氧化应激晶状体上皮细胞中黏着斑激酶的表达

发布时间：2018-07-07 11:58

本文选题：过氧化氢 + 黏着斑激酶　；参考：《南方医科大学》2013年硕士论文

【摘要】：背景晶状体上皮细胞(lens epithelial cells, LECs)是位于晶状体前囊膜下的一层单层立方上皮细胞,其代谢活跃,具有生长、分化和创伤刺激后发生愈合反应的能力。晶状体上皮细胞起到对晶状体的营养、代谢、损伤修复作用,是维持晶状体透明性的最重要的防线,任何原因(如衰老、营养代谢异常、中毒变性、外伤等)破坏LECs的正常结构和功能,都可能导致不同类型的晶状体混浊。在既往的研究中,人们通过对比正常人与白内障患者的LECs,发现白内障患者LECs有三个特点：形态改变、密度下降、局部增殖导致细胞复层排列或向后囊移行。这些形态结构的异常,是晶状体发生浑浊的机制之一。因此,研究晶状体上皮细胞的形态、结构、凋亡等生物学特性是研究晶状体疾病的重要基础。白内障的发生发展与晶状体长期处于氧化应激状态有密切的联系。通常认为,过量的氧化应激是外界各种致白内障因素作用的共同途径,它激活一系列的细胞内信号通路,最终因损伤LECs而导致晶状体的浑浊。在众多导致细胞氧化应激的物质中,过氧化氢(hydrogen peroxide, H2O2)是最重要的物质。正常人的晶状体及房水中存在一定数量的自由基,其中H2O2的浓度约为20-30μmol/L,而在白内障患者的房水中可高达660μmol/L,为正常患者的30倍。由于氧化应激,LECs膜通透性改变,细胞内的蛋白质漏出,房水成分发生改变,细胞内环境改变,稳定性下降；细胞内蛋白质结构发生改变,细胞生理功能、透明度受到影响；DNA受损,晶状体上皮细胞凋亡,进而无法供给晶状体代谢所需的营养物质,加重氧化应激的损害。这一系列的变化都能导致晶体的浑浊。氧化应激激起了白内障发生、发展过程中恶性循环。可见,H202诱导LECs的氧化损伤是白内障发生发展的起始途径,研究晶状体上皮细胞的氧化应激机制是研究年龄相关性白内障发生发展机制的重要方面之一。黏着斑激酶(Focal adhesion kinase, FAK)是一种位于细胞质的非受体型酪氨酸激酶。FAK自发现起至今已近20年,目前认为FAK在诱导细胞增殖、细胞粘附、迁移、抗凋亡、纤维化、分化方面都起到了一定的作用。(1)在细胞增殖方面,细胞与ECM的连接是细胞增殖的必要条件。整合素与细胞外基质连接后,在生长因子的刺激下,FAK被激活,通过MAPK或P13K的激活促进细胞增殖。(2)在细胞粘附移行方面,已有大量研究证实FAK与细胞的移行有重要的关系,而且细胞的移行依赖于细胞外基质(extracellular matrixc,ECM)-整合素-FAK这一系列因素的相互作用。(3)在抑制凋亡方面,FAK家族对细胞有不同的影响。抑制FAK能导致细胞的凋亡,这可能与PI3-K/Akt-1和MEK/Erk信号通路相关。总而言之,由于FAK位于多条信号通路的上游,其对细胞生物行为的各个方面都起到一定的调节作用。目的通过过氧化氢处理晶状体上皮细胞,制造氧化应激模型,研究氧化应激晶状体上皮细胞的增殖、移行、凋亡、及形态学的改变,同时,观察细胞内黏着斑激酶的动态表达及活化程度,初步探讨黏着斑激酶是否对氧化应激晶状体上皮细胞的调控功能。方法： 1、晶状体上皮细胞培养与处理：人晶状体上皮细胞(HLECs)细胞株,购自美国ATCC。细胞用含有10%FBS的低糖DMEM培养基培养,于37℃、体积分数5%的CO2饱和湿度的细胞培养箱内培养。细胞达到80%-90%融合后,用不含血清、H202含量分别为0,30,50,70,100,300,500,700,1000μmol/L的低糖DMEM细胞0,30min,3h,6h,12h,24h。 2、CCK-8法检测细胞存活率：将细胞密度调至1×108/L,并将细胞悬液接种于96孔培养板,每孔100μL,置37℃,体积分数5%C02培养箱中孵育,24h细胞贴壁后弃上清,对照组(H202浓度为0μmol/L)加100μL低糖DMEM培养液,处理组分别加入H202浓度为30,50,70,100,300,500,700,1000μmol/L的低糖DMEM100μL,每组设六个复孔,继续孵育30min,3h,6h,12h,24h。避光取出,弃上清,PBS洗涤两次,每孔加入100μL培养基和10μL CCK-8,再加入一组空白对照组(为无细胞组,仅加入培养基与CCK-8)至于培养箱内2h,全自动酶标仪进行比色,波长为450nm,测每孔吸光度A值。计算药物对细胞的生存率。生存率(%)=(实验组A值-空白组A值)／(对照组A值-空白组A值)×100%。 3、细胞划痕实验检验细胞移行能力：将进入对数生长期的检测细胞用100μL无菌枪头在每个孔中长满的单层细胞上迅速而轻轻地划1-2道痕,弃培养基,PBS冲洗3遍以去除掉脱落的细胞及培养基中的细胞因子。对照组加入不含H202的培养基,处理组分别加入H202浓度为100,300,500,700,1000μmol/L的低糖DMEM,每组设4个复孔,测量8个值,分别于0时,12h,24h拍照观察,通过图像处理系统测量细胞爬行的距离,比较不同细胞划痕修复速度。 4、流式细胞仪检测细胞凋亡情况：取对数生长期HLECs,接种于6孔板,长至90%融合后弃上清,移液管吸净培养液,无菌PBS液洗细胞3次。对照组加入不含H202的培养基,处理组分别加入H202浓度为100μmol/L、1000μmol/L的低糖DMEM处理24h。按照美国eBioscience公司Annexin V-FITC细胞凋亡检测试剂盒步骤进行细胞凋亡流式细胞仪检测。 