微囊泡在介导急性中长波紫外线辐射“旁观者效应”中的作用

发布时间：2018-06-01 06:15

本文选题：紫外线 + 成纤维细胞　；参考：《南京医科大学》2016年硕士论文

【摘要】：研究背景及目的日光中紫外线所致的皮肤光损伤疾病一直是人们关注的一类热点疾病。研究表明,紫外线除了引起受辐射细胞的直接损伤外,还可以通过紫外线辐射旁效应(Ultraviolet irradiation induced bystander effects, UV-BE)引起临近细胞的间接损伤。近年来,有关紫外线辐射旁效应的研究,已经覆盖体内外多种光损伤模型。研究者发现不同波段的紫外线均可产生多种旁观者效应。此外,不同剂量紫外线也可诱导不同强度的旁观者效应。种种证据表明,紫外线辐射旁效应可能在加剧皮肤光损伤和光致癌等重要病理过程中发挥重要作用。既往旁效应的研究主要集中于离子辐射,尽管紫外线作为一种非离子射线,其诱发旁效应的证据已经建立得很充分,而这一机制发生的生化、分子机制研究目前却刚刚起步,这一方面的研究已经得到众多学者的关注。近年来,微囊泡(microvesicles, MVs)的功能和其携带的信息物质是生物学研究一大热点。研究报道受到离子辐射的细胞可向外分泌微囊泡,它是细胞出芽与细胞膜融合后直接脱落形成的囊性结构,并携带蛋白质、mRNA、miRNA和DNA,参与细胞间的信息传递,介导旁效应的产生。本研究的目的在于观察急性中长波紫外线辐射诱导皮肤成纤维细胞微囊泡的生成及其对人皮肤成纤维细胞形态,增殖活性,氧化损伤及凋亡的影响。方法1.中长波紫外线辐射诱导皮肤成纤维细胞微囊泡的生成及鉴定分别采用不同剂量中长波紫外线(Ultraviolet A/Ultraviolet B, UVA/UVB)辐射人皮肤成纤维细胞(human skin fibroblasts, HSF),建立急性光损伤细胞模型。本研究中采用的UVA辐射剂量为10J/cmm2和20J/cm2;UVB辐射剂量为30mJ/cm2和60mJ/cm2。用特别配置的无微囊泡(microvesicles, MVs)培养基孵育HSF,经上述剂量UVA和UVB辐射细胞后,于照射后0.5h、24h、48h、72h分离提取细胞上清液中的MVs。随后使用透射电镜及光散射分析技术检测所获得的MVs大小及含量,BCA蛋白定量法测定各组细胞上清中MVs蛋白的含量,蛋白质免疫印迹法(Western blotting, WB)检测MVs表面蛋白。2.急性中长波紫外线辐射诱导生成的微囊泡对人皮肤成纤维细胞形态、增殖活性、氧化损害及凋亡的影响采用UVA(20J/cm2)及UVB(60mJ/cm2)对HSF进行急性辐射,收集培养24h后上清液中的MVs。将上述收集的MVs用PKH26染料标记后与正常HSF共孵育,观察HSF对MVs的摄取情况；倒置显微镜下观察旁观者细胞形态,细胞计数CCK-8法及EDU染色法检测旁观者细胞增殖率。ROS检测试剂盒检测旁观者细胞的氧化损伤,使用线粒体膜电位检测试剂盒检测旁观者细胞的早期凋亡,膜联蛋白V(Annexin V)/碘化丙啶(PI)双染法流式细胞仪检测旁观者细胞的凋亡率。结果1.中长波紫外线辐射诱导人皮肤成纤维细胞微囊泡的生成及鉴定中长波紫外线辐射诱导生成的MVs明显高于正常成纤维细胞的释放量。透射电镜结果示MVs是一种膜性微囊结构,圆形或椭圆形,有完整的包膜,腔内为低电子密度成分：光散射分析技术提示紫外线辐射后诱导生成的MV8的直径多聚集在150-300nm,而正常HSF分泌的MVs直径多在120-200nm,且辐射后HSF分泌的MVs高于正常HSF所分泌的数量；BCA蛋白定量结果示,紫外线辐射后HSF上清液中MVs在辐射后24h-48h间有一个分泌高峰,而正常细胞在不同时间点分泌MVs的数量相对恒定；WB结果提示紫外线辐射组诱导生成的MVs,其表面蛋白Alix、tsg101及CD9表达阳性,显著表达Alix。2急性中长波紫外线辐射诱导生成的微囊泡对人皮肤成纤维细胞形态、增殖活性、氧化损害及凋亡的影响PKH26荧光标记的MVs在加入培养基30min后即可被旁观者HSF摄取,在共孵育的48h内,细胞核周边聚集的MVs逐渐增多。紫外线辐射诱导生成的MVs较正常HSF分泌的MVs被旁观者细胞摄取速度更快。旁观者细胞与紫外线辐射诱导生成的MVs共孵育后,倒置显微镜下观察出现较多的细胞碎片,细胞体积变大,过度伸展；CCK-8结果提示旁观者HSF增殖减缓；EDU染色法提示出现旁观者细胞周期阻滞,S期细胞增多；ROS结果提示,旁观者细胞活性氧荧光强度高于未辐射组；线粒体膜电位结果提示旁观者HSF表现为出现较多的细胞碎片,并出现较多绿色荧光,表明细胞出现早期凋亡；AV/PI双染法结果提示旁观者HSF表现出凋亡率升高；而细胞的氧化损害和凋亡率在加入抗氧化剂NAC后,上述旁观者效应能够被部分逆转。结论急性中长波紫外线辐射可诱导人皮肤成纤维细胞释放微囊泡,微囊泡能够被旁观者成纤维细胞所摄取,导致后者出现细胞形态变化,细胞增殖减缓,细胞周期阻滞,出现细胞氧化损伤和凋亡。
[Abstract]:The study of background and purpose of ultraviolet radiation caused by ultraviolet radiation in the sun has been a hot spot of concern. The study shows that the ultraviolet radiation can also cause the adjacent cells through the Ultraviolet irradiation induced bystander effects (UV-BE) in addition to direct damage to the irradiated cells. Indirect damage. In recent years, research on the effect of ultraviolet radiation has covered a variety of light damage models in the body and outside. Researchers have found that ultraviolet radiation in different bands can produce a variety of bystander effects. In addition, different doses of ultraviolet radiation can also induce the bystander effect of different intensities. Various evidence suggests that the effect of ultraviolet radiation on the ultraviolet radiation may be possible. It plays an important role in aggravating the important pathological processes such as skin light damage and light carcinogenesis. The previous study of side effects is mainly focused on ion radiation. Although ultraviolet radiation is a nonionic ray, the evidence of its induced side effects has been established sufficiently, and the molecular mechanism of this mechanism is now just starting, In recent years, the functions of microvesicles (MVs) and the information they carry are a hot spot in biological research. The purpose of this study is to observe the formation of microvesicles induced by acute ultraviolet radiation induced skin fibroblasts and their effects on the morphology, proliferation, oxidative damage and apoptosis of human skin fibroblasts. Method 1. long wave in 1. The formation and identification of microvesicles of skin fibroblasts induced by ultraviolet radiation, using Ultraviolet A/Ultraviolet B (UVA/UVB) in different doses, irradiated human skin fibroblasts (human skin fibroblasts, HSF) to establish an acute light damage cell model. The dose of UVA radiation used in this study is 10J/cmm2 and 20J/cm. 2; UVB radiation dose of 30mJ/cm2 and 60mJ/cm2. were incubated with a specially configured free microvesicle (microvesicles, MVs) medium for incubating HSF. After