Q开关Nd:YAG1064nm激光照射对人表皮黑素细胞生物学及黑素合成的影响

发布时间：2018-05-04 18:09

本文选题：Q开关Nd:YAG + 1064nm激光　；参考：《第四军医大学》2010年硕士论文

【摘要】： 近年来随着“选择性光热理论”的提出,医用激光技术在临床治疗和皮肤美容领域的发展十分迅速,其中Q开关Nd:YAG1064nm激光在治疗太田痣、黄褐斑、雀斑等色素增加性皮肤病取得了比较满意的效果。然而,目前Q开关激光治疗表皮色素增加性皮肤病还存在一定的副作用,究其原因一方面可能是表皮色素增加性皮肤病病因复杂,另一方面是由于Q开关激光对表皮黑素细胞黑素合成的影响及其分子机制目前尚不十分清楚,从而限制了Q开关激光的广泛应用。目前人们对Q开关Nd:YAG1064nm激光的治疗原理的认识尚仅局限于黑素颗粒对光的选择性吸收,而除此之外对激光照射引起的光生物学作用并不清楚。因此,我们通过实验研究初步探讨Q开关Nd:YAG1064nm激光照射对人表皮黑素细胞的生物学及黑素合成的影响。实验目的:研究Q开关Nd:YAG1064nm激光照射对人表皮黑素细胞的生物学及黑素合成的影响。实验方法:在本研究中,我们选择目前最先进的MedliteC6Q开关Nd:YAG激光照射体外培养的人表皮黑素细胞,参数选择为:波长1064nm,光斑直径6mm,频率2Hz。研究内容有:①光学显微镜下观察Q开关Nd:YAG1064nm激光照射后细胞形态的变化;②MTT法检测Q开关Nd:YAG1064nm激光照射对人表皮黑素细胞细胞活性的影响;③流式细胞仪检测Q开关Nd:YAG1064nm激光照射对人表皮黑素细胞周期与凋亡的影响;④多巴染色法测定Q开关Nd:YAG1064nm激光照射后人表皮黑素细胞树突数量及长度的变化;⑤多巴氧化法检测Q开关Nd:YAG1064nm激光照射后酪氨酸酶(Tyr)活性的变化;⑥荧光定量反转录(RT)-PCR和Westenblot分别检测Q开关Nd:YAG1064nm激光照射后Tyr和酪氨酸酶相关蛋白(TRP-1和TRP-2)基因转录和蛋白表达水平的变化。实验结果:1.不同能量密度的Q开关Nd:YAG1064nm激光照射对人表皮黑素细胞活性及形态的影响:①MTT结果表明:人表皮黑素细胞对Q开关Nd:YAG1064nm激光照射的能量密度有一定的耐受范围,在本实验中不影响细胞存活率的最高照射能量密度为3J/cm2,即人表皮黑素细胞可以耐受的照射能量密度阈值为3J/cm2。我们以此为依据将细胞分为对照组,1J/cm2照射组(代表本实验中不影响细胞生存率的最低照射能量密度)与5J/cm2照射组(代表本实验中超出黑素细胞可以耐受的阈值范围的最高照射能量密度),继续下面的实验。②不同能量密度的Q开关Nd:YAG1064nm激光照射后人表皮黑素细胞活性的时效性分析结果表明:与对照组比较,1J/cm2组黑素细胞活性在各时间点均无明显差异(P0.05),而5J/cm2组人表皮黑素细胞活性分别在照射后0h时、24h时与48h时这三个时间点明显降低(P0.05)。5J/cm2照射后,人表皮黑素细胞增殖曲线在48h内逐渐下降至最低点后又显著上升。③我们在光学显微镜下观察到各组细胞的形态在Q开关Nd:YAG1064nm激光照射后即刻无明显改变,但接受照射后24h时与对照组比较1J/cm2照射组黑素细胞大小无明显变化,但是树突长度增加;而3J/cm2照射组黑素细胞体积明显变小,树突数量减少并且长度缩短。 2.Q开关Nd:YAG1064nm激光照射对人表皮黑素细胞周期与凋亡的影响:①在不影响细胞存活率的能量密度范围内,将黑素细胞分为对照组、1J/cm2照射组(代表本实验中不影响细胞生存率的最低照射能量密度)与3J/cm2照射组(代表在本实验中不影响细胞存活率的最高照射能量密度,即黑素细胞可以耐受的照射能量密度阈值)②。细胞周期结果表明:1J/cm2照射组人表皮黑素细胞S期细胞数量轻度增加(P0.05),而3J/cm2照射组人表皮黑素细胞S期细胞数量则显著减少(P0.05)。③细胞凋亡结果表明:与对照组细胞凋亡率相比,1J/cm2照射组无明显差异(P0.05),而3J/cm2照射组细胞凋亡率明显增加(P0.05)。 3.Q开关Nd:YAG1064nm激光照射对人表皮黑素细胞黑素合成的影响:①在不影响黑素细胞存活率的范围内,能量密度为1J/cm2的激光照射可以刺激Tyr活性,增加黑素细胞树突的数量和长度;上Ty调r与TRP-1的mRNA表达,而TRP-2mRNA表达并无明显变化,其蛋白水平的变化与转录水平相符。②在不影响黑素细胞活性的范围内,能量密度为3J/cm2的激光照射能明显抑制Tyr活性,减少黑素细胞树突的数量和长度;同时也不同程度的抑制了Try、TRP-1和TRP-2的mRNA表达与蛋白合成。主要结论: 1.人表皮黑素细胞对Q开关Nd:YAG1064nm激光照射的能量密度存在一定的耐受范围。当能量密度大于一定值(人表皮黑素细胞能耐受的值)阈时,Q开关Nd:YAG1064nm激光的照射对细胞活性的抑制作用会随着能量密度的增加而逐渐增强,但这种抑制作用并不持续存在,黑素细胞的活性经过一段时间后可以逐渐恢复。 2.在不影响黑素细胞存活率的范围内,其中较低的能量密度的Q开关Nd:YAG1064nm激光照射能够刺激人表皮黑素细胞DNA合成,表现出生物刺激作用。当选择人表皮黑素细胞可以耐受的能量密度阈值照射时,Q开关Nd:YAG1064nm激光可以明显抑制人表皮黑素细胞DNA合成并促进其凋亡。 3.在不影响黑素细胞存活率的范围内,Q开关Nd:YAG1064nm激光照射可以影响黑素合成。其中较低的能量密度的激光照射能够通过刺激酪氨酸酶家族蛋白(TRPs)从而促进黑素的合成,而选择较高能量密度(人表皮黑素细胞可以耐受的能量密度阈值)的Q开关Nd:YAG1064nm激光照射则起着抑制黑素合成的作用。 4.在不影响黑素细胞存活率的范围内,Q开关Nd:YAG1064nm激光照射可以影响人表皮黑素细胞树突的形成。 5.Q开关Nd:YAG1064nm激光照射对人表皮黑素细胞存在光生物学作用。