5、激光共聚焦显微镜观察细胞内黏着斑激酶的表达与分布：细胞爬片后,对照组加入1000μL低糖DMEM培养液,处理组分别加入H202浓度为100,300,500,700,1000μmol/L的低糖DMEM1000μL置37℃,体积分数5%C02培养箱中孵育24h。弃去上清,PBS清洗三次,4%多聚甲醛室温固定5min, PBS清洗三次,-80℃甲醇-20℃固定15min, PBS清洗三次。山羊血清封闭1h, PBS清洗后加入1：200兔抗人FAK一抗4℃湿盒中孵育过夜。PBS清洗3次后,Hoechst33258染核1小时,FITC荧光二抗孵育30min后,PBS清洗多余二抗,甘油封片。避光保存,激光共聚焦显微镜下观察。 6、western blot检测细胞内FAK和磷酸化FAK的动态表达：细胞接种于6孔板上,长至90%融合,弃上清。PBS清洗后,对照组加1000μL低糖DMEM培养液,处理组(实验组)分别加入H2O2浓度为100,300,500,700,1000μmol/L的低糖DMEM1000μL,二氧化碳培养箱内培养30min,3h,6h,12h,24h。分别收集细胞,提取蛋白质,取15μl蛋白上样于8%聚丙烯酰胺凝胶进行电泳；转移样品蛋白于PVDF膜上；3%BSA室温封闭1h,1:1000FAK一抗4℃孵育过夜；TBST洗膜3次,加1：5000的辣根过氧化物酶标记的二抗室温孵育2h; TBST洗膜3次后,浸入增强化学发光试剂,暗室X线片压片曝光,洗片。图片经光密度图像扫描仪扫描,Flour Chem程序测定条带光密度值。 7统计处理采用SPSS13.0软件对实验数据进行统计分析。变量采用(x±s)描述。通过One-way ANOVA法分析比较CCK-8各个时间点中不同浓度组间的细胞存活率的差异,比较细胞划痕实验中不同浓度组中细胞的移行速度的差异,比较流式细胞结果中不同浓度处理组间细胞凋亡、死亡、存活率的差异,比较western blot实验中不同浓度组间FAK表达量的差异,LSD法(方差齐)或Dunnelt's T3法(方差不齐)对组间进行两两比较。采用Two-way ANOVA分析不同浓度过氧化氢和不同处理时间对细胞移行速度是否存在交互效应。P0.05表示有统计学意义。结果 1、高浓度过氧化氢处理晶状体细胞24h后,细胞形态发生改变：贴壁细胞变得稀疏,细胞皱缩、轮廓增强、边缘僵硬,并由原来的多边形变成细长型,形成伪足,细胞核与细胞浆界限不明显。而FAK分布也集中至细胞拉长部位。 2、处理12小时以内,500μmol/L浓度以上的过氧化氢对细胞有杀伤作用；处理24h时,不同浓度处理组间细胞存活率差异有统计学意义F=17.96,p0.01。30,70,100,300μmol/L组的细胞较对照组增殖明显,其存活率分别达到1.24±0.03%(p0.01),1.35±0.08%(p0.01),1.75±0.19%(p0.01)与1.37±0.17%(p=0.04),但100μmol/L组与50,70,300μmol/L之间没有明显差异(p50=0.20,p70=0.051,p300=0.10)。而500,700,1000μmol/L组细胞存活率较之对照组降低(p5000.01,p7000.01,plooo0.010) 3、在无血清情况下培养细胞24h,100μmol/L组细胞移行速度最快,达到62.23±1.99单位/小时(p0.01),对前、后12个小时移行速度进行两组间比较(two-way ANOVA)发现,细胞移行速度除受到H2O2浓度影响外(F=23.34,p0.01),还受到时间因素的影响(F=27.76,p0.01)。此外,时间与浓度之间还存在交互效应(F=9.61,p0.01)。 4、细胞凋亡率总体差异有统计学意义(F=7.49,p=0.02)。对组间进行两两比较发现,100μmol/L浓度处理组细胞总体凋亡率为2.40±0.01%,低于对照组的5.04±0.00%(p=0.01)及1000μmol/L浓度组的4.61±0.01%(p=0.02)比。而1000μmol/L浓度组细胞的凋亡程度与对照组比差异无统计学意义。 5、0,100,300,500,700及1000μmol/L的H202处理细胞24h后,细胞中FAK含量发生改变。其中,100,300μmol/L浓度组处理24小时后,FAK表达量增加,而700,1000浓度处理过后FAK表达量降低,差异有统计学意义(p0.05)。在此过程中,FAK磷酸化被激活,随处理浓度和处理时间的改变而改变。结论过氧化氢对晶状体上皮细胞有着双重影响：1000μmol/L的H202能提高细胞内FAK的表达,促进细胞增殖、移行,抑制细胞的凋亡。而1000μmol/L的过氧化氢抑制细胞内FAK的表达,对细胞生理功能产生抑制作用,并能导致细胞死亡。
[Abstract]:background
Lens epithelial cells (LECs) is a layer of monolayer cuboid epithelial cells located in the anterior capsule of the lens. Its metabolism is active and has the ability of growth, differentiation and healing after traumatic stimulation. Lens epithelial cells play the role of nutrition, metabolism, repair and repair of the lens, and maintain the transparency of the lens. The