本文選題：UVA + UVB�。� 參考：《南方醫(yī)科大學(xué)》2015年碩士論文

【摘要】：背景來自太陽的紫外線輻射按其波長可分為315-400nm的紫外線A (ultraviolet A, UVA)、280～315nm的紫外線B (UVB)和100-280nm的紫外線C (UVC).太陽輻射通過地球大氣層時,其中所有的UVC和90%以上的UVB能被臭氧和水汽等吸收,UVA則較少受影響。因此到達(dá)地面的紫外線主要是UVA和少量UVB�，F(xiàn)階段,隨工業(yè)發(fā)展地球臭氧層空洞面積的不斷增加,導(dǎo)致大氣對太陽輻射特別是UVB的吸收減少,使地球上的人類受到的UVB輻射增加。人類皮膚覆蓋于機體的表面,是抵抗外界刺激的第一道防線,照射到皮膚的紫外線95%左右被角質(zhì)細(xì)胞所吸收。過量的UVB照射可引起皮膚出現(xiàn)紅斑、炎癥、老化甚至皮膚癌。以往研究表明,UVB可以通過以下主要四種途徑破壞細(xì)胞,影響細(xì)胞正常功能和生存狀況,導(dǎo)致細(xì)胞壞死或凋亡：1直接損傷細(xì)胞DNA：2激活神經(jīng)鞘磷脂酶,使之降解神經(jīng)鞘磷脂,增加“第二信使”神經(jīng)酞胺及其衍生物水平；3活化CD95等細(xì)胞膜表面死亡受體；4經(jīng)細(xì)胞膜和線粒體膜作用產(chǎn)生脂質(zhì)過氧化產(chǎn)物和自由基。無論是通過哪一種途徑,就純粹的分子之間的相互作用而言,紫外線作用于細(xì)胞是通過紫外光子和細(xì)胞的分子成分之間發(fā)生了能量轉(zhuǎn)移,能量轉(zhuǎn)移的結(jié)果根據(jù)相互作用的能量高低分為兩種：高于“電離能”時,直接破壞介質(zhì)的原子和分子；低于“電離能”時則在使介質(zhì)電子發(fā)生躍遷的同時被介質(zhì)吸收。紫外線對機體皮膚細(xì)胞的光生物學(xué)作用主要是皮膚細(xì)胞發(fā)色基團對紫外光譜能量的選擇吸收后,引起細(xì)胞內(nèi)分子的激發(fā),被激發(fā)的分子經(jīng)過光輻射、內(nèi)轉(zhuǎn)換、碰撞等非輻射衰變方式、化學(xué)反應(yīng)方式、能量傳遞方式等將能量轉(zhuǎn)換回到基態(tài)。三種不同波長的紫外線其能量差異較大,損傷DNA的方式以及結(jié)果也有較大區(qū)別：UVB和UVC波長范圍正好是在DNA的吸收峰附近,DNA就能夠直接吸收其能量,形成環(huán)丁烷嘧啶二聚體、嘧啶酮光產(chǎn)物、嘧啶的單加和物和非二聚堿基損傷、嘌呤光產(chǎn)物等形式的損傷；UVA不能被DNA直接吸收,而是通過細(xì)胞中的光增敏劑將能量傳遞給DNA引起損傷,損傷的類型有堿基損傷、DNA鏈交聯(lián)、DNA鏈斷裂等。拉曼光譜(Ramanspectra)是一種散射光譜：當(dāng)單色的入射光投射到物質(zhì)中產(chǎn)生散射時,其中有一部分散射光與入射光頻率不同,稱為拉曼光譜。這一現(xiàn)象于1928年由印度物理學(xué)家拉曼首先發(fā)現(xiàn)。拉曼光譜技術(shù)應(yīng)用于DNA大分子構(gòu)象研究始于20世紀(jì)70年代初期,根據(jù)拉曼譜線(峰)的特征和位置(拉曼頻移)來確定其所屬的DNA功能基團或某一個分子鍵,以及判斷哪些DNA功能基團和化學(xué)鍵發(fā)生改變而引起物質(zhì)結(jié)構(gòu)和功能上的損傷。本研究采用UVA和UVB照射HaCaT細(xì)胞后,提取細(xì)胞DNA進(jìn)行拉曼光譜分析,根據(jù)拉曼光譜相應(yīng)特征譜線的位置和強度變化情況研究UVA和UVB對HaCaT細(xì)胞DNA的損傷形式,進(jìn)一步分析損傷過程中通過哪一具體的分子結(jié)構(gòu)進(jìn)行能量交換和作用。白藜蘆醇是從百合科菝葜屬植物菝葜的干燥根莖中提取出來的一種天然的二苯乙烯類化合物,可以降低丙二醛(MDA)的生成量,增加超氧化物歧化酶(SOD)、谷胱甘肽過氧化物酶(GSH-px)及過氧化氫酶(CAT)的活性,清除自由基。白藜蘆醇本身也對紫外線有較好的吸收作用,且含有的多酚結(jié)構(gòu)能與DNA通過靜電吸引和氫鍵作用。本試驗通過拉曼光譜技術(shù)分析白藜蘆醇在UVB致HaCaT細(xì)胞DNA損傷過程中,對DNA二級結(jié)構(gòu)的保護作用。細(xì)胞自噬是廣泛存在于真核細(xì)胞內(nèi)的溶酶體依賴的降解途徑,細(xì)胞內(nèi)損傷的細(xì)胞器和蛋白質(zhì)等可通過自噬作用被降解,降解產(chǎn)生的氨基酸、脂肪酸等產(chǎn)物再重新利用。生長因子缺乏、大量氧自由基、DNA損傷等皆可誘導(dǎo)自噬。根據(jù)細(xì)胞內(nèi)的物質(zhì)運送到溶酶體的途徑,分為三種自噬途徑：1巨自噬,自噬最主要的形式,細(xì)胞質(zhì)中產(chǎn)生雙層膜結(jié)構(gòu)形成自噬小體,隨后結(jié)合溶酶體形成自噬性囊泡,通過這一途徑降解自噬內(nèi)容物主要有線粒體、內(nèi)質(zhì)網(wǎng)以及核糖體等；2微自噬,溶酶體膜直接內(nèi)陷包裹周圍的待降解物質(zhì),然后在水解酶的作用下進(jìn)行降解；3分子伴侶介導(dǎo)自噬,與巨自噬、微自噬的最大區(qū)別是沒有膜性結(jié)構(gòu)形成,而是細(xì)胞質(zhì)內(nèi)具有特殊基序的蛋白被分子伴侶識別后,與溶酶體膜上的特殊受體--溶酶體相關(guān)膜蛋白結(jié)合后進(jìn)入溶酶體被降解。自噬是細(xì)胞對于環(huán)境變化的有效、快速反應(yīng),當(dāng)細(xì)胞營養(yǎng)缺失時,細(xì)胞立即啟動自噬以維持胞質(zhì)中氨基酸池的平衡,可通過合成新的蛋白質(zhì)、能量生成和促進(jìn)糖異生來避免細(xì)胞“餓死”。小鼠胚胎成纖維細(xì)胞在饑餓誘導(dǎo)下,三十分鐘后既可以檢測出自噬特異性蛋白LC3-II的改變,而其他細(xì)胞的自噬水平的變化從開始到結(jié)束經(jīng)歷的時間各有不同,半衰期從8分鐘到幾小時。為研究HaCaT細(xì)胞受刺激后的自噬變化規(guī)律,本實驗選用人永生化上皮細(xì)胞(HaCaT)作為研究對象,以波長為305nm的UVB作為刺激,觀察照射后5h內(nèi)的自噬變化。目的1、研究UVA和UVB對HaCaT細(xì)胞DNA二級結(jié)構(gòu)的損傷情況。2、研究白藜蘆醇在UVB致HaCaT細(xì)胞DNA損傷過程中對DNA二級結(jié)構(gòu)的保護作用。