多巴色素异构酶调节DHICA介导的抗氧化作用机制研究

发布时间：2018-04-09 23:24

本文选题：多巴色素异构酶　切入点：黑素细胞　出处：《武汉大学》2011年博士论文

【摘要】：人类表皮黑素细胞通过产生黑素,从而对紫外线辐射和氧化应急具有关键的防护作用。黑素在黑素小体(melanosome)中合成。酪氨酸酶(Tyr)、酪氨酸酶相关蛋白1(Tyrp1)和多巴色素异构酶(Dct)以多酶复合体的形式存在于黑素小体膜,共同参与对黑素细胞黑素生成的调节。酪氨酸酶家族包括三种成员：酪氨酸酶、酪氨酸酶相关蛋白1、多巴色素异构酶,它们在黑素合成过程中具有不同的作用。Tyr基因与Tyrp1基因发生突变可引起1型或3型眼皮肤型白化病,但很少有关于Dct基因突变直接导致人类色素性疾病的报道。位于小鼠14号染色体上的Dct基因编码区第194位精氨酸被谷氨酰胺置换(R194Q)发生slaty突变,使Dct酶活性降为野生型的36%。但salty突变黑素细胞中黑素小体的成熟发育过程是否受到影响尚不清楚。本研究通过比较salty突变黑素细胞与野生型melan-a黑素细胞中黑素生成蛋白表达与黑素小体的发育形态,阐述Dct基因突变对黑素细胞结构及发育的影响。尽管人们早就认识到一些黑素生成中间产物,如L-3,4-左旋多巴和DHI,氧化时能产生大量活性氧基使细胞造成损伤。然而在正常生理状态下,并未发现有任何中间产物对黑素细胞产生毒性。这提示黑素细胞除了为避免毒性中间产物泄漏到胞质中,从而机械性地将黑素合成反应限制在黑素小体内以外,还具有强大的防护机制来清除中间产物的毒性,抵抗氧化应激造成的细胞损伤。目前认为,Dct能将中间产物L-多巴色素(L-dopachrome)异构成DHICA,从而减少Dct酶活性不足时L-多巴色素自发脱羧反应生成DHI的毒性作用。Dct通过影响优黑素中DHICA单体掺入到DHI多聚链中的不同比率,从而控制着天然优黑素中的羧基基团的含量。但目前Dct调节的DHICA/DHI比率具有何种生理学意义仍然待确定。Dct-/-基因敲除小鼠,其小鼠体内编码Dct的基因中启动序列内的外显子1(exonl)的缺失,使Dct mRNA完全不表达,因而也完全缺乏Dct蛋白。当Dct基因被敲除后,Dct-/-基因敲除小鼠的表型仅发现有轻度被膜颜色稀释,并不像Tyr和Tyrp1基因突变能引起明显的眼皮肤白化病表型。我们推测Tyr和Tyrpl可能在功能上对Dct缺陷给予一定程度的补偿。本研究通过将Dct-/-基因敲除小鼠与正常野生型小鼠进行慢性UVA照射后,观察表皮细胞内ROS水平、SBC数量以及细胞凋亡的情况,旨在研究Dct参于UVA诱导皮肤细胞凋亡的作用机制。同时,我们也为优黑素的抗氧化作用受DHICA/DHI比率的影响提供了新的证据。第一部分多巴色素异构酶突变对黑素细胞发育的影响研究目的研究多巴色素异构酶突变对黑素细胞黑素小体发育成熟的影响。方法用透射电子显微镜技术和Fontana-Masson嗜银染色观察Dct突变slaty小鼠的黑素细胞和野生型melan-a黑素细胞胞内黑素小体发育情况及嗜银着色的优黑素颗粒分布状况；用分光光度计法和蛋白印迹技术测定Tyr活性和Tyr、Tyrp1和Dct蛋白表达水平。结果透射电子显微镜观察发现salty细胞内缺乏成熟的Ⅳ期黑素小体,Fontana-Masson嗜银染色也证实slaty黑素细胞胞浆几乎完全缺乏嗜银染阳性的优黑素颗粒。与野生型melan-a黑素细胞相比,slaty黑素细胞色素化程度明显减低,这种细胞表型改变与Tyr活性和蛋白质表达水平无关。结论Dct突变影响黑素细胞Dct酶活性及蛋白表达水平,影响黑素小体的发育成熟,导致黑素细胞色素化程度减低。第二部分多巴色素异构酶参与UVA诱导皮肤凋亡的作用机制目的观察慢性UVA照射对Dct-/-基因敲除小鼠与野生型C57BL/6J小鼠表皮细胞凋亡的影响。方法将Dct基因敲除小鼠(Dcttml(Cre)Bee/Dcttml(Cre)Bee,Dct-/-)和野生型小鼠C57BL/6J进行总剂量为60 J/cm2(每周三次,每次5 J/cm2,共四周)的慢性UVA照射后,检测各组小鼠表皮内SBC的数量、细胞凋亡的情况,以及测定皮肤组织内ROS水平。结果UVA照射组SBC、凋亡细胞明显增多(P0.01),未照射组之间小鼠SBC、凋亡细胞无明显差别(P0.05),但照射组内Dct-/-基因敲除小鼠较C57BL/6J小鼠SBC、凋亡细胞明显增多(P0.01)。慢性UVA照射后,Dct-/-基因敲除小鼠较C57BL/6J小鼠皮肤组织内ROS水平明显增高。结论Dct可能参与氧化应激的清除和皮肤光保护的作用。体内Dct的缺陷可导致ROS的大量聚集,可能是UVA诱导Dct-/-基因敲除小鼠表皮细胞发生凋亡的原因。第三部分多巴色素异构酶调节DHICA介导的抗氧化作用目的探讨Dct对黑素组成成分的影响及其调节DHICA介导的抗氧化作用机制。方法将Dct基因敲除小鼠(Dcttml(Cre)Bee/Dcttml(Cre)Bee,Dct-/-)和野生型小鼠C57BL/6J进行总剂量为60 J/cm2(每周三次,每次5 J/cm2,共四周)的慢性UVA照射后,观察各组小鼠皮肤组织中的黑素分布及形态的变化,检测小鼠表皮中优黑素(EM)及褐黑素(PM)的含量。用电子自旋共振光谱(ESR)测定DHICA-黑素、DHI-黑素及它们的混合物在Fenton反应中对羟基产生的作用。结果慢性UVA照射后,C57BL/6J小鼠皮肤组织较Dct-/-小鼠皮肤组织黑素颗粒增多、均匀,且在表皮的最外层角质形成细胞更容易观察到形成特殊的帽状保护结构；C57BL/6J小鼠皮肤组织中EM/PM的比率升高两倍,而Dct-/-小鼠皮肤中仅轻度升高,两组之间有显著性差异。DHICA-黑素在Fenton反应中具有羟基清除能力,而DHI-黑素没有羟基清除能力。结论Dct增加黑素组成成分中EM/PM的比率。DHICA-黑素在Fenton反应中具有羟基清除能力。Dct具有调节DHICA介导的抗氧化作用。