most important line of defense, such as aging, abnormal metabolism, toxic degeneration, trauma, etc., destroys the normal structure and function of LECs, which can lead to different types of lens opacities. In the past study, people found three characteristics of LECs in cataract patients by comparing the LECs of normal and cataract patients: morphological changes, The density decreases and the local proliferation leads to the arrangement of cell layers or the migration of the posterior capsule. These abnormalities are one of the mechanisms of turbidity in the lens. Therefore, the study of the morphology, structure and apoptosis of lens epithelial cells is an important basis for the study of lens diseases.
The development of cataract is closely related to the oxidative stress state of the lens for a long time. It is generally believed that excessive oxidative stress is the common way of various external cataract factors. It activates a series of intracellular signaling pathways and eventually causes turbidity of the crystalline body due to the damage of LECs. In the substance, hydrogen peroxide (H2O2) is the most important substance. There are a certain number of free radicals in the lens and aqueous humor of normal people, of which the concentration of H2O2 is about 20-30 mu mol/L, and in the aqueous humor of the cataract patients up to 660 mu, 30 times as high as that of the normal patients. Because of oxidative stress, the permeability of the LECs membrane changes, cells are changed. Protein leakage in the chamber, changes in the composition of aqueous humor, changes in the intracellular environment, and the decline in stability; the changes in the protein structure in the cells, the physiological function of the cells, the transparency of the cells; the damage of DNA, the apoptosis of the lens epithelial cells, which can not supply the nutrients needed for the metabolism of the lens and aggravate the damage of oxidative stress. The changes in the column can cause the turbidity of the crystal. Oxidative stress arouses the occurrence of cataract and the vicious cycle in the process of development. It can be seen that H202 induced oxidative damage of LECs is the beginning of the development of cataract. The study of the oxidative stress mechanism of lens epithelial cells is an important aspect of the study of the mechanism of the development of age related cataract. 1.