3、研究UVB輻射對HaCaT細(xì)胞自噬影響的劑量反應(yīng)關(guān)系和時間效應(yīng)關(guān)系。方法1、細(xì)胞培養(yǎng)HaCaT細(xì)胞接種于60mm×15mm培養(yǎng)皿中,培養(yǎng)皿含體積分?jǐn)?shù)為10%的小牛血清(Fetal bovine serum, FBS),單位為1×105U/L青霉素、質(zhì)量濃度為100mg/L鏈霉素的DMEM高糖培養(yǎng)基,于37℃、體積分?jǐn)?shù)為5%的CO2培養(yǎng)箱中常規(guī)貼壁培養(yǎng)。2、紫外輻射上海顧村光電儀器廠生產(chǎn),并經(jīng)上海市計量局檢測,其發(fā)射的紫外線波峰為305nm。培養(yǎng)皿中細(xì)胞長至80%-90%融合時棄培養(yǎng)基,用1mlPBS漂洗2遍后加入1.8mlPBS覆蓋細(xì)胞,放在距光源垂直距離為40Cmm位置進(jìn)行照射。3、白藜蘆醇預(yù)處理HaCaT細(xì)胞于UVB照射前加入白藜蘆醇(使用終濃度0.1μpmol/ml)培養(yǎng)6小時,棄去含有白藜蘆醇的培養(yǎng)基,1mlPBS漂洗2遍后照射方法同前。4、細(xì)胞全基因組DNA提取82.6mJ/cm2UVA和29.7mJ/cm2UVB照射細(xì)胞后繼續(xù)培養(yǎng)至照射后0.5、1.0、2.0、3.0、4.0和5.0h時間點收獲HaCaT細(xì)胞,按照QIAamp DNA Mini Kit操作說明書步驟,分別對各組收獲的HaCaT細(xì)胞進(jìn)行全基因組DNA提取。5、激光共焦拉曼光譜測量采用激光共焦拉曼光譜倒置顯微鏡系統(tǒng)檢測各組HaCaT細(xì)胞全基因組DNA的拉曼光譜：測量前采用標(biāo)準(zhǔn)硅片對激光共焦拉曼光譜倒置顯微鏡系統(tǒng)的波數(shù)軸和激光功率進(jìn)行校準(zhǔn),以確保每次測量時照射到樣品處的激光功率相同。實驗時使用20倍物鏡,光柵采用6001ines/mm,共焦孔徑為500μm,采集的頻率范圍為600-2000cm-1,光譜分辨率為lcm-1,樣品掃描曝光積分時間為30s,重復(fù)10次。6、吖啶橙(Acridine orange, AO)染色不同劑量UVB照射HaCaT細(xì)胞后,分別在照后1.0、2.0、3.0、4.0、5.0h收集細(xì)胞進(jìn)行染色,具體方法是：棄去培養(yǎng)皿中培養(yǎng)基,用1mlPBS清洗2次,再加入3m1濃度為5μg/ml的AO染液,置于C02培養(yǎng)箱中繼續(xù)培養(yǎng)15min, PBS清洗3次后。熒光倒置顯微鏡下觀察染色。7、Western blot檢測細(xì)胞蛋白不同劑量的UVB照射HaCaT細(xì)胞后,分別在照后0.5、1.0、2.0、3.0、4.0、5.0h收集細(xì)胞,具體方法是：棄去培養(yǎng)皿中培養(yǎng)基,用上海博彩生物工程公司的試劑盒提取細(xì)胞總蛋白并定量后進(jìn)行SDS-PAGE電泳,轉(zhuǎn)膜、5%脫脂奶粉進(jìn)行封閉,然后室溫下一抗孵育2h、TBST漂洗3次,室溫二抗孵育2h、TBST漂洗3次后化學(xué)發(fā)光反應(yīng),最后進(jìn)行顯影、定影。8、統(tǒng)計學(xué)分析采用SPSS 19.0軟件進(jìn)行統(tǒng)計分析。計量資料經(jīng)正態(tài)性檢驗符合正態(tài)分布或近似正態(tài)分布,以x±s描述,多組均數(shù)比較采用單因素方差分析；多重比較,方差齊時采用LSD法,方差不齊時采用Dunnett-T3法,檢驗水準(zhǔn)a=0.05。結(jié)果1、經(jīng)UVA和UVB照射后DNA拉曼光譜的主要譜線輪廓未發(fā)生明顯變化；三組細(xì)胞鳥嘌呤氫鍵、磷酸基靜電斥力、腺嘌呤堿基分子內(nèi)能和腺嘌呤堿基堆積力特征譜線強度變化差異有統(tǒng)計學(xué)意義(P0.05)；多重比較結(jié)果中,僅UVA組與對照組鳥嘌呤氫鍵特征譜線強度變化差異不明顯(P0.05),其余各組各指標(biāo)兩兩相比差異均有統(tǒng)計學(xué)意義(P0.05)；各組與照后時間呈現(xiàn)交互效應(yīng)(P0.05)。2、經(jīng)UVB和UVB+白藜蘆醇照射后DNA拉曼光譜的主要譜線輪廓未發(fā)生明顯變化；三組鳥嘌呤氫鍵、磷酸基靜電斥力、腺嘌呤堿基分子內(nèi)能和腺嘌呤堿基堆積力特征譜線強度變化差異有統(tǒng)計學(xué)意義(P0.05)；多重比較結(jié)果中,三組細(xì)胞鳥嘌呤氫鍵特征頻率兩兩相比差異均有統(tǒng)計學(xué)差異(P0.01),三組細(xì)胞磷酸基靜電斥力特征頻率兩兩相比僅UVB組和對照組差異具有統(tǒng)計學(xué)意義(P0.05),三組細(xì)胞腺嘌呤堿基分子內(nèi)能特征頻率兩兩相比僅UVB組和UVB+白藜蘆醇差異無統(tǒng)計學(xué)意義(P0.05),三組細(xì)胞腺腺嘌呤堿基堆積力特征頻率兩兩相比僅對照組和UVB+白藜蘆醇差異無統(tǒng)計學(xué)意義(P0.05)；各組與照后時間呈現(xiàn)交互效應(yīng)(P0.05)。3、HaCaT細(xì)胞經(jīng)49.5mJ/cm2劑量UVB照射后,繼續(xù)培養(yǎng)1.0h.2.0h、3.0h、 4.0h、5.0h后觀察自噬,結(jié)果顯示自噬細(xì)胞比例從照后1.0h的20.15±2.10%先是增長到最高50.67士7.33%,然后下降直至照后5.0h還保持在21.39士3.75%,自噬細(xì)胞比例峰值出現(xiàn)在照后3.0h(P0.05)。HaCaT細(xì)胞經(jīng)0mJ/cm2(對照)、9.9mJ/cm2、29.7mJ/cm2、49.5mJ/cm2劑量UVB照射后,繼續(xù)培養(yǎng)3.0h后觀察自噬,結(jié)果顯示HaCaT細(xì)胞自噬比例隨UVB照射劑量增加而上升,從4.13士1.02%增長到65.78±6.14%(P0.05)。結(jié)論1、UVA和UVB可通過作用于鳥嘌呤氫鍵、磷酸基靜電斥力、腺嘌呤堿基分子內(nèi)能和腺嘌呤堿基堆積力等分子結(jié)構(gòu)損傷HaCaT細(xì)胞DNA, UVB的損傷作用更大。2、白藜蘆醇可通過保護HaCaT細(xì)胞DNA鳥嘌呤氫鍵、磷酸基靜電斥力和腺嘌呤堿基堆積力等分子結(jié)構(gòu)而減少UVB所致?lián)p傷。3. HaCaT細(xì)胞經(jīng)49.5mJ/cm2劑量UVB照射并培養(yǎng)至1-5h,自噬細(xì)胞比例先上升然后下降,峰值出現(xiàn)在照后3h左右。HaCaT細(xì)胞經(jīng)9.9～49.5mJ/cm2劑量UVB照射并培養(yǎng)至3.0h,自噬細(xì)胞比例隨照射劑量增加而上升。
