abstract】 Vitiligo is acquired skin pigment and its main symptoms are skin pigment loss.The clinical manifestation of vitiligo is relatively simple, so it’s easy to diagnose but difficult to treat. The incidence of vitiligo is in upward tendency for the past few years. The etiological factors and pathogenesis of vitiligo, which aren’t yet understood,are considered to be concerned with immunity,endocrine,heredity,consciousness,microelement and free radicals. The pathomechanism of vitiligo aren’t yet final conclusions. The research of anti-oxidant for vitiligo increase in recent years.More and more evidence prompt. The dysequilibrium of oxidationand and anti-oxidant lead to free radicals of microenvironment are abundantly accumulated, then induce oxidative stress and cell damage eventually,which become an important pathogenic factor of vitiligo. Mitochondrion is a master switch in apoptosis.At present,curative effect of Western medicine treatment is not very good, high recurrence and side effect. In contrast, curative effect of Chinese medicine treatment is good, with low recurrence and side effects. Chinese medicine et iology of vitiligo as wind stroke skin, blood disharmony, deficiency of liver and kidney. ZBGSF reinforces liver and kidney, nourishes blood,dispels wind, which is an effective prescription. This research is to explore the action target point of Chinese medicine for vitiligo by observing the protective effect of ZBGSF and peoniflorin in vitro formelanocyte apoptosis which can provide theoretical basis for Clinical application.The research include two parts.The first part is about research of ZBGSF’s protective action for melanocyte oxidative damage. To observe the protective effect of ZBGSF conditioning against H2O2 induced damage on Mice B16 Melanocytes, the experiments consist of three parts. MTT was used to set up B16 melanocyte oxidative damage model:B16 Melanocytes were stimulated with lmm/1 H2O2 for half hour after pre-treatment with 10% different serum groups for 24h. MTT method was adopted to determine cellular activity. The activity of SOD, CAT, GSH-Px, MDA content and the inhibition of OH" were detected by biochemical methods. The results prompt that B16 cell activity reduce after H2O2 stimulating and model group (P<0.01) was lowest. Medcine groups improved the cell survival rates, increased SOD, CAT, GSH-Px activity(P<0.05), reduced OH- releasing and MDA content (P<0.05), compared with model group, and the effect of ZBGSF group was best. The B16 apoptosis rate was detected by flow cytometry, the B16 nucleus change was detected by Hoechst 33342 dyeing, the B16 mitochondrial membrane potential was detected by laser confocal microscopy, the activity of Caspase-3/9 were detected by substrate fluorescence method and the protein expression of Bcl-2、Cyt-c were detected by Western blot, the results display:①Compared with normal control group, the apoptosis rate of model group (29.50±3.49) was higher (P<0.01); we discovered that H2O2 caused nucleus enrich and sapphirine, emerged apoptotic body, mitochondrial membrane potential reducing, mitochondrion present dot shape or granular and gather toward nucleus, and H2O2 induced the activity of Caspase-3/9 increasing, increased the protein expression of Cyt-c and reduce Bcl-2.②Compared with model group, the apoptosis rate of ZBGSF groups was lower (P<0.01); we discovered that H2O2 caused only part cells of ZBGSF groups emerged nucleus enrich and sapphirine, emerged apoptotic body. After treatment of ZBGSF groups, mitochondrion still present line pipe or reticular and diffused distribution in cytoplasm. The membrane potential reducing was eased. The Caspase-3/9 activity of ZBGSF groups was induced compared with model group (P<0.01 separately). we discovered that ZBGSF groups reduced the protein expression of Cyt-c and increased the protein expression of Bcl-2 compared with model group (P<0.01).The effect of BGS group is best.The second part is about molecular mechanism research of peoniflorin’s protective action for melanocyte oxidative damage.The method ibid. MTT was used to set up B16 melanocyte oxidative damage model:B16 Melanocytes were stimulated with lmm/1 H2O2 for four hours after pre-treatment with different peoniflorin groups for 24h. The results were:①Compared with normal control group, the apoptosis rate of model group (28.13±3.31) was higher (P<0.01); we discovered that H2O2 caused nucleus enrich and sapphirine, emerged apoptotic body, mitochondrial membrane potential reducing, mitochondrion present dot shape or granular and gather toward nucleus. H2O2 induced the activity of Caspase-3/9 increasing and increased the protein expression of Cyt-c and reduced Bcl-2.②Compared with model group, the apoptosis rates of High, Middle, low dose peoniflorin groups were lower than model group (P<0.01) and present dose dependent. We discovered that H2O2 caused only part cells of High, Middle, low dose peoniflorin groups emerge a nucleus enrich and sapphirine, emerged apoptotic body. After treatment of High, Middle, low dose peoniflorin groups, mitochondrion still present line pipe or reticular and diffused distribution in cytoplasm. The membrane potential reducing was eased. The Caspase-3/9 activity of High, Middle, low dose peoniflorin groups induced compared with model group (P<0.01 separately) and present dose dependent. High, Middle, low dose peoniflorin groups reduced the protein expression of Cyt-c and increased the protein expression of Bcl-2 compared with model group (P<0.01). The protective effect of peoniflorin for melanocytes present dose dependent.To sum up, The thesis invest igated from B16 melanocyte oxidat ive damage model to research, the inhibiting oxidative stress mechanism of ZBGSF groups and peoniflorin groups possibly works by elevating the protein expression of Bcl-2, inhibiting mitochondrial membrane permeability, preventing the Cyt-c releasing and activating caspase cascade reaction to inhibit apoptosis. In conclusion,The thesis revealed that the mitochondrial signaling pathway is the regulation mechanism that ZBGSF groups and peoniflorin groups interfere in B16 Melanocytes apoptosis by oxidative stress.更多还原