Focal adhesion kinase (FAK), a non receptor tyrosine kinase.FAK located in cytoplasm, has been found for nearly 20 years since it has been found. Now, FAK has been considered to play a definite role in inducing cell proliferation, cell adhesion, migration, anti apoptosis, fibrosis and differentiation. (1) in cell proliferation, the connection between cells and ECM is The necessary condition of cell proliferation. After the integrin is connected with the extracellular matrix, FAK is activated by growth factor stimulation, and the activation of MAPK or P13K promotes cell proliferation. (2) there is a large number of studies on cell adhesion and migration that the migration of FAK and cells is important, and the migration of cells depends on the extracellular matrix (extrace Llular matrixc, ECM) - the interaction of integrin -FAK this series of factors. (3) in the inhibition of apoptosis, the FAK family has a different effect on cells. Inhibition of FAK can lead to cell apoptosis, which may be associated with PI3-K/Akt-1 and MEK/Erk signaling pathways. In a word, because FAK is upstream of multiple signal pathways, it is responsible for cellular biological behavior. All aspects play a certain role in regulating.
objective
By treating the lens epithelial cells by hydrogen peroxide, the oxidative stress model was made to study the proliferation, migration, apoptosis and morphological changes of the epithelial cells of the oxidative stress lens epithelial cells. At the same time, the dynamic expression and activation degree of the focal adhesion kinase in the cells were observed, and the modulation of the focal adhesion kinase on the epithelial cells of the oxidative stress lens epithelial cells was preliminarily discussed. Control function.
Method:
1, lens epithelial cell culture and processing: human lens epithelial cell (HLECs) cell line, purchased from American ATCC. cells using a low sugar DMEM medium containing 10%FBS, cultured in a cell culture box with a volume fraction of 5% CO2 saturated humidity. After 80%-90% fusion, the content of H202 content is 0,30,50,70100,30, respectively. 05007001000 - mol/L low sugar DMEM cells 0,30min, 3h, 6h, 12h, 24h.
2, CCK-8 method detected cell survival rate: the cell density was adjusted to 1 x 108/L, and the cell suspension was inoculated to 96 hole culture plate, 100 mu L per pore, 37 centigrade, the volume fraction 5%C02 incubator, the 24h cells after adherence to the supernatant, the control group (H202 concentration was 0 mol/L) and 100 u L low sugar DMEM culture liquid, and the treatment group was added H202 concentration to 30,50,70100 3005007001000 mu mol/L low sugar DMEM100 mu L, each set of six compound holes, continue to incubate 30min, 3h, 6h, 12h, 24h. to take out the light, abandon the supernatant, PBS washing two times, add 100 mu L culture medium and 10 micron L CCK-8, and then add a group of blank control group (for no cell group, only add medium with the culture) as to the incubator Colorimetry, the wavelength was 450nm, and the absorbance of each hole was A. The survival rate of the cell was calculated. The survival rate (%) = (the A value of the experimental group - the blank group A value) / (the A value of the control group - the blank group A value) * 100%.
3, cell scratch test test cell migration ability: to enter the logarithmic growth period of the detection of cells with 100 mu L aseptic gun head in each hole full of the monolayer quickly and gently stroke 1-2 trace, discard medium, PBS rinse for 3 times to remove the cells and cytokines in the culture medium. The control group is added without H202 medium, The treatment group was added to the low sugar DMEM with the concentration of 1003005007001000 H202 mol/L respectively. Each group had 4 compound holes, and 8 values were measured. At 0, 12h and 24h were photographed, and the distance of the cell crawling was measured by the image processing system, and the rate of repair of different cells was compared.
4, flow cytometry detected the cell apoptosis: take the logarithmic growth period HLECs, inoculate the 6 hole plate, long to 90% fusion, remove the supernatant, the pipette suction culture solution, the aseptic PBS liquid washing cell for 3 times. The control group added the medium without H202, and the treatment group was added to the H202 concentration of 100 u mol/L, and the low sugar DMEM processing 24h. of 1000 u mol/L respectively according to American eBiosc Ience Annexin V-FITC apoptosis detection kit was used to detect apoptosis by flow cytometry.
5, the expression and distribution of intracellular sticky kinases were observed by laser confocal microscope: after the cell crawling, the control group was added 1000 L low sugar DMEM culture medium, and the treatment group was added to the low sugar DMEM1000 mu L with the concentration of 1003005007001000 mu mol/L, respectively, and the volume fraction 5%C02 incubator was incubated with 24h. abandoned to the supernatant, PBS cleaning three times, 4% more. Polyoxymethylene was fixed at room temperature for 5min, PBS was cleaned three times, 15min was fixed at -20 C at -80 C -20 C, and PBS was cleaned. The goat serum closed 1H, PBS was cleaned and incubated in the wet box of anti human FAK one anti 4 C for 3 times after cleaning for 1 hours, and after fluorescent two was incubated for 1 hours. It is observed under laser confocal microscope.
