紫外线诱导HaCaT细胞自噬及其拉曼光谱特性变化研究

发布时间：2018-05-06 09:11

本文选题：UVA + UVB　；参考：《南方医科大学》2015年硕士论文

【摘要】：背景来自太阳的紫外线辐射按其波长可分为315-400nm的紫外线A (ultraviolet A, UVA)、280～315nm的紫外线B (UVB)和100-280nm的紫外线C (UVC).太阳辐射通过地球大气层时,其中所有的UVC和90%以上的UVB能被臭氧和水汽等吸收,UVA则较少受影响。因此到达地面的紫外线主要是UVA和少量UVB。现阶段,随工业发展地球臭氧层空洞面积的不断增加,导致大气对太阳辐射特别是UVB的吸收减少,使地球上的人类受到的UVB辐射增加。人类皮肤覆盖于机体的表面,是抵抗外界刺激的第一道防线,照射到皮肤的紫外线95%左右被角质细胞所吸收。过量的UVB照射可引起皮肤出现红斑、炎症、老化甚至皮肤癌。以往研究表明,UVB可以通过以下主要四种途径破坏细胞,影响细胞正常功能和生存状况,导致细胞坏死或凋亡：1直接损伤细胞DNA：2激活神经鞘磷脂酶,使之降解神经鞘磷脂,增加“第二信使”神经酞胺及其衍生物水平；3活化CD95等细胞膜表面死亡受体；4经细胞膜和线粒体膜作用产生脂质过氧化产物和自由基。无论是通过哪一种途径,就纯粹的分子之间的相互作用而言,紫外线作用于细胞是通过紫外光子和细胞的分子成分之间发生了能量转移,能量转移的结果根据相互作用的能量高低分为两种：高于“电离能”时,直接破坏介质的原子和分子；低于“电离能”时则在使介质电子发生跃迁的同时被介质吸收。紫外线对机体皮肤细胞的光生物学作用主要是皮肤细胞发色基团对紫外光谱能量的选择吸收后,引起细胞内分子的激发,被激发的分子经过光辐射、内转换、碰撞等非辐射衰变方式、化学反应方式、能量传递方式等将能量转换回到基态。三种不同波长的紫外线其能量差异较大,损伤DNA的方式以及结果也有较大区别：UVB和UVC波长范围正好是在DNA的吸收峰附近,DNA就能够直接吸收其能量,形成环丁烷嘧啶二聚体、嘧啶酮光产物、嘧啶的单加和物和非二聚碱基损伤、嘌呤光产物等形式的损伤；UVA不能被DNA直接吸收,而是通过细胞中的光增敏剂将能量传递给DNA引起损伤,损伤的类型有碱基损伤、DNA链交联、DNA链断裂等。拉曼光谱(Ramanspectra)是一种散射光谱：当单色的入射光投射到物质中产生散射时,其中有一部分散射光与入射光频率不同,称为拉曼光谱。这一现象于1928年由印度物理学家拉曼首先发现。拉曼光谱技术应用于DNA大分子构象研究始于20世纪70年代初期,根据拉曼谱线(峰)的特征和位置(拉曼频移)来确定其所属的DNA功能基团或某一个分子键,以及判断哪些DNA功能基团和化学键发生改变而引起物质结构和功能上的损伤。本研究采用UVA和UVB照射HaCaT细胞后,提取细胞DNA进行拉曼光谱分析,根据拉曼光谱相应特征谱线的位置和强度变化情况研究UVA和UVB对HaCaT细胞DNA的损伤形式,进一步分析损伤过程中通过哪一具体的分子结构进行能量交换和作用。白藜芦醇是从百合科菝葜属植物菝葜的干燥根茎中提取出来的一种天然的二苯乙烯类化合物,可以降低丙二醛(MDA)的生成量,增加超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)及过氧化氢酶(CAT)的活性,清除自由基。白藜芦醇本身也对紫外线有较好的吸收作用,且含有的多酚结构能与DNA通过静电吸引和氢键作用。本试验通过拉曼光谱技术分析白藜芦醇在UVB致HaCaT细胞DNA损伤过程中,对DNA二级结构的保护作用。