UVB照射对黑素小体转运的影响及其机制

发布时间：2018-07-03 07:39

本文选题：UVB照射 + 黑素细胞　；参考：《第四军医大学》2011年硕士论文

【摘要】：人体皮肤的颜色主要受色素沉着的影响,它不仅决定皮肤、头发和眼睛的颜色,还能够保护皮肤免受紫外线的过度照射。其过程非常复杂,包括:1、黑素细胞内黑素小体的装配与黑素合成;2、成熟的黑素小体沿细胞树突伸展方向向树突远端转移;3、而后黑素小体脱离黑素细胞进入周围的角质形成细胞;4、黑素小体在角质形成细胞内再分布和降解。通常将黑素小体从核周向树突末梢转移并传递至邻近角质形成细胞的过程称为黑素小体的转运[1]。研究表明黑素小体的转运在皮肤色素沉着过程中发挥极为重要的作用,但其机制尚未被完全认识。紫外线是电磁波谱中波长为0.01～0.40微米辐射的总称。皮肤的色素沉着主要由UVB引起,UVB的生物学效应强度是UVA的800～1000倍,是引起皮肤光损伤的最重要波段[2]。研究证明,UVB照射可以引起皮肤色素沉着,一定剂量的UVB照射可以促进黑素细胞增殖,增加黑素合成~([3])。已有研究证实UVB照射体外重建表皮类似物时可以增强黑素小体的转运。发现在表皮类似物的基底层,及角质层都有黑素小体~([4-5])。以上提示UVB照射可以促进黑素小体的转运。但是对于UVB促黑素转运的机制并不清楚。因此,我们通过实验研究初步探讨UVB照射促进黑素小体向角质形成细胞转运的机制。实验目的:研究UVB照射对黑素小体向角质形成细胞转运的影响及其机制。实验方法:本研究使用上海希格玛公司生产的紫外线光疗仪,辐照度以UVB辐照度监示器标定,UVB剂量=UVB辐照度×时间(s)。研究内容有:①不同能量UVB照射对黑素细胞株PIG1增殖活性的影响;②UVB照射对PIG1细胞的形态学影响及细胞树突变化;③UVB照射对酪氨酸酶Tyr的mRNA及蛋白影响的时间规律性;④gp100免疫荧光法观察UVB照射对黑素合成的影响;⑤RT-PCR方法检测UVB照射对F-actin、Rac、Rho及PAR-2的mRNA表达的影响;⑥Western Blot方法检测UVB照射对黑素小体转运相关蛋白F-actin、Rac、Rho及PAR-2蛋白表达的影响。实验结果: 1.不同能量UVB照射对黑素细胞株PIG1活性的影响; MTT结果表明:UVB照射后24h,照射能量在50、100mJ/cm~2时,细胞增殖活性高于照射前水平(P0.05),50mJ/cm~2时细胞增殖活性最强;而照射能量高于200mJ/cm~2时,细胞增殖活性低于照射前水平,并随着能量升高呈逐渐下降趋势。UVB照射后48h,照射能量在100mJ/cm~2时细胞活性有一定程度的恢复,而到400mJ/cm~2后呈下降趋势。100mJ/cm~2时的细胞增殖活性较24h升高,200 mJ/cm~2时细胞活性达最大值,而24h则从200 mJ/cm~2时开始下降。 2. UVB照射对PIG1细胞的形态学影响及树突变化:由于UVB照射能量在100mJ/cm~2时能够较稳定增加细胞活性,因此选择100mJ/cm~2 UVB照射细胞后48h观察了细胞的形态变化。①选择100mJ/cm~2 UVB照射PIG1后48h,光学显微镜下观察细胞形态的变化。对照组PIG1细胞形态:细胞贴壁后很快进入对数生长期,生长状况良好,细胞呈多分支树突状,轮廓清楚折光性好。照射组形态:PIG1细胞数量明显增多,树突显著延长。③UVB照射对PIG1细胞树突的影响统计学方法计数PIG1细胞的树突数量及长度。用100mJ/cm~2的UVB照射PIG1后48h,细胞树突相对于未照射组明显增多延长,具有统计学意义。 3. UVB照射对酪氨酸酶Tyr的mRNA及蛋白影响的时间规律性:用100mJ/cm~2的UVB照射PIG1后48h,酪氨酸酶Tyr的mRNA和蛋白表达在UVB照射后24h后显著增加,并呈上升趋势。 4. gp100免疫荧光法观察UVB照射对黑素合成的影响:UVB能量为50 mJ/cm~2时,照射后48h时荧光强度最强。UVB能量为100mJ/cm~2时同样在照射后48h时荧光强度最强。 5.RT-PCR方法检测UVB照射对F-actin、Rac1、RhoA及PAR-2的mRNA表达的影响:100mJ/cm~2 UVB照射48h后Rac1、F-actin和PAR-2的mRNA表达相对于未照射组明显增加,而RhoA的表达下降。6.Western Blot方法检测UVB照射对黑素小体转运相关蛋白F-actin、Rac1、RhoA及PAR-2蛋白表达的影响:100mJ/cm~2 UVB照射48h后Rac1、F-actin和PAR-2的表达升高,而RhoA的表达下降。主要结论: 1. UVB照射能量在100mJ/cm~2时能够较稳定增加细胞活性。提示一定剂量的UVB照射有刺激PIG1细胞增殖的作用。而选择超过100mJ/cm~2的能量照射时,PIG1细胞的活性受到抑制。而这种抑制作用并不是持续性的,细胞活性会在一段时间后恢复,因此在这种情况下UVB对PIG1细胞表现出的损伤作用是可逆性的。 2.一定剂量的UVB照射有促进PIG1细胞增殖和刺激树突生长的作用。 3.UVB照射可以影响酪氨酸酶Tyr的mRNA及蛋白表达,在UVB照射后24h后显著增加,并呈上升趋势。 4. UVB照射后用gp100这一黑素小体特异性抗体进行免疫荧光染色,可以观察黑素合成情况。黑素合成在100mJ/cm~2 UVB照射后48h时达到最大值。 5.UVB照射可以促进树突形态调控因子Rac1、丝状肌动蛋白F-actin及蛋白酶活性受体PAR-2的mRNA及蛋白表达,抑制RhoA的表达。