the above doses of UVA and UVB irradiated cells, 0.5h, 24h, 48h, and 72h separation and extraction of cell supernatants after irradiation of UVA and UVB cells were followed by transmission and light scattering analysis. The content of MVs protein in cell supernatant of each group was measured by BCA protein quantitative method. Protein immunoblotting (Western blotting, WB) was used to detect the effects of MVs surface protein.2. on the morphology, proliferation, oxidative damage and apoptosis of human skin fibroblasts induced by acute ultraviolet ultraviolet radiation (UVB), and UVA (20J/cm2) and UVB (60mJ/cm2) were used. The acute radiation of HSF was carried out, and the MVs. in the supernatant after 24h was collected and cultured. The above collected MVs was labeled with PKH26 dye and was incubated with the normal HSF. The uptake of MVs by HSF was observed. The bystander cell morphology was observed under the inverted microscope. The cell count CCK-8 method and EDU staining method were used to detect the.ROS detection kit for the proliferation rate of bystander cells. Oxidative damage of bystander cells, the early apoptosis of bystander cells was detected by the mitochondrial membrane potential detection kit. The apoptosis rate of bystander cells was detected by the membrane associated protein V (Annexin V) / PI double staining flow cytometry. Results the formation and identification of the microvesicles of human dermal fibroblasts induced by UV radiation in 1. The MVs generated by UV radiation was significantly higher than that of normal fibroblasts. The transmission electron microscopy showed that MVs was a membrane microcapsule structure, round or elliptical, with a complete envelope and low electron density in the cavity. The light scattering analysis technique suggested that the diameter of MV8 induced by ultraviolet radiation was more concentrated in 150-300. Nm, while normal HSF secreted more MVs in 120-200nm, and the MVs secreted after radiation was higher than the number of normal HSF secreted; BCA protein quantitative results showed that MVs in the HSF supernatant after ultraviolet radiation had a secretory peak in 24h-48h after radiation, while the number of normal cells secreting MVs at different time points was relatively constant; the results suggest that the number of normal cells at different time points is relatively constant. MVs induced by ultraviolet radiation group, its surface protein Alix, TSG101 and CD9 are positive, and the expression of microvesicles induced by ultraviolet radiation induced by Alix.2 in acute Alix.2 on human skin fibroblast morphology, proliferation activity, oxidative damage and apoptosis, PKH26 fluorescence labeled MVs can be observed by bystander H after adding medium 30min. In the SF uptake, in the co incubated 48h, the aggregation of MVs around the nucleus increased gradually. The MVs induced by ultraviolet radiation was faster than the normal HSF secreted by the bystander cells. After the bystander cells were reincubated with the MVs induced by ultraviolet radiation, more cell fragments were observed under the inverted microscope, and the cell volume became larger. CCK-8 results suggested that bystander HSF proliferation was slowed down, and EDU staining suggested that bystander cell cycle arrest and S cell increase; ROS results suggested that bystander cell activated oxygen fluorescence intensity was higher than that of unirradiated group; mitochondrial membrane potential results suggested that bystander HSF table appeared more cell fragments and appeared more. The green fluorescence showed that the cell appeared early apoptosis, and the AV/PI double staining showed that the bystander HSF showed an increase in the apoptosis rate, and the oxidative damage and apoptosis rate of the cells could be partially reversed after the addition of antioxidant NAC. Conclusion the acute ultraviolet radiation can induce the release of microcapsules from human skin fibroblasts. Vesicles, microvesicles, can be taken by bystander fibroblasts, resulting in changes in cell morphology, slow cell proliferation, cell cycle arrest, and cell oxidative damage and apoptosis.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R758.1

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