细胞自噬是广泛存在于真核细胞内的溶酶体依赖的降解途径,细胞内损伤的细胞器和蛋白质等可通过自噬作用被降解,降解产生的氨基酸、脂肪酸等产物再重新利用。生长因子缺乏、大量氧自由基、DNA损伤等皆可诱导自噬。根据细胞内的物质运送到溶酶体的途径,分为三种自噬途径：1巨自噬,自噬最主要的形式,细胞质中产生双层膜结构形成自噬小体,随后结合溶酶体形成自噬性囊泡,通过这一途径降解自噬内容物主要有线粒体、内质网以及核糖体等；2微自噬,溶酶体膜直接内陷包裹周围的待降解物质,然后在水解酶的作用下进行降解；3分子伴侣介导自噬,与巨自噬、微自噬的最大区别是没有膜性结构形成,而是细胞质内具有特殊基序的蛋白被分子伴侣识别后,与溶酶体膜上的特殊受体--溶酶体相关膜蛋白结合后进入溶酶体被降解。自噬是细胞对于环境变化的有效、快速反应,当细胞营养缺失时,细胞立即启动自噬以维持胞质中氨基酸池的平衡,可通过合成新的蛋白质、能量生成和促进糖异生来避免细胞“饿死”。小鼠胚胎成纤维细胞在饥饿诱导下,三十分钟后既可以检测出自噬特异性蛋白LC3-II的改变,而其他细胞的自噬水平的变化从开始到结束经历的时间各有不同,半衰期从8分钟到几小时。为研究HaCaT细胞受刺激后的自噬变化规律,本实验选用人永生化上皮细胞(HaCaT)作为研究对象,以波长为305nm的UVB作为刺激,观察照射后5h内的自噬变化。目的1、研究UVA和UVB对HaCaT细胞DNA二级结构的损伤情况。2、研究白藜芦醇在UVB致HaCaT细胞DNA损伤过程中对DNA二级结构的保护作用。3、研究UVB辐射对HaCaT细胞自噬影响的剂量反应关系和时间效应关系。方法1、细胞培养HaCaT细胞接种于60mm×15mm培养皿中,培养皿含体积分数为10%的小牛血清(Fetal bovine serum, FBS),单位为1×105U/L青霉素、质量浓度为100mg/L链霉素的DMEM高糖培养基,于37℃、体积分数为5%的CO2培养箱中常规贴壁培养。2、紫外辐射上海顾村光电仪器厂生产,并经上海市计量局检测,其发射的紫外线波峰为305nm。培养皿中细胞长至80%-90%融合时弃培养基,用1mlPBS漂洗2遍后加入1.8mlPBS覆盖细胞,放在距光源垂直距离为40Cmm位置进行照射。3、白藜芦醇预处理HaCaT细胞于UVB照射前加入白藜芦醇(使用终浓度0.1μpmol/ml)培养6小时,弃去含有白藜芦醇的培养基,1mlPBS漂洗2遍后照射方法同前。4、细胞全基因组DNA提取82.6mJ/cm2UVA和29.7mJ/cm2UVB照射细胞后继续培养至照射后0.5、1.0、2.0、3.0、4.0和5.0h时间点收获HaCaT细胞,按照QIAamp DNA Mini Kit操作说明书步骤,分别对各组收获的HaCaT细胞进行全基因组DNA提取。5、激光共焦拉曼光谱测量采用激光共焦拉曼光谱倒置显微镜系统检测各组HaCaT细胞全基因组DNA的拉曼光谱：测量前采用标准硅片对激光共焦拉曼光谱倒置显微镜系统的波数轴和激光功率进行校准,以确保每次测量时照射到样品处的激光功率相同。实验时使用20倍物镜,光栅采用6001ines/mm,共焦孔径为500μm,采集的频率范围为600-2000cm-1,光谱分辨率为lcm-1,样品扫描曝光积分时间为30s,重复10次。6、吖啶橙(Acridine orange, AO)染色不同剂量UVB照射HaCaT细胞后,分别在照后1.0、2.0、3.0、4.0、5.0h收集细胞进行染色,具体方法是：弃去培养皿中培养基,用1mlPBS清洗2次,再加入3m1浓度为5μg/ml的AO染液,置于C02培养箱中继续培养15min, PBS清洗3次后。