[Abstract]:The color of the human skin is mainly affected by the pigmentation, which not only determines the color of the skin, hair and eyes, but also protects the skin from excessive ultraviolet radiation. The process is very complex, including: 1, the assembly of melanosomes in melanocytes and melanin synthesis; and 2, the mature melanosomes extend to the distal dendrites along the dendritic direction. 3, the melanosomes are separated from the melanocytes into the surrounding keratinocytes; 4, the melanosomes are redistributed and degraded in the keratinocytes. Usually the process of transferring melanosomes from the nucleus to the dendritic cells and transferring to the adjacent keratinocytes is called the transport of melanin corpuscle in the transport of [1].. The mechanism of skin pigmentation plays a very important role, but its mechanism has not been fully understood. Ultraviolet radiation is the general name of 0.01 to 0.40 microns in the electromagnetic wave spectrum. The pigmentation of the skin is mainly caused by UVB, and the biological effect of UVB is 800~1000 times that of UVA, and it is the most important band of [2]. to cause skin light damage. It has been shown that UVB irradiation can cause pigmentation in the skin, and a certain dose of UVB can promote the proliferation of melanocytes and increase melanin synthesis ~ ([3]). It has been proved that UVB irradiation can enhance the transport of melanosomes in the reconstruction of epidermal analogues in vitro. It is found that there are melanosomes in the basal layer of the epidermal analogues and in the cuticle layer ~ ([4-5]). These findings suggest that UVB irradiation can promote the transport of melanosomes. However, the mechanism for the transport of melanin in UVB is not clear. Therefore, we have preliminarily explored the mechanism of UVB irradiation promoting the transport of melanosomes to keratinocytes.
Objective: To study the effect of UVB irradiation on the transport of melanosomes to keratinocytes and its mechanism.
Experimental method: This study used the UV light therapy instrument produced by Shanghai Hege Ma company, irradiance was calibrated with UVB irradiance monitor, and UVB dose =UVB irradiance x time (s). The study contents were: (1) the effect of different energy UVB irradiation on the proliferation of PIG1 in melanocyte strain; the morphological influence of UVB irradiation on PIG1 cells and the change of cell dendrites (3) the time regularity of the effect of UVB irradiation on the mRNA and protein of tyrosinase Tyr; (4) the effect of UVB irradiation on the synthesis of melanin by gp100 immunofluorescence; and (5) RT-PCR method to detect the effect of UVB irradiation on F-actin, Rac, Rho and PAR-2 mRNA expression. And the effect of PAR-2 protein expression.