[Abstract]:The ultraviolet radiation from the sun can be divided into 315-400nm A (ultraviolet A, UVA), 280 to 315nm ultraviolet B (UVB) and 100-280nm ultraviolet C (UVC). When solar radiation passes through the earth's atmosphere, all UVC and more than 90% can be absorbed by ozone and water vapor. The ultraviolet rays reaching the ground are mainly UVA and a small amount of UVB. at the present stage. As the industrial development of the hole area of the earth's ozone layer increases, the absorption of the atmosphere to the solar radiation, especially the UVB, is reduced, and the human UVB radiation on the earth is increased. Human skin is covered by the surface of the body and is the first line of defense against external stimuli. More than 95% of the ultraviolet rays of the skin are absorbed by the keratinocytes. Excessive UVB exposure can cause erythema, inflammation, aging and even skin cancer in the skin. Previous studies have shown that UVB can destroy cells through the following main four ways, affect normal function and survival of the cells, lead to cell necrosis or apoptosis: 1 directly damage the cell DNA:2 Activation of neurileinenzyme to degrade neurileinomyelin, increase the level of "second messenger" neurophthalamidin and its derivatives; 3 activate the death receptors on the surface of cell membrane such as CD95, and produce lipid peroxidation products and free radicals through the action of the membrane and mitochondrial membrane. No matter which pathway, the intermolecular interaction is pure. In terms of use, ultraviolet radiation acts on cells through the transfer of energy between ultraviolet light and molecular components of the cell. The result of energy transfer is divided into two types according to the energy of the interaction: higher than the "ionization energy", which directly destroys the atoms and molecules of the medium, and the transition occurs when the ionization energy is lower than that of the ionization energy. The photo biological effect of ultraviolet radiation on the skin cells of the body is mainly caused by the selection and absorption of the ultraviolet spectral energy by the chromophore groups of the skin cells, and the excitation of the molecules in the cells. The excited molecules pass through the light radiation, internal conversion, collision and other non radiation failure modes, chemical reactions, and energy transfer modes. The energy conversion goes back to the ground state. The energy difference between the three different wavelengths is different. The way and the result of the damage to the DNA are different: the wavelength range of UVB and UVC is just near the