低剂量UVA诱导人皮肤成纤维细胞出现适应性反应中自噬的变化观察

发布时间：2018-09-08 15:23

【摘要】：近年来,紫外线与人皮肤健康的关系一直是研究的热点。紫外线是把“双刃剑”,一方面,紫外线一直被认为是皮肤的杀手,除了可能导致各种光敏性皮肤病,过多的紫外线照射也是造成皮肤光老化及暴露部位皮肤癌多发的最重要因素；另一方面,紫外线也存在有益的生物学效应,接受适当的紫外线照射可以提高机体免疫力,预防某些自身免疫性疾病,促进骨骼的生长发育,减少肿瘤的发生。这些有益的生物效应可能与其低剂量照射诱导的兴奋效应存在一致的关系。适应性反应是辐射兴奋效应的重要内容。我们前期实验已证实低剂量的长波紫外线(UVA)辐射可以诱导培养皮肤细胞出现适应性反应,这种适应性反应表现为单次或多次低剂量UVA预照射能减轻随后致死剂量UVA照射对培养皮肤成纤维细胞形态学上的毒性反应,并使细胞凋亡的比例下降、DNA链断裂减少及修复加快,但这种适应性反应的机制仍有待进一步阐明。自噬(autophagy)是真核细胞中普遍存在的生命现象,是将细胞内变形、衰老或损伤的蛋白质和细胞器转运到溶酶体腔中消化降解的一种代谢过程。自噬有利于为此细胞和组织的动态平衡,是维持真核细胞内环境的自我稳定并实现更新的一种有效途径,也是其适应环境变化的一种重要的防御机制,但低剂量UVA照射诱导的适应性反应与自噬之间的相关性目前仍末见报道。本文观察了低剂量UVA诱导人皮肤成纤维细胞适应性反应中自噬的变化,探讨其在诱导培养人皮肤成纤维细胞适应性反应的可能作用,为全面阐明低剂量UVA诱导人皮肤细胞适应性反应的机制提供更多的实验依据。目的：1.观察低剂量长波紫外线诱导人皮肤成纤维细胞出现适应性反应中自噬的形态及数量的变化,对比不同组细胞的自噬形态及数量的差异。2.通过检测自噬相关蛋白LC3、Beclinl来探讨低剂量UVA诱导人皮肤成纤维细胞适应性反应中自噬的可能机制。3.探讨自噬在诱导培养人皮肤成纤维细胞适应性反应的可能作用。方法：1.取18岁健康男性阴茎包皮环切术后包皮(取自广州军区广州总医院泌尿外科门诊手术室)采用组织消化法培养人皮肤成纤维细胞。2.分组：假照射组：为正常的培养细胞,与照射组同样处理后置于照射装置下进行假照射；低剂量照射组：(1)低剂量照射A组：每天以单次剂量分别为7.2J/cm2、14.4 J/cm2、 21.6J/cm2、28.8J/cm2、36J/cm2照射进行照射；(2)低剂量照射B组：每天以剂量为7.2 J/cm2进行照射,累积剂量分别为7.2J/cm2、14.4J/cm2、21.6J/cm2、28.8 J/cm2、36 J/cm2。高剂量照射组：(1)高剂量照射A组：每天以单次剂量分别为7.2J/cm2、14.4J/cm2、21.6 J/cm2、28.8 J/cm2、36 J/cm2照射后,随后以86.4J/cm2 UVA照射细胞。(2)高剂量照射B组：每天为先以累积剂量分别为7.2 J/cm2、14.4 J/cm2、21.6 J/cm2、 28.8 J/cm2、36 J/cm2低剂量UVA预照射后,再以86.4J/cm2 UVA照射细胞。3.运用激光共聚焦扫描显微镜观察MDC染色后各组细胞内自噬形态及数量的变化,对比不同组细胞的自噬形态及数量的差异；利用流式细胞术检测各组自噬相关蛋白LC3、Beclinl自噬阳性表达百分比。4.统计学方法：用GraphPad Prism 5软件进行统计学分析,所有数据均采用均数±标准差(x±s)。方差齐性检验后,进行双因素方差分析,以P0.05为差异有统计学意义。结果：1.组织消化法培养的人皮肤成纤维细胞24h内贴壁,刚开始贴壁时细胞多数呈圆形,随贴壁时间延长逐渐伸展成梭形或多边形。5-6天进行传代,传代后约5天长满。取第3-10代人皮肤成纤维细胞进行实验研究。2.采用激光共聚焦显微镜观察,假照射组的细胞自噬体阳性表达率在1d、2d、3d、4d、5d时分别为4.333±0.577、4.333±1.528、5.000±1.155、7.000±1.000、5.333±1.155,假照射组组内的细胞自噬体阳性表达率无显著差异(P0.05)；低剂量照射A组的细胞自噬体阳性表达率在1d、2d、3d、4d、5d时分别为13.677±1.528、37.333±1.528、57.333±2.082、79.333±1.528、36.330±1.528,与假照射组相比较有显著差异(P0.01)；低剂量照射B组的细胞自噬体阳性表达率在1d、2d、3d、4d、5d时分别为13.677±1.284、32.000±2.000、46.677±1.528、57.000±2.000、30.000±1.000,与假照射组相比较有显著差异(P0.01)；低剂量照射A组和低剂量照射B组的细胞自噬体阳性表达率均随着照射剂量的增加而增加,但在28.8 J/cm2后细胞自噬体阳性表达率出现减少,低剂量照射A组细胞自噬体阳性表达率比低剂量照射B组高,低剂量照射A组与低剂量照射B组相比较有显著差异(P0.01)。高剂量照射A组的细胞自噬体阳性表达率在1d、2d、3d、4d、5d时分别为29.360±2.020、80.130±3.096、45.140±1.845、19.630±0.772、7.537±0.463,与假照射组相比较有显著差异(P0.01)；高剂量照射B组的细胞自噬体阳性表达率在1d、2d、3d、4d、5d时分别为29.360±2.020、35.850±1.513、56.100±1.365、76.000±1.579、31.440±1.662,与假照射组相比较有显著差异(P0.01)；高剂量照射A组在14.4 J/cm2接受86.4J/cm2照射后,细胞自噬体阳性表达率出现减少,高剂量照射B组细胞自噬体阳性表达率在28.8 J/cm2接受86.4J/cm2照射后出现减少,高剂量照射A组与高剂量照射B组相较有显著差异(P0.01)。3.与细胞自噬体阳性表达率检测结果相一致,低剂量照射A组和低剂量照射B组的Beclinl、LC3表达均随着照射剂量的增加而升高,但在28.8 J/cm2后Beclinl、LC3表达水平下降,低剂量照射A组Beclinl、LC3表达水平均比低剂量照射B组高,低剂量照射A组与低剂量照射B组相比较有显著差异(P0.01)；高剂量照射A组与高剂量照射B组相比,高剂量照射A组Beclinl、LC3表达很快达到高峰值然后迅速下调,而高剂量照射B组Beclinl、LC3表达仍能慢慢随着剂量的累积而增加高峰值才出现下调。高剂量照射A组与高剂量照射B组相比较有显著差异(P0.01)。结论：1.低剂量UVA照射诱导人皮肤成纤维细胞出现适应性反应同时可引起人皮肤成纤维细胞自噬的改变,其变化的规律与剂量密切相关,其调控与LC3、Beclinl表达有关。2.结果表明,自噬在低剂量UVA照射诱导人皮肤成纤维细胞出现适应性反应可能起重要作用。