[Abstract]:In recent years, with the "selective photothermal theory", medical laser technology has developed rapidly in the field of clinical treatment and skin beauty, in which Q switched Nd:YAG1064nm laser has achieved satisfactory results in the treatment of pigmented dermatosis such as nevus of Ota, chloasma, freckles and other pigmented dermatosis. However, the Q switch laser is currently used to treat epidermal pigments. There is also some side effects of additive dermatosis, which may be due to the complicated cause of epidermal pigmented dermatosis, and the other is that the effect of Q switched laser on melanin synthesis in epidermal melanocytes and its molecular mechanism are still not very clear, thus limiting the wide application of Q switched laser. At present, people have opened Q. The understanding of the principle of the treatment of Nd:YAG1064nm laser is still limited to the selective absorption of light by melanin particles, but it is not clear that the effect of laser irradiation is not clear. Therefore, we have preliminarily studied the biological and melanogenesis of human surface melanocytes by the Q switch Nd:YAG1064nm laser irradiation. Ringing.
Objective: To study the effects of Q switch Nd:YAG1064nm laser irradiation on the biological and melanin synthesis of human epidermal melanocytes.
Experimental methods: in this study, we chose the most advanced MedliteC6Q switched Nd:YAG laser to irradiate human epidermal melanocytes in vitro. The parameters were as follows: wavelength 1064nm, spot diameter 6mm, frequency 2Hz. research content: (1) observation of cell morphology changes after Q switch Nd: YAG1064nm laser irradiation under optical microscope; (2) MTT method detection The effect of Q switch Nd:YAG1064nm laser irradiation on the cell activity of human epidermal melanocytes; and (3) flow cytometry to detect the effect of Q switch Nd:YAG1064nm laser irradiation on the cell cycle and apoptosis of human epidermal melanocytes; (4) DOPA staining method was used to determine the number and length of dendritic cells of human epidermal melanocytes after Q switch Nd:YAG1064nm laser irradiation; 5 The changes of tyrosinase (Tyr) activity after Q switch Nd:YAG1064nm laser irradiation were detected by Ba. (6) fluorescence quantitative reverse transcription (RT) -PCR and Westenblot were used to detect the changes in the transcriptional and egg white expression of Tyr and tyrosinase related proteins (TRP-1 and TRP-2) after Q switch Nd:YAG1064nm laser irradiation, respectively.