absorption peak of DNA, and DNA can absorb its energy directly, forming the CyA pyrimidine, the pyrimidine light products, the single addition and the non two of pyrimidine. The damage of POLYBASE damage, purine light products, and so on; UVA can not be absorbed directly by DNA, but through the light sensitizer in the cell to transfer energy to DNA, and the types of damage are base damage, DNA chain crosslinking, DNA chain fracture, and so on. The Raman spectrum (Ramanspectra) is a kind of scattering spectrum, when the monochromatic incident light is projected into the substance. When the scattering is produced, there is a difference between the scattered light and the incident light frequency, called the Raman spectrum. This phenomenon was first discovered by the India physicist Raman in 1928. The application of Raman spectroscopy to the study of the conformation of DNA macromolecules began in the early 1970s, and is determined by the characteristics and positions of the Raman spectrum (peak) (Raman frequency shift). The DNA functional group or a molecular bond of the genus, and what DNA functional groups and chemical bonds change to cause material structural and functional damage. In this study, HaCaT cells were irradiated by UVA and UVB to extract the cell DNA for Raman spectrum analysis, according to the position and intensity changes of the characteristic spectral lines of the Raman spectra. Investigate the damage form of UVA and UVB to HaCaT cell DNA, and further analyze the specific molecular structure of energy exchange and action in the process of damage. Resveratrol is a natural two styrene compound extracted from the dry rhizome of Smilax China L., which can reduce the production of malondialdehyde (MDA). With the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT), free radicals are scavenged. Resveratrol itself also has a good absorption effect on ultraviolet light, and the polyphenol structure containing DNA can be used by electrostatic attraction and hydrogen bond. The experiment was conducted by Raman spectroscopy to analyze the results of resveratrol in UVB. In the process of HaCaT cell DNA damage, the protective effect of the DNA two grade structure. Cell autophagy is a lysosome dependent degradation pathway that widely exists in eukaryotic cells. The intracellular organelles and proteins damaged by the cells can be degraded by autophagy, the degraded amino acids, fatty acids and other products are reused. The oxygen free radicals, DNA damage and so on can induce autophagy. According to the pathway of the intracellular material to the lysosome, it is divided into three autophagy pathways: 1 gigantic autophagy, the most important form of autophagy, the formation of autophagic body in the cytoplasm and the formation of autophagic vesicles with the lysosome, and the degradation of