[Abstract]:In recent years, the relationship between ultraviolet radiation and human skin health has been a research hotspot. Ultraviolet radiation is a "double-edged sword". On the one hand, ultraviolet radiation has been regarded as a killer of skin. In addition to possibly causing various photosensitive skin diseases, excessive ultraviolet radiation is also the most important factor causing skin photoaging and skin cancer in exposed areas. On the other hand, ultraviolet radiation also has beneficial biological effects. Appropriate ultraviolet irradiation can improve the immunity of the body, prevent some autoimmune diseases, promote bone growth and development, and reduce the incidence of tumors. Adaptive response is an important part of the excitatory effect of radiation. Previous studies have shown that low-dose long-wave ultraviolet radiation (UVA) can induce adaptive response of cultured skin cells. This adaptive response is manifested by single or multiple low-dose UVA pre-irradiation that can reduce the subsequent lethal dose of UVA radiation on cultured skin fibroblast fineness. Autophagy is a ubiquitous life phenomenon in eukaryotic cells, which transports deformed, senescent or damaged proteins and organelles to lysosomes. Autophagy is a metabolic process of intracavitary digestion and degradation. It is an effective way to maintain the homeostasis and renewal of eukaryotic cells. It is also an important defense mechanism for eukaryotic cells to adapt to environmental changes. In this paper, we observed the changes of autophagy in the adaptive response of human skin fibroblasts induced by low dose UVA, and discussed its possible role in inducing the adaptive response of cultured human skin fibroblasts. AIM: 1. To observe the morphological and quantitative changes of autophagy in the adaptive response of human skin fibroblasts induced by low-dose long-wave ultraviolet radiation, and to compare the morphological and quantitative differences of autophagy in different groups. 2. To explore the autophagy in the adaptive response of human skin fibroblasts induced by low-dose UVA by detecting autophagy-related protein LC3 and Beclinl. To explore the possible mechanism of phagocytosis. 3. To explore the possible role of autophagy in inducing adaptive response of cultured human skin fibroblasts. Methods: 1. Human skin fibroblasts were cultured by tissue digestion method after penile circumcision in 18-year-old healthy men. Group A was irradiated with a single dose of 7.2J/cm2, 14.4J/cm2, 21.6J/cm2, 28.8J/cm2, 36J/cm2, respectively. Group B was irradiated with a low dose of 7.2J/cm2 daily. The cumulative doses were 7.2J/cm2, 14.4J/cm2, 21.6J/cm2, 28.8J/cm2, 36 J/cm2. High dose group: (1) High dose group A: Single dose was 7.2J/cm2, 14.4J/cm2, 21.6J/cm2, 28.8J/cm2, 36 J/cm2, and then 86.4J/cm2 UVA was used to irradiate the cells. (2) High dose group B: The cumulative doses were 7.2J/cm2, 14.4J/cm2, 21.6J/cm2, 28.8J/cm2, 36 J/cm2, respectively. The amount of autophagy was 7.2 J/cm2,14.4 J/cm2,21.6 J/cm2,28.8 J/cm2,36 J/cm2 after low-dose UVA pre-irradiation, and then 86.4 J/cm2 UVA was used to irradiate the cells. Autophagy-related protein LC3 and Beclinl autophagy positive expression percentage were detected by operation. 