[Abstract]:Human epidermal melanocytes produce melanin through, which has protective effect on key ultraviolet radiation and oxidative stress. Melanin in melanosomes (melanosome) (Tyr). In the synthesis of tyrosinase, tyrosinase related protein 1 (Tyrp1) and dopachrome isomerase (Dct) with multi enzyme complex exists in the form of melanin corpuscle membrane, might participate in the regulation of melanogenesis of melanocytes. The tyrosinase family consists of three members: tyrosinase, tyrosinase related protein 1, dopachrome isomerase, as they have different mutations can cause type 1 or type 3 eye skin type albinism with.Tyr gene and Tyrp1 gene in melanin synthesis process. But there are a few mutations in the Dct gene resulted in human pigmented diseases reported. Located on mouse chromosome 14 194th encoding region of Dct gene was an arginine glutamine replacement (R194Q) slaty process Change, the Dct activity reduced to wild-type 36%. but salty mutation in melanosome maturation development process is affected is unclear. In this study, through the comparison of the salty mutant melanocytes and wild type Melan-A in melanocytes produce protein expression and morphological development of melanosomes, elaborate effect of Dct gene mutation on the structure and the development of melanocytes.
Although it is well known that some melanogenesis intermediates, such as L-3,4- and DHI oxidation of levodopa, can produce large amounts of reactive oxygen radicals that damage cells. However, under normal physiological conditions, has not found any toxic intermediates on melanocytes. This suggests that the melanocytes in addition to avoid toxic intermediate product leakage to the cytoplasm, thus the mechanical melanin synthesis reaction limited in melanosomes in the outside, also has a powerful protective mechanism to eliminate the intermediate product toxicity, cell damage caused by oxidative stress resistance. At present, Dct can be the intermediate product of L- (L-dopachrome) of dopachrome DHICA, thus reducing Dct the enzyme activity is insufficient L- DOPA toxicity of.Dct spontaneous decarboxylation of DHI generated by influencing eumelanin DHICA monomer incorporation into DHI polymer chains in different ratio, so as to control natural advantages The carboxyl group content of melanin in. But the Dct adjusted DHICA/DHI ratio is still to be determined what the physiological significance of.Dct-/- gene knockout mice, the promoter sequence in mice with the gene encoding Dct in exon 1 (exonl) of the Dct mRNA deletion, no expression, thus completely lacking Dct protein. When the Dct gene knockdown phenotype of Dct-/- knockout mice found only mild film color dilution, and unlike the Tyr and Tyrp1 gene mutation can cause oculocutaneous albinism phenotype obviously. We speculate that Tyr and Tyrpl may be given a certain degree of Dct defects in the function of compensation. This study of the Dct-/- gene knockout mice and wild type mice were chronic UVA after irradiation to observe the level of ROS in epidermal cells, the number of SBC and cell apoptosis, UVA induced apoptosis of skin cells to study Dct parameters for machine At the same time, we also provide new evidence for the effect of the antioxidant activity of the DHICA/DHI on the antioxidation of the pheromone.