autophagic contents by this pathway. There are mitochondria, endoplasmic reticulum, and ribosomes; 2 micro autophagy, the lysosome membrane is directly entrapped in the surrounding parcels, and then degradates under the action of hydrolase; the 3 molecular chaperone mediates autophagy, with giant autophagy, and the biggest difference between the micro autophagy is the non membranous structure, but the protein with special order of the cytoplasm in the cytoplasm. After the subchaperone is identified, the lysosome related membrane protein, a special receptor on the lysosome membrane, is degraded into the lysosome. Autophagy is an effective and rapid response to environmental changes. When the cell nutrition is missing, the cell immediately starts autophagy to maintain the balance of the amino acid pool in the cytoplasm, and can generate energy by synthesizing new proteins. The mouse embryonic fibroblasts can not only detect the change of the autophagic protein LC3-II after thirty minutes, but the changes in the autophagy level of other cells vary from the beginning to the end, from 8 minutes to a few hours, for the study of HaCaT fines. The regularity of autophagy changes after the stimulation, the experiment selected human immortalized epithelial cells (HaCaT) as the research object, with the wavelength of 305nm UVB as the stimulation, observe the autophagy changes in 5h after irradiation. Objective 1, to study the damage of UVA and UVB on the DNA two structure of HaCaT cells, and to study the process of HaCaT cell DNA damage by UVB. The protective effect of.3 on the DNA two level structure was used to study the dose response relationship and time effect relationship of UVB radiation on the autophagy of HaCaT cells. Method 1, cell culture HaCaT cells were inoculated in 60mm x 15mm culture dish, and the culture dish contains calf serum (Fetal bovine serum, FBS) with volume fraction of 10% (Fetal bovine serum, FBS), and the mass concentration is 1 * 105U/L penicillin. DMEM high sugar medium for 100mg/L streptomycin, at 37 C and 5% in CO2 culture box, was routinely cultured.2, ultraviolet radiation was produced in Shanghai Gu Cun optoelectronic instrument factory and tested by Shanghai metrology Bureau. The ultraviolet wave peak was abandoned in 305nm. culture dish when the cell was long to 80%-90% fusion, and after 2 times rinsed with 1mlPBS 1.8mlPBS covered cells and irradiated.3 from the vertical distance from the light source to 40Cmm. The resveratrol pretreated HaCaT cells added resveratrol (with the final concentration of 0.1 u pmol/ml) for 6 hours before UVB irradiation, and abandoned the culture medium containing resveratrol, 1mlPBS rinse 2 times after 2 times the same.4, and the whole genome DNA extracted 82.6mJ/. HaCaT cells were harvested after irradiated by cm2UVA and 29.7mJ/cm2UVB, and HaCaT