环氧合酶在UVB诱导黑素细胞树突形成中的作用及其机制研究

发布时间：2018-05-06 09:09

本文选题：UVB照射 + 黑素小体转运　；参考：《第四军医大学》2012年硕士论文

【摘要】：人体皮肤颜色多样，主要是由表皮细胞中的色素沉着决定的。色素沉着是一个复杂而精密的多步骤过程，目前研究认为主要可以分为四步，首先是黑素在黑素细胞中的黑素小体内合成，，接着是由核周沿着树突延伸的方向向远端运输，然后是向周围的角质形成细胞转运，最后在角质形成细胞中再分布、降解[1]。黑素小体由核周向树突远端转移转移至周围的角质形成细胞的过程被人们称为黑素小体的转运[2]。在色素沉着的过程中，黑素合成固然重要，但黑素小体转运也必不少的。树突不仅是黑素细胞的形态学标志，更重要是黑素小体转运的中介点，其数量及形态的变化在黑素转运中起着极为重要的作用，调控树突的形成可以对色素沉着产生明显的影响[3]。研究发现，很多因素能够影响树突的形成。Scott等研究发现，Rho家族小GTP酶，尤其是Rac1和RhoA在黑素细胞树突形成方面起重要作用[4]，其中Rac1起着促进树突形成与延伸的作用，而RhoA则具有相反的作用。我们前期研究发现，UVB照射可以通过调控树突形成相关因子Rac1和RhoA的表达明显促进黑素细胞树突的形成和延长，但是，UVB照射调控Rac1和RhoA表达的机制尚不清楚。近年来，环氧合酶(cyclooxygenase，COX)在肿瘤的形成和发展中的作用成为人们关注的热点。有研究显示，COX-2抑制剂能抑制αvβ3调节的Cdc42/Rac1依赖的内皮细胞的迁移[5]，并且，COX-2特异性抑制剂NS-398能够使肿瘤细胞丝状伪足消失及细胞侵袭能力下降，表明COX-2和Cdc42/Rac1都是NSAIDs引起细胞丝状伪足消失和体外侵袭能力下降的重要分子[6]。这些研究提示环氧合酶与树突的形成可能具有一定的关系。因此，我们通过检测UVB照射后黑素细胞COX水平的变化及观察抑制COX活性对黑素细胞树突形态及Rac1、RhoA的影响，深入探讨环氧合酶在UVB照射诱导黑素细胞树突形成中的作用及机制，为阐明UVB照射后色素沉着的发生机制以及防止提供新思路和新策略。实验方法：本研究采用永生化的人表皮黑素细胞系PIG1，常规培养后分为5组，分别为：①对照组；②100mJ/3潂2UVB照射组；③100mJ/3潂2UVB照射+ASA；④100mJ/3潂2UVB照射+NS-398；⑤100mJ/3潂2UVB照射+SC-560。首先，应用Western blot和RT-PCR检测UVB照射对黑素细胞中COX-1、COX-2表达的影响；然后，显微镜下观察不同处理对黑素细胞树突形态的影响；最后，提取蛋白和RNA应用Western blot和PT-PCR方法检测不同处理对黑素细胞Rac1和RhoA表达的影响。实验结果： 1．100mJ/3潂2UVB照射后COX-1和COX-2mRNA及蛋白的表达均明显增高； 2．100μmol/L ASA及30μmol/L NS-398均可以抑制UVB照射诱导的人黑素细胞系PIG1树突的形成和延长，而10μmol/L SC-560对UVB照射诱导的人黑素细胞系PIG1树突的形成和延长没有明显的抑制作用； 3．ASA浓度在50~150μmol/L内时，可翻转UVB照射引起的人黑素细胞系PIG1中Rac1的高表达以及RhoA的低表达，并呈剂量依赖关系；100μmol/L ASA和30μmol/L NS-398均可以抑制UVB照射诱导的人黑素细胞系PIG1树突形态相关分子Rac-1的高表达及RhoA的低表达,但SC-560并没有类似效果。结论： 1．一定剂量UVB照射可促进黑素细胞表达COX-1和COX-2； 2．环氧合酶在树突的形成及延长中起一定作用，其中，COX-2起着主导作用。
[Abstract]:The color of human skin is varied, mainly determined by the pigmentation in the epidermal cells. Pigmentation is a complex and precise multistep process. It is considered to be mainly divided into four steps. First, melanin is synthesized in melanin corpuscle in melanocytes, then it is transported to the distal end from the direction of the dendrite along the dendrite. Later, the keratinocytes are transported to the surrounding keratinocytes, and then redistributed in the keratinocytes, and the process of degrading the [1]. melanosomes from the perinuclear to the distal dendrites to the surrounding keratinocytes is called the transport of the melanosomes of the [2]..
In the process of pigmentation, melanin synthesis is important, but the transport of melanosomes must be quite a few. Dendrites are not only the morphological markers of melanocytes, but also the mediating point of melanosomes transport. The changes in quantity and morphology play an important role in melanin transport, and the regulation of dendrites can produce pigmentation. [3]. studies have found that many factors can affect the formation of dendrites, such as.Scott, and other studies have found that small GTP enzymes in the Rho family, especially Rac1 and RhoA, play an important role in the formation of dendrites of melanocytes, in which Rac1 plays the role of promoting the formation and extension of dendrites, while RhoA has the opposite effect. Our previous study found UV, UV. B irradiation can promote the formation and prolongation of the dendrites of melanocytes by regulating the expression of Rac1 and RhoA by regulating the dendrites. However, the mechanism of UVB irradiation to regulate the expression of Rac1 and RhoA is still unclear.