The study of the effect of DOPA isomerase mutation on the development of melanocyte in part 1
Objective to study the effect of dopamine isomerase mutation on the development and maturation of melanocyte melanosomes.
Methods by using transmission electron microscopy and Fontana-Masson staining observation of Dct mutant slaty mouse melanocytes and wild-type Melan-A melanoma cells intracellular melanin development and argyrophilic bodies and black coloring pigment particle distribution; Determination of the activity of Tyr and Tyr by spectrophotometry and Western blot technique level Tyrp1 and Dct protein expression.
The results of transmission electron microscopy observation showed that salty cells lacking mature stage melanosomes, Fontana-Masson silver staining confirmed that slaty melanocyte cytoplasm almost complete lack of silver staining and melanin granules. Compared with wild-type Melan-A melanoma cells, melanocytes cytochtome slaty level decreased significantly, the the cell phenotype change and Tyr activity and protein expression level.
Conclusion Dct mutation affects the activity of Dct and protein expression in melanocytes, which affects the maturation of melanosomes and reduces the pigmentation of melanocytes.
The second part of dopamine isomerase involved in the mechanism of UVA induced skin apoptosis
Objective To observe the effect of chronic UVA irradiation on the apoptosis of epidermal cells in Dct-/- knockout mice and wild type C57BL/6J mice.
Methods Dct gene knockout mice (Dcttml (Cre) Bee/Dcttml (Cre) Bee, Dct-/-) and wild type mice C57BL/6J to a total dose of 60 J/cm2 (three times a week, each time 5 J/cm2, four weeks) of chronic UVA after irradiation, the number of mice within the epidermis SBC detection, cell apoptosis, and determination of skin tissue level ROS.
Results UVA SBC irradiation group, the apoptotic cells increased significantly (P0.01), non irradiated group mice had no significant difference between SBC and apoptosis cells (P0.05), but Dct-/- gene knockout mice in irradiated group compared with the C57BL/6J mice, SBC, apoptotic cells increased significantly (P0.01). Chronic UVA irradiation, Dct-/- gene knockout mice obviously the level of ROS C57BL/6J in mouse skin tissue increased.
Conclusion Dct may be involved in the clearance of oxidative stress and the effect of skin protection. The deficiency of Dct in vivo can lead to a large accumulation of ROS, which may be the cause of UVA induced apoptosis in epidermal cells of Dct-/- knockout mice.
The third part of dopamine isomerase mediated antioxidant activity mediated by DHICA
Objective to investigate the effect of Dct on the composition of melanin and the mechanism of regulating the antioxidant activity mediated by DHICA.
Methods Dct gene knockout mice (Dcttml (Cre) Bee/Dcttml (Cre) Bee, Dct-/-) and wild type mice C57BL/6J to a total dose of 60 J/cm2 (three times a week, each time 5 J/cm2, four weeks) of chronic UVA after irradiation, to observe the change of skin tissue of mice in the melanin distribution and morphology. In the detection of mouse epidermal eumelanin and pheomelanin (EM) (PM). The contents of electron spin resonance (ESR) spectra of DHICA- DHI- determination of melanin, melanin and mixtures of hydroxyl in Fenton reaction.
Results chronic skin tissue after UVA irradiation, C57BL/6J mice compared with Dct-/- mice skin black pigment particles increased, uniform, and more easily observed cells form a cap protection special structure formed in the outermost cuticle; ratio of EM/PM skin tissue in C57BL/6J mice increased two times, while the skin of Dct-/- mice only mildly elevated. There was significant difference in.DHICA- melanoma with hydroxyl scavenging ability in the Fenton reaction between the two groups, while DHI- melanin no hydroxyl scavenging ability.
Conclusion Dct increases the ratio of EM/PM in melanin components..DHICA- melanin has hydroxyl clearance ability in Fenton reaction,.Dct regulates DHICA mediated antioxidant activity.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R751

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