Experimental results:
1. the effect of different energy UVB irradiation on the activity of PIG1 in melanocyte strain. The MTT results showed that the proliferation activity of cells was higher than before irradiation (P0.05) and 50mJ/cm~2 was the strongest when UVB irradiation was 50100mJ/cm~2, while the irradiation energy was higher than that of 200mJ/cm~ 2, and the cell proliferation activity was lower than that before irradiation, and with the energy of irradiation, the cell proliferation activity was lower than that before irradiation. When the amount of.UVB decreased gradually, the cell activity was recovered to a certain extent at 100mJ/cm~2, and the cell proliferation activity was higher than that of 24h at.100mJ/cm~2 after 400mJ/cm~2, and the cell activity reached the maximum at 200 mJ/cm~2, while 24h began to decline from 200 mJ/cm~2.
The morphological influence of 2. UVB irradiation on the morphology of PIG1 cells and the change of dendrites: because UVB irradiated energy at 100mJ/cm~2, the cell activity was more stable, so the morphological changes of the cells were observed after 100mJ/cm~2 UVB irradiated the cells. (1) the 100mJ/cm~2 UVB irradiated PIG1 48h, the morphological changes of the cells were observed under the light microscope. Group PIG1 cell morphology: the cells quickly entered the logarithmic growth period after being adhered to the wall. The growth condition was good, the cells showed a multi branch dendritic shape, and the contour clearly refracted. The number of PIG1 cells increased significantly and the dendrites were significantly prolonged. (3) the number and length of the dendrites of the PIG1 cells were counted by the effect of UVB irradiation on the dendrites of the PIG1 cells. The number and length of the dendritic cells of PIG1 cells were counted. When 00mJ/cm~2 was irradiated with UVB for PIG1, the 48h cell dendrites increased significantly compared with the unirradiated group, with statistical significance.
The time regularity of the effect of 3. UVB irradiation on the mRNA and protein of tyrosinase Tyr: 48h after PIG1 irradiation with 100mJ/cm~2, mRNA and protein expression of tyrosinase Tyr significantly increased after 24h after UVB, and showed an upward trend.
The effect of UVB irradiation on the synthesis of melanin was observed by 4. gp100 immunofluorescence. When the energy of UVB was 50 mJ/cm~2, the strongest.UVB energy of the fluorescence intensity was 100mJ/cm~2 after 48h, and the intensity of the fluorescence was the strongest at 48h after irradiation.
The effect of UVB irradiation on the mRNA expression of F-actin, Rac1, RhoA and PAR-2: 100mJ/cm~2 UVB irradiated 48h Rac1, F-actin and F-actin expression were significantly increased compared to those in the unirradiated group. The effect of 100mJ/cm~2 UVB and 48h on Rac1, F-actin and PAR-2 increased, while RhoA expression decreased.
The main conclusions are as follows:
1. UVB irradiate energy at 100mJ/cm~2 can increase cell activity more steadily. It suggests that a certain dose of UVB irradiation stimulates the proliferation of PIG1 cells. While the activity of PIG1 cells is inhibited when the energy exceeds 100mJ/cm~2, and this inhibition is not persistent, and the cell activity will recover after a period of time. Therefore, the activity of the cells will be recovered after a period of time. In this case, the damaging effect of UVB on PIG1 cells is reversible.
2. a certain dose of UVB irradiation can promote the proliferation of PIG1 cells and stimulate the growth of dendrites.
3.UVB irradiation could affect the expression of tyrosinase Tyr mRNA and protein, and increased significantly after UVB irradiation, and showed an upward trend.
4. UVB irradiated with gp100, a melanosomes specific antibody for immunofluorescence staining, can be used to observe the synthesis of melanin. Melanin synthesis reached the maximum value at 48h after 100mJ/cm~2 UVB irradiation.
5.UVB irradiation can promote the expression of mRNA and protein of dendritic morphology regulator Rac1, filamentous actin F-actin and protease active receptor PAR-2, and inhibit the expression of RhoA.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.14

【参考文献】