热处理对UVB辐射后人表皮黑素细胞活性影响的研究

发布时间：2018-02-21 06:22

本文关键词： 热 UVB 黑素细胞黑素合成酪氨酸酶活性细胞周期　出处：《第四军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的白癜风是一种常见的以局部或全身皮肤黏膜色素脱失为特征的皮肤病。人群患病率约为0.1～2%,多于幼年起病，无明显种族及性别差异。皮损可发生于任何部位，皮肤病理显示皮损处表皮黑素细胞明显减少或消失。该病病因不明，目前认为可能与遗传、自身免疫异常、氧化应激、神经精神等因素有关。白癜风临床治疗较为棘手，主要包括药物治疗、光疗、手术治疗等。光疗，特别是波长为311～313nm的中波紫外线，由于疗效确切、安全可靠，现已成为白癜风临床治疗的首选方法之一。近年来，人们逐渐发现热处理(42℃,1h）与紫外线相似，能够增加黑素细胞的酪氨酸酶活性及黑素合成，促使黑素细胞和角质形成细胞共培养体系中酪氨酸酶相关蛋白-1阳性的黑素细胞明显增多；同时，以热效应为主的红外线可明显减少大剂量UVB辐射诱导的角质形成细胞凋亡。以上结果提示我们热与UVB的联合治疗可能将成为白癜风治疗的重要手段。本实验研究热处理对UVB辐射后人表皮黑素细胞的增殖活性、酪氨酸酶活性、黑素合成及细胞周期的影响，并初步探讨其机制，为今后热疗与UVB联合应用治疗白癜风等色素脱失性皮肤病提供理论依据。方法 1.以体外培养的正常人黑素细胞作为研究对象，分别以20、30、50、70、90、120、180mJ/cm~2NB-UVB辐射，采用四甲基偶氮唑蓝比色法（MTT）检测细胞存活率，并选择最佳照射剂量进行后续实验。 2.以42℃,1h为热处理剂量，将黑素细胞分为四组：正常对照组、UVB组、加热组、UVB+热组，，分别给予不同干预，连续3d，末次干预24h后以MTT法检测细胞增殖活性，左旋多巴(L-Dopa)为底物测定酪氨酸酶活性，NAOH法测定黑素含量，流式细胞仪检测细胞周期变化。结果 1.分别以20、30、50、70、90、120、180mJ/cm~2UVB连续照射黑素细胞3d后，细胞存活率随着剂量的增加而降低，选择50mJ/cm~2作为最佳照射剂量。 2.黑素细胞经不同干预后比较细胞增殖活性，UVB组较对照组减少7.1%（P0.05），加热组、UVB+热组细胞较对照组分别增加25.3%和58.1%（P0.05）。热处理与UVB共同干预具有协同作用。 3.黑素细胞经不同干预后，UVB组、加热组、UVB+热组细胞的酪氨酸酶活性较对照组分别增长3.8%、11.5%和31.9%（P0.05），三组细胞的黑素含量较对照组分别增加17.5%、31.6%和67.3%（P0.05）。热处理与UVB辐射联合干预具有协同作用。 4.黑素细胞给予不同干预后，UVB组、加热组、UVB+热组处于G1期的细胞较对照组分别下降24.61%、13.16%和32.02%（P0.05）；处于S期的细胞较对照组分别增加15.02%、8.58%和17.14%（P0.05），处于G2期的细胞分别增加12.93%、7.52%、23.60%（P0.05）。热处理与UVB辐射联合干预具有协同作用。结论 1.热处理（42℃,1h）能有效促进黑素细胞增殖，与UVB辐射（50mj/cm2）联合干预具有协同作用。 2.热处理（42℃,1h）和UVB辐射（50mj/cm2）均可显著增加黑素细胞内酪氨酸酶活性及黑素合成，两者联合干预具有协同作用。 3.热处理（42℃,1h）及UVB辐射（50mj/cm2）干预均可促使细胞周期阻滞在S期和G2期，减少处于G1期的细胞数量，两者联合干预具有协同作用。
[Abstract]:objective
Vitiligo is a common skin disease to local or generalized depigmentation of skin and mucosa characterized. The prevalence is about 0.1 ~ 2%, more than the juvenile onset, no obvious racial and gender differences. The lesions can occur in any part of the skin lesions, pathological examination showed epidermal melanocytes decreased or disappeared. The cause of this disease currently unknown, it may be related to genetic, autoimmune disorders, oxidative stress and related neuropsychiatric factors. The clinical treatment of vitiligo is difficult, mainly including drug therapy, phototherapy, phototherapy. Surgical treatment, especially at the wavelength of 311 ~ 313nm due to ultraviolet ray, curative effect, safe and reliable, has become one of the preferred methods of clinical the treatment of vitiligo.
In recent years, people gradually found that the heat treatment (at 42 1H) and ultraviolet similar, can increase the tyrosinase activity and melanin synthesis in black cells, promote the melanocytes and keratinocytes of tyrosinase related protein -1 positive cells co cultured black pigment cells increased significantly; at the same time, the thermal effect of the infrared ray reduce the dose of UVB radiation induced apoptosis of keratinocytes. The above results suggest that our combined treatment of heat and UVB will likely become an important means in the treatment of vitiligo. The experimental study on heat treatment of irradiation on Human Epidermal Melanocytes Proliferation and tyrosinase activity of UVB, effects of melanin synthesis and cell cycle, and to explore the mechanism, provide a theoretical basis for future hyperthermia combined with UVB treatment of depigmentation skin disease.