The results were as follows: 1. the effects of different energy density Q switch Nd:YAG1064nm laser irradiation on the activity and morphology of human epidermal melanocytes: (1) MTT results showed that human epidermal melanocytes had a certain range of tolerance to the energy density of Q switched Nd:YAG1064nm laser irradiation, and the highest energy density of cell survival rate was not affected in this experiment. 3J/cm2, the threshold of radiation energy density that the human epidermal melanocytes can tolerate, is 3J/cm2.. We divide the cells into the control group based on this, and the 1J/cm2 irradiation group (representing the minimum irradiation energy density that does not affect the cell survival rate in this experiment) and the 5J/cm2 irradiation group (representing the threshold range beyond the tolerance of melanocytes in this test) The results of the aging analysis of the activity of human epidermal melanocytes after Q switch Nd:YAG1064nm laser irradiation with different energy density showed that there was no significant difference in the activity of melanocyte in the 1J/cm2 group at all time points compared with the control group (P0.05), while the activity of human epidermal melanocytes in the 5J/cm2 group was in the 5J/cm2 group, respectively. After 0h, the three time points of 24h and 48h decreased significantly (P0.05).5J/cm2 irradiation, and the proliferation curve of human epidermal melanocytes decreased gradually to the lowest point in 48h and then increased significantly. Compared with the control group, the size of the melanocytes in the 1J/cm2 irradiated group was not significantly changed, but the length of the dendritic cells increased, while the volume of melanocytes in the 3J/cm2 group decreased obviously, the number of dendrites decreased and the length shortened in the 3J/cm2 irradiation group.
The effect of 2.Q switch Nd:YAG1064nm laser irradiation on the cell cycle and apoptosis of human epidermal melanocytes: (1) the melanocytes were divided into the control group in the range of energy density that did not affect the cell survival rate, and the 1J/cm2 irradiation group (representing the minimum irradiation energy density that does not affect the cell survival rate in this experiment) and the 3J/cm2 irradiation group (represented in this experiment) The maximum radiation energy density that affects the cell survival rate, that is, the threshold of the radiation energy density that the melanocytes can tolerate. The cell cycle results show that the number of S cells in the human epidermal melanocytes in the 1J/cm2 irradiation group is slightly increased (P0.05), while the number of S cells in the 3J/cm2 irradiated group is significantly reduced (P0.05). (3) cell apoptosis The results showed that: compared with the control group, the apoptosis rate of 1J/cm2 group was not significantly different (P0.05), while the apoptosis rate of 3J/cm2 irradiation group increased significantly (P0.05).
The effect of 3.Q switch Nd:YAG1064nm laser irradiation on melanin synthesis in human epidermal melanocytes: (1) laser irradiation with energy density of 1J/cm2 can stimulate the activity of Tyr, increase the number and length of the dendrites of melanocytes in the range of the survival rate of melanocytes; the expression of R and TRP-1 in the upper Ty is expressed, and the expression of TRP-2mRNA is not significantly changed. The changes in the protein level are consistent with the transcriptional level. In the range of the activity of melanocytes, the laser irradiation with energy density of 3J/cm2 can significantly inhibit the activity of Tyr, reduce the number and length of the dendrites of melanocytes, and inhibit the mRNA expression and protein synthesis of Try, TRP-1 and TRP-2 to varying degrees.
The main conclusions are as follows:
1. human epidermal melanocytes have a certain range of tolerance to the energy density of Q switched Nd:YAG1064nm laser irradiation. When the energy density is greater than a certain value (the tolerance value of human epidermal melanocytes), the inhibitory effect of the Q switch Nd:YAG1064nm laser irradiation on the cell activity will gradually increase with the increase of the energy density, but this inhibition is inhibited. The production does not persist. The activity of melanocytes can recover gradually after a period of time.
2. in the range that does not affect the survival rate of melanocytes, the low energy density Q switch Nd:YAG1064nm laser irradiation can stimulate the DNA synthesis of human epidermal melanocytes and show biological stimulation. When the energy density threshold of human epidermal melanocytes can be tolerated, the Q switch Nd:YAG1064nm laser can obviously inhibit people. Epidermal melanocyte DNA synthesizes and promotes its apoptosis.
3. the Q switch Nd:YAG1064nm laser irradiation can affect melanin synthesis without affecting the survival rate of melanocytes, in which low energy density laser irradiation can stimulate the synthesis of melanin by stimulating the tyrosinase family protein (TRPs) and select the higher energy density (the energy density tolerable by human epidermal melanocytes). The Q switch Nd:YAG1064nm laser irradiation plays a role in inhibiting melanin synthesis.
4. the Q switch Nd:YAG1064nm laser irradiation can affect the formation of human epidermal melanocyte dendrites without affecting the survival rate of melanocytes.
5.Q switch Nd:YAG1064nm laser irradiation has a photo biologic effect on human epidermal melanocytes.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R454.2;R758.4

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