本文選題：紫外線 + 成纖維細(xì)胞��； 參考：《南京醫(yī)科大學(xué)》2016年碩士論文

【摘要】：研究背景及目的日光中紫外線所致的皮膚光損傷疾病一直是人們關(guān)注的一類熱點疾病。研究表明,紫外線除了引起受輻射細(xì)胞的直接損傷外,還可以通過紫外線輻射旁效應(yīng)(Ultraviolet irradiation induced bystander effects, UV-BE)引起臨近細(xì)胞的間接損傷。近年來,有關(guān)紫外線輻射旁效應(yīng)的研究,已經(jīng)覆蓋體內(nèi)外多種光損傷模型。研究者發(fā)現(xiàn)不同波段的紫外線均可產(chǎn)生多種旁觀者效應(yīng)。此外,不同劑量紫外線也可誘導(dǎo)不同強度的旁觀者效應(yīng)。種種證據(jù)表明,紫外線輻射旁效應(yīng)可能在加劇皮膚光損傷和光致癌等重要病理過程中發(fā)揮重要作用。既往旁效應(yīng)的研究主要集中于離子輻射,盡管紫外線作為一種非離子射線,其誘發(fā)旁效應(yīng)的證據(jù)已經(jīng)建立得很充分,而這一機制發(fā)生的生化、分子機制研究目前卻剛剛起步,這一方面的研究已經(jīng)得到眾多學(xué)者的關(guān)注。近年來,微囊泡(microvesicles, MVs)的功能和其攜帶的信息物質(zhì)是生物學(xué)研究一大熱點。研究報道受到離子輻射的細(xì)胞可向外分泌微囊泡,它是細(xì)胞出芽與細(xì)胞膜融合后直接脫落形成的囊性結(jié)構(gòu),并攜帶蛋白質(zhì)、mRNA、miRNA和DNA,參與細(xì)胞間的信息傳遞,介導(dǎo)旁效應(yīng)的產(chǎn)生。本研究的目的在于觀察急性中長波紫外線輻射誘導(dǎo)皮膚成纖維細(xì)胞微囊泡的生成及其對人皮膚成纖維細(xì)胞形態(tài),增殖活性,氧化損傷及凋亡的影響。方法1.中長波紫外線輻射誘導(dǎo)皮膚成纖維細(xì)胞微囊泡的生成及鑒定分別采用不同劑量中長波紫外線(Ultraviolet A/Ultraviolet B, UVA/UVB)輻射人皮膚成纖維細(xì)胞(human skin fibroblasts, HSF),建立急性光損傷細(xì)胞模型。本研究中采用的UVA輻射劑量為10J/cmm2和20J/cm2;UVB輻射劑量為30mJ/cm2和60mJ/cm2。用特別配置的無微囊泡(microvesicles, MVs)培養(yǎng)基孵育HSF,經(jīng)上述劑量UVA和UVB輻射細(xì)胞后,于照射后0.5h、24h、48h、72h分離提取細(xì)胞上清液中的MVs。隨后使用透射電鏡及光散射分析技術(shù)檢測所獲得的MVs大小及含量,BCA蛋白定量法測定各組細(xì)胞上清中MVs蛋白的含量,蛋白質(zhì)免疫印跡法(Western blotting, WB)檢測MVs表面蛋白。2.急性中長波紫外線輻射誘導(dǎo)生成的微囊泡對人皮膚成纖維細(xì)胞形態(tài)、增殖活性、氧化損害及凋亡的影響采用UVA(20J/cm2)及UVB(60mJ/cm2)對HSF進(jìn)行急性輻射,收集培養(yǎng)24h后上清液中的MVs。將上述收集的MVs用PKH26染料標(biāo)記后與正常HSF共孵育,觀察HSF對MVs的攝取情況；倒置顯微鏡下觀察旁觀者細(xì)胞形態(tài),細(xì)胞計數(shù)CCK-8法及EDU染色法檢測旁觀者細(xì)胞增殖率。ROS檢測試劑盒檢測旁觀者細(xì)胞的氧化損傷,使用線粒體膜電位檢測試劑盒檢測旁觀者細(xì)胞的早期凋亡,膜聯(lián)蛋白V(Annexin V)/碘化丙啶(PI)雙染法流式細(xì)胞儀檢測旁觀者細(xì)胞的凋亡率。結(jié)果1.中長波紫外線輻射誘導(dǎo)人皮膚成纖維細(xì)胞微囊泡的生成及鑒定中長波紫外線輻射誘導(dǎo)生成的MVs明顯高于正常成纖維細(xì)胞的釋放量。透射電鏡結(jié)果示MVs是一種膜性微囊結(jié)構(gòu),圓形或橢圓形,有完整的包膜,腔內(nèi)為低電子密度成分：光散射分析技術(shù)提示紫外線輻射后誘導(dǎo)生成的MV8的直徑多聚集在150-300nm,而正常HSF分泌的MVs直徑多在120-200nm,且輻射后HSF分泌的MVs高于正常HSF所分泌的數(shù)量；BCA蛋白定量結(jié)果示,紫外線輻射后HSF上清液中MVs在輻射后24h-48h間有一個分泌高峰,而正常細(xì)胞在不同時間點分泌MVs的數(shù)量相對恒定；WB結(jié)果提示紫外線輻射組誘導(dǎo)生成的MVs,其表面蛋白Alix、tsg101及CD9表達(dá)陽性,顯著表達(dá)Alix。2急性中長波紫外線輻射誘導(dǎo)生成的微囊泡對人皮膚成纖維細(xì)胞形態(tài)、增殖活性、氧化損害及凋亡的影響PKH26熒光標(biāo)記的MVs在加入培養(yǎng)基30min后即可被旁觀者HSF攝取,在共孵育的48h內(nèi),細(xì)胞核周邊聚集的MVs逐漸增多。紫外線輻射誘導(dǎo)生成的MVs較正常HSF分泌的MVs被旁觀者細(xì)胞攝取速度更快。旁觀者細(xì)胞與紫外線輻射誘導(dǎo)生成的MVs共孵育后,倒置顯微鏡下觀察出現(xiàn)較多的細(xì)胞碎片,細(xì)胞體積變大,過度伸展；CCK-8結(jié)果提示旁觀者HSF增殖減緩；EDU染色法提示出現(xiàn)旁觀者細(xì)胞周期阻滯,S期細(xì)胞增多；ROS結(jié)果提示,旁觀者細(xì)胞活性氧熒光強度高于未輻射組；線粒體膜電位結(jié)果提示旁觀者HSF表現(xiàn)為出現(xiàn)較多的細(xì)胞碎片,并出現(xiàn)較多綠色熒光,表明細(xì)胞出現(xiàn)早期凋亡；AV/PI雙染法結(jié)果提示旁觀者HSF表現(xiàn)出凋亡率升高；而細(xì)胞的氧化損害和凋亡率在加入抗氧化劑NAC后,上述旁觀者效應(yīng)能夠被部分逆轉(zhuǎn)。結(jié)論急性中長波紫外線輻射可誘導(dǎo)人皮膚成纖維細(xì)胞釋放微囊泡,微囊泡能夠被旁觀者成纖維細(xì)胞所攝取,導(dǎo)致后者出現(xiàn)細(xì)胞形態(tài)變化,細(xì)胞增殖減緩,細(xì)胞周期阻滯,出現(xiàn)細(xì)胞氧化損傷和凋亡。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R758.1

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