In recent years, the role of cyclooxygenase (COX) in the formation and development of tumors has become a focus of attention. Some studies have shown that COX-2 inhibitors can inhibit the migration of [5] in the Cdc42/Rac1 dependent endothelial cells of the Cdc42/Rac1 dependent V beta 3, and the COX-2 specific inhibitor NS-398 can cause the disappearance of filamentous pseudo foot and cell invasion in tumor cells. The decrease in capacity indicates that both COX-2 and Cdc42/Rac1 are important molecules that cause the disappearance of cell filamentous pseudo foot and the decrease of invasion ability in vitro. These studies suggest that the cyclooxygenase may have a certain relationship with the formation of dendrites.
Therefore, by detecting the changes in the COX level of melanocytes after UVB irradiation and observing the effect of inhibition of COX activity on the dendritic morphology of melanocytes and the effect of Rac1 and RhoA, we explored the role and mechanism of cyclooxygenase in the formation of melanocyte dendritic cells induced by UVB irradiation, in order to clarify the mechanism of pigmentation after UVB irradiation and to provide a new way to prevent it. Ideas and new strategies.
Experimental methods:
The immortalized human epidermal melanocyte line PIG1 was used in this study and was divided into 5 groups after routine culture: (1) the control group, (2) 100mJ/3 2UVB irradiation group; (3) 100mJ/3 2UVB irradiation +ASA; (4) 100mJ/3 2UVB irradiation +NS-398; (5) 100mJ/3 2UVB 2UVB irradiation +SC-560. first. The effects of COX-1 and COX-2 expression were observed. Then, the effects of different treatments on the dendritic morphology of melanocytes were observed under the microscope. Finally, the effects of different treatments on the expression of Rac1 and RhoA in melanocytes were detected by extracting protein and RNA using Western blot and PT-PCR methods.
Experimental results:
The expression of COX-1 and COX-2mRNA and protein increased significantly after irradiation with 1.100mJ/3 2UVB.
2.100 mol/L ASA and 30 mol/L NS-398 inhibited the formation and extension of PIG1 dendritic in human melanocyte line induced by UVB irradiation, while 10 mu mol/L SC-560 had no obvious inhibitory effect on the formation and extension of PIG1 dendrites in the human melanocyte line induced by UVB irradiation.
When the concentration of 3.ASA is within 50~150 mol/L, the high expression of Rac1 and the low expression of RhoA in human melanocyte line PIG1 caused by flipping UVB irradiation are in a dose-dependent manner, and 100 u mol/L ASA and 30 micron mol/L NS-398 can inhibit the high expression of the dendritic form related molecules of the human melanocyte line induced by UVB irradiation and the low table Yes, but SC-560 does not have a similar effect.
Conclusion:
1. certain doses of UVB irradiation can promote the expression of COX-1 and COX-2 in melanocytes.
2. cyclooxygenase plays a role in the formation and extension of dendrites, and COX-2 plays a leading role.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R751

【参考文献】