cells were harvested at the time point of 0.5,1.0,2.0,3.0,4.0 and 5.0h after irradiation. According to the QIAamp DNA Mini Kit operation instructions, the whole genome DNA.5 was carried out on the HaCaT cells harvested in each group, and laser confocal Raman spectroscopy was used to reverse laser confocal Raman spectroscopy. The Raman spectrum of the whole genome DNA of HaCaT cells in each group is detected by a mirror system. Before measurement, the standard silicon chip is used to calibrate the wave number axis and laser power of the laser confocal Raman spectroscopy inverted microscope system to ensure that the laser power of the sample is the same at each measurement. The 20 times objective lens is used in the experiment, and the grating is used in 6001ines/mm. The focal aperture is 500 mu m, the frequency range is 600-2000cm-1, the spectral resolution is LCM-1, the scanning exposure integration time is 30s, the 10 times.6 is repeated, the acridine orange (Acridine orange, AO) is stained with the HaCaT cells with different doses of UVB, and the cells are dyed 1.0,2.0,3.0,4.0,5.0h after the illumination. The specific method is to discard the culture dish. The culture medium was cleaned 2 times with 1mlPBS, then the AO dye with the concentration of 5 g/ml was added to the AO dye solution, and the 15min was continued in the C02 culture box. After the PBS cleaning for 3 times, the staining.7 was observed under the fluorescence inverted microscope. The Western blot was used to detect the UVB irradiated HaCaT cells at different doses of the cell protein. The specific methods were collected after the illumination. It is: discard the culture medium in the culture dish, extract the total cell protein with the reagent box of Shanghai gambling bio engineering company, and then carry out SDS-PAGE electrophoresis, transfer film and 5% skimmed milk powder to close it, and then incubate 2h, TBST rinse 3 times at room temperature, two anti incubating 2H at room temperature, 3 times after TBST rinsing, and finally develop and shadow.8, Statistical analysis used SPSS 19 software for statistical analysis. The measurement data were tested in normal distribution or approximate normal distribution through normal test, which was described by X + s, and the multiple groups were compared with single factor analysis of variance; multiple comparison, LSD method was used for the homogeneous variance, Dunnett-T3 method was adopted when the variance was uneven, and the result of a=0.05. was tested by UVA and UVB. There was no obvious change in the main line profile of DNA Raman spectrum after irradiation. The three groups of guanine hydrogen bond, phosphoric acid based electrostatic repulsion, adenine base molecular internal energy and adenine base accumulation power spectrum intensity variation have statistical significance (P0.05); in multiple comparison fruit, only UVA group and control group of guanine hydrogen bond characteristic spectrum There was no significant difference in line intensity variation (P0.05), and the difference of the other indexes 22 was statistically significant (P0.05); each group had an interaction effect (P0.05).2, and the main spectral lines of the DNA Raman spectrum after UVB and UVB+ resveratrol did not change clearly; the three group of guanine hydrogen bonds, phosphoric acid static repulsion, and glands were found. There was a significant difference in the intensity variation of the intrinsic and adenine base accumulation force of the purine base molecules and adenine bases (P0.05). In the multiple comparison results, there were significant differences in the frequency of the three groups of guanine hydrogen bond characteristics (P0.01), and the frequency of the phosphoric acid based static repulsion of the three groups was 22 compared to that of the UVB group and the control group. The difference was statistically significant (P0.05). There was no statistical difference between the three groups of adenine base molecular internal energy characteristics (22) and UVB+ resveratrol (P0.05). There was no significant difference between the three groups of adenine base accumulation frequency 22 (P0.05) compared with the control group and UVB+ resveratrol (P0.05). Time presented interaction effect (P0.05).3. After 49.5mJ/cm2 dose UVB irradiation, HaCaT cells continued to cultivate 1.0h.2.0h, 3.0h, 4.0h, and 5.0h to observe autophagy. The results showed that the proportion of autophagic cells increased from 20.15 + 2.10% to the maximum of 50.67, 7.33%, and then decreased to 3.75%, and the proportion of autophagic cells was at 21.39. After irradiation, 3.0h (P0.05).HaCaT cells were exposed to 0mJ/cm2 (control), 9.9mJ/cm2,29.7mJ/cm2,49.5mJ/cm2 dose UVB irradiated, and continued to cultivate 3.0h to observe autophagy. The results showed that the percentage of autophagy increased with the increase of UVB irradiation dose, from 4.13 to 65.78 + 6.14% (P0.05). Conclusion 1, UVA and UVB can act on guanine. The hydrogen bond, the electrostatic repulsion of phosphoric acid, the internal energy of adenine base molecules and the accumulation of adenine base damage the HaCaT cells DNA, and the damage of UVB is more.2. The resveratrol can reduce the UVB induced.3. HaCa by protecting the DNA guanine hydrogen bond of HaCaT cells, the phosphoric base electrostatic repulsion and the adenine base accumulation force. T cells were irradiated with 49.5mJ/cm2 dose UVB and cultured to 1-5h. The percentage of autophagic cells increased first and then decreased. The peak value appeared at about 3h after.HaCaT.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R594.8

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