Method
1., the normal human melanocytes were cultured in vitro. The cell survival rate was detected by 20,30,50,70,90120180mJ/cm~2NB-UVB radiation and four MZR blue colorimetric assay (MTT). The best dose of radiation was selected for subsequent experiments.
2. to 42 DEG C, 1H heat treatment dose, melanocytes were divided into four groups: normal control group, UVB group, UVB+ group of heating, heat group, given different interventions, respectively for 3D, 24h after the last intervention by MTT was used to detect the proliferation activity of levodopa (L-Dopa) for determination of activity of casein substrate tyrosinase, melanin content was measured by NAOH assay, flow cytometry to detect the cell cycle.
Result
1., after continuously irradiating melanocytes 3D with 20,30,50,70,90120180mJ/cm~2UVB, the cell survival rate decreases with the increase of dose. 50mJ/cm~2 is chosen as the best dose.
2., compared with the control group, the proliferative activity of the melanocytes was decreased by 7.1% (P0.05) compared with the control group after different intervention. In the heating group, the UVB+ fever group increased by 25.3% and 58.1% (P0.05) respectively compared with the control group. The co intervention of heat treatment and UVB had synergistic effect.
3. melanocytes after different intervention, UVB group, UVB+ group, heat heating group, cell tyrosinase activity increased by 3.8% compared with the control group, 11.5% and 31.9% (P0.05), three groups of melanocytes was increased by 17.5% compared with the control group, 31.6% and 67.3% (P0.05). Heat treatment and UVB radiation combined with the intervention has a synergistic effect.
4. melanocytes give different intervention, UVB group, UVB+ group, thermal heating group, cells in G1 phase decreased by 24.61% respectively compared with the control group, 13.16% and 32.02% (P0.05); the cells at S phase increased by 15.02% respectively compared with the control group, 8.58% and 17.14% (P0.05), the cells at G2 phase increased respectively 12.93%, 7.52%, 23.60% (P0.05). The joint intervention of heat treatment and UVB radiation has a synergistic effect.
conclusion
1. heat treatment (42 centigrade, 1H) can effectively promote the proliferation of melanocytes and have synergistic effect with UVB radiation (50mj/cm2).
2. heat treatment (42, 1H) and UVB radiation (50mj/cm2) significantly increased tyrosinase activity and melanin synthesis in melanocytes, and the combined intervention had synergistic effects.
3., heat treatment (42 C, 1H) and UVB radiation (50mj/cm2) intervention can promote cell cycle arrest in S and G2 phase, reduce the number of cells in G1 phase, and the combination intervention has synergistic effect.

【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】：R758.41

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