4. Statistical method: GraphPad Prism 5 software was used for statistical analysis. All data were analyzed by means of mean (+) standard deviation (x (+) s). After homogeneity test of variance, two-way ANOVA was performed, and the difference was statistically significant with P 0.05. Results: 1. The cultured human skin fibroblasts adhered to the wall within 24 hours. Most of the cells were round when they first adhered to the wall. The cells gradually expanded into spindle or polygon with the prolongation of adherence time. After passage, they grew up about 5 days. The human skin fibroblasts of the 3rd to 10th generations were taken for experimental study. 2. The cells in the sham irradiation group were observed by laser confocal microscope. The positive rate of phage expression was 4.333+0.577,4.333+1.528,5.000+1.155,7.000+1.000,5.333+1.155 at day 1,2,3,4 and 5,respectively. There was no significant difference in the positive rate of autophagy expression in sham irradiation group (P 0.05); the positive rate of autophagy expression in low dose irradiation group A was 13.677+1.528,37.333+1.528 at day 1,4,5 and 5 respectively. The positive rates of autophagy in low dose irradiation group B were 13.677 [1.284], 32.000 [2.000], 46.677 [1.528], 57.000 [2.000] and 30.000 [1.000] at 1 day, 2 days, 3 days, 4 days and 5 days respectively (P 0.01). The positive expression rate of autophage in group A and group B increased with the increase of irradiation dose, but decreased after 28.8 J/cm 2. The positive expression rate of autophage in group A was higher than that in group B after low dose irradiation. The positive rates of autophagy in group A were 29.360 (+ 2.020), 80.130 (+ 3.096), 45.140 (+ 1.845), 19.630 (+ 0.772) and 7.537 (+ 0.463) on the 1st, 2nd, 3rd, 4th and 5th day, respectively, which were significantly different from those in sham irradiation group A (P 0.01). Compared with sham irradiation group, the positive expression rate of autophagy decreased in group A after 14.4 J/cm2 irradiation and 86.4 J/cm2 irradiation. The positive expression rate of autophagy in group B was 28.8 J/cm2 irradiation and 86.4 J/cm2 irradiation respectively. The expression of Beclinl and LC3 in low dose group A and low dose group B increased with the increase of irradiation dose, but the expression of Beclinl and LC3 decreased after 28.8 J/cm 2. The expression levels of Beclinl and LC3 in low dose group A were higher than those in low dose group B, and there was significant difference between low dose group A and low dose group B (P 0.01). Conclusion: 1. Low dose UVA irradiation can induce the adaptive response of human skin fibroblasts and induce the change of autophagy of human skin fibroblasts. The results suggest that autophagy may play an important role in the adaptive response of human skin fibroblasts induced by low dose UVA irradiation.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R758.1

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