荧光倒置显微镜下观察染色。7、Western blot检测细胞蛋白不同剂量的UVB照射HaCaT细胞后,分别在照后0.5、1.0、2.0、3.0、4.0、5.0h收集细胞,具体方法是：弃去培养皿中培养基,用上海博彩生物工程公司的试剂盒提取细胞总蛋白并定量后进行SDS-PAGE电泳,转膜、5%脱脂奶粉进行封闭,然后室温下一抗孵育2h、TBST漂洗3次,室温二抗孵育2h、TBST漂洗3次后化学发光反应,最后进行显影、定影。8、统计学分析采用SPSS 19.0软件进行统计分析。计量资料经正态性检验符合正态分布或近似正态分布,以x±s描述,多组均数比较采用单因素方差分析；多重比较,方差齐时采用LSD法,方差不齐时采用Dunnett-T3法,检验水准a=0.05。结果1、经UVA和UVB照射后DNA拉曼光谱的主要谱线轮廓未发生明显变化；三组细胞鸟嘌呤氢键、磷酸基静电斥力、腺嘌呤碱基分子内能和腺嘌呤碱基堆积力特征谱线强度变化差异有统计学意义(P0.05)；多重比较结果中,仅UVA组与对照组鸟嘌呤氢键特征谱线强度变化差异不明显(P0.05),其余各组各指标两两相比差异均有统计学意义(P0.05)；各组与照后时间呈现交互效应(P0.05)。2、经UVB和UVB+白藜芦醇照射后DNA拉曼光谱的主要谱线轮廓未发生明显变化；三组鸟嘌呤氢键、磷酸基静电斥力、腺嘌呤碱基分子内能和腺嘌呤碱基堆积力特征谱线强度变化差异有统计学意义(P0.05)；多重比较结果中,三组细胞鸟嘌呤氢键特征频率两两相比差异均有统计学差异(P0.01),三组细胞磷酸基静电斥力特征频率两两相比仅UVB组和对照组差异具有统计学意义(P0.05),三组细胞腺嘌呤碱基分子内能特征频率两两相比仅UVB组和UVB+白藜芦醇差异无统计学意义(P0.05),三组细胞腺腺嘌呤碱基堆积力特征频率两两相比仅对照组和UVB+白藜芦醇差异无统计学意义(P0.05)；各组与照后时间呈现交互效应(P0.05)。3、HaCaT细胞经49.5mJ/cm2剂量UVB照射后,继续培养1.0h.2.0h、3.0h、 4.0h、5.0h后观察自噬,结果显示自噬细胞比例从照后1.0h的20.15±2.10%先是增长到最高50.67士7.33%,然后下降直至照后5.0h还保持在21.39士3.75%,自噬细胞比例峰值出现在照后3.0h(P0.05)。HaCaT细胞经0mJ/cm2(对照)、9.9mJ/cm2、29.7mJ/cm2、49.5mJ/cm2剂量UVB照射后,继续培养3.0h后观察自噬,结果显示HaCaT细胞自噬比例随UVB照射剂量增加而上升,从4.13士1.02%增长到65.78±6.14%(P0.05)。结论1、UVA和UVB可通过作用于鸟嘌呤氢键、磷酸基静电斥力、腺嘌呤碱基分子内能和腺嘌呤碱基堆积力等分子结构损伤HaCaT细胞DNA, UVB的损伤作用更大。2、白藜芦醇可通过保护HaCaT细胞DNA鸟嘌呤氢键、磷酸基静电斥力和腺嘌呤碱基堆积力等分子结构而减少UVB所致损伤。3. HaCaT细胞经49.5mJ/cm2剂量UVB照射并培养至1-5h,自噬细胞比例先上升然后下降,峰值出现在照后3h左右。HaCaT细胞经9.9～49.5mJ/cm2剂量UVB照射并培养至3.0h,自噬细胞比例随照射剂量增加而上升。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R594.8

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