6, Western blot detected the dynamic expression of FAK and phosphorylated FAK in cells: the cells were inoculated on the 6 hole plate, long to 90% fusion, and after the cleaning of the supernatant.PBS, the control group was added with 1000 mu L low sugar DMEM culture solution, and the treatment group (experimental group) was added to the H2O2 concentration of 1003005007001000 micron DMEM1000 u L, the incubator of carbon dioxide was incubated for 30min, 3 H, 6h, 12h, 24h., respectively collect cells, extract protein, take 15 mu L protein on 8% polyacrylamide gel electrophoresis; transfer sample protein on PVDF membrane; 3%BSA room temperature closed 1H, 1:1000FAK one anti 4 C incubation for night; TBST washing 3 times, 1:5000 horseradish peroxidase labelled two at room temperature incubating 2H; baptised membrane after 3 times, soak. Enhanced chemiluminescence reagents, darkroom X-ray films, exposures and films. The images were scanned by optical density scanner and the optical density values were measured by Flour Chem program.
7 the statistical analysis of the experimental data was carried out by SPSS13.0 software. The variables were described by (x + s). The difference of cell survival rate between different concentration groups in each time point of CCK-8 was analyzed by One-way ANOVA method, and the difference in the migration velocity of cells in different concentration groups was compared, and the results of flow cytometry were compared. The difference of apoptosis, death and survival rate between different concentration groups was compared, and the difference of FAK expression between different concentration groups in Western blot experiment was compared. The LSD method (Fang Chaqi) or Dunnelt's T3 method (variance uneven) was compared between the 22 groups. The cell migration velocity of different concentration of hydrogen peroxide and different treatment time was analyzed by Two-way ANOVA Whether there was interaction effect,.P0.05 showed statistical significance.
Result
1, after the high concentration of hydrogen peroxide treated the lens cell 24h, the cell morphology changes: the adherent cells become sparse, the cell crinkle, the contour is strengthened, the edge is rigid, and the original polygon becomes slender, forming the pseudo foot, the nucleus and the cytoplasm boundary are not obvious. And the FAK distribution is also concentrated to the elongated part of the cell.
2, under the treatment of 12 hours, the concentration of hydrogen peroxide above 500 mu mol/L had a killing effect on the cells. When treating 24h, the difference of cell survival rate between different concentration treated groups was statistically significant F=17.96, the cells of p0.01.30,70100300 mu mol/L group proliferated significantly compared with the control group, the survival rate was 1.24 + 0.03% (P0.01), 1.35 + 0.08% (P0.01), 1.7 5 + 0.19% (P0.01) and 1.37 + 0.17% (p=0.04), but there was no significant difference between the 100 mu mol/L group and 50,70300 mu mol/L (p50=0.20, p70=0.051, p300=0.10). The survival rate of the 5007001000 micron group was lower than that of the control group (p5000.01, p7000.01, plooo0.010).
3, in serum-free cell culture, cell 24h was cultured, and the speed of cell migration was the fastest, reaching 62.23 + 1.99 units / hours (P0.01). Before and after 12 hours, the rate of migration was compared between the two groups (two-way ANOVA). The rate of cell migration was not affected by H2O2 concentration (F=23.34, P0.01), but also influenced by time factors (F=27.76, P0.01). In addition, there is an interaction effect between time and concentration (F=9.61, P0.01).
4, the overall difference of apoptosis rate was statistically significant (F=7.49, p=0.02). The total apoptosis rate of 100 mu mol/L concentration treatment group was 2.40 + 0.01%, compared with 5.04 + 0% (p=0.01) and 1000 mu mol/L concentration group of 4.61 + 0.01% (p= 0.02) ratio in the control group, and the degree of apoptosis and the control of cells in the 1000 mu mol/L concentration group were compared with those of the control group. There was no significant difference in the group ratio.
After 5,0100300500700 and 1000 mol/L H202 treated cells 24h, the FAK content in the cells changed. Among them, the expression of FAK increased after 24 hours treatment with 100300 mol/L concentration group, and the expression of FAK decreased after 7001000 concentration treatment. The difference was statistically significant (P0.05). In this process, FAK phosphorylation was activated, with treatment concentration and treatment. Change of time.
conclusion
Hydrogen peroxide has a dual effect on lens epithelial cells: 1000 mol/L H202 can increase the expression of FAK in cells, promote cell proliferation, move and inhibit the apoptosis of cells. The 1000 mol/L hydrogen peroxide inhibits the expression of FAK in cells, inhibits the physiological functions of cells and causes cell death.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R776

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