烟草烟雾冷凝物和大气细颗粒物对BEAS-2B细胞的毒性及机制研究

发布时间：2018-01-18 05:02

  本文关键词：烟草烟雾冷凝物和大气细颗粒物对BEAS-2B细胞的毒性及机制研究　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 烟草烟雾冷凝物 细颗粒物 人支气管上皮细胞 毒作用机制

【摘要】：目的利用体外培养人支气管上皮细胞(BEAS-2B),观察并比较两种空气颗粒物烟草烟雾冷凝物(cigarette smoke condensate,CSC)和天津市大气细颗粒物(fine particulate matter,PM_(2.5))对BEAS-2B细胞的毒性效应,揭示相关毒作用机制,为预防和控制两种典型空气颗粒物所致呼吸系统疾病提供科学基础。方法1.两种颗粒物染毒液的制备和处理:CSC:选用3R4F参比卷烟为样品。按GB/T5606.1-2004的规定取样,捕集烟草烟雾冷凝物并调整浓度得10mg/ml的CSC样品母液,-80℃保存备用。PM_(2.5):利用崂山2030型中流量智能TSP采样器的第三级采集细颗粒物,随后进行超声洗脱处理,制成细颗粒物干粉,配成终浓度为100mg/ml的细颗粒物母液,-80℃保存备用。2.利用CCK-8法检测不同浓度颗粒物对BEAS-2B细胞存活率的影响,明确CSC和PM_(2.5)的半数抑制浓度并确定后续实验的干预剂量;利用倒置显微镜观察两种颗粒物对BEAS-2B细胞形态的影响;采用Annexin V-FITC/PI双染试剂盒,利用流式细胞仪分析两种颗粒物对细胞凋亡的影响。3.利用酶联免疫(ELISA)法检测颗粒物暴露后BEAS-2B细胞裂解液中MDA含量、GSH、8-OHd G表达水平以及细胞培养上清中SOD水平,明确颗粒物暴露导致的细胞氧化应激反应;利用酶联免疫(ELISA)法检测颗粒物暴露后BEAS-2B细胞培养液中IL-1β、TNF-α、IL-6和IL-8的含量,明确颗粒物暴露导致的炎症反应。4.利用定制的凋亡抗体芯片Ray Bio?Human Apoptosis Antibody Array G1高通量筛选CSC和PM_(2.5)诱导BEAS-2B细胞凋亡时涉及的凋亡蛋白,并利用RT-PCR和Western blotting方法对差异的凋亡蛋白及其所在通路蛋白进行验证。5.利用定制的炎症抗体芯片Ray Bio?Human Inflammation Antibody Array G Series III筛选PM_(2.5)暴露导致BEAS-2B细胞中炎症因子表达的变化,并利用ELISA方法对差异炎症因子及其所在通路进行验证。结果1、CSC和PM_(2.5)的成分分析。CSC中共检测出13种多环芳烃类有机物,含量由多到少依次为:蒽(18.99%)、苯并蒽(11.57%)、苯并k荧蒽(8.52%)、苯并b荧蒽(8.29%)、屈(8.17%)、菲(6.97%)、苯并傒(6.83%)、茚并芘(5.13%)、芘(3.57%)、二苯并蒽(3.53%)、荧蒽(3.23%)、芴(2.13%)、苯并芘(0.81%);另外检出5种重金属类物质,含量由多到少依次为:Zn(8.86%)、Cu(2.65%)、Cd(0.44%)、Mn(0.22%)、Hg(0.09%)。PM_(2.5)中共检测出11种多环芳烃类有机物,含量由多到少依次为:苯并b荧蒽(6.50%)、苯并k荧蒽(5.62%)、屈(4.71%)、苯并芘(4.53%)、苯并傒(4..05%)、苯并蒽(3.92%)、荧蒽(3.87%)、茚并芘(3.21%)、芘(2.82%)、菲(0.69%)、蒽(0.26%);另外检出8种重金属类物质,含量由多到少依次为:Zn(28.95%)、Pb(11.47%)、Mn(9.80%)、Cu(9.29%)、Cr(0.13%)、Ni(0.13%)、Cd(0.06%)、Hg(0.002%)。2、CSC和PM_(2.5)对BEAS-2B细胞的毒性效应。(1)根据CCK-8实验结果得出CSC对BEAS-2B细胞的半数抑制浓度(IC50)约为80μg/ml,即设置CSC染毒剂量为(0μg/ml、10μg/ml、20μg/ml、40μg/ml);PM_(2.5)对BEAS-2B细胞的半数抑制浓度(IC50)约为165μg/ml,因此设置PM_(2.5)染毒剂量为(0μg/ml、17.5μg/ml、35μg/ml、70μg/ml)。(2)BEAS-2B细胞经CSC暴露24h,可见细胞发生形态学改变。与对照组相比,细胞胞浆疏松,触角变狭长,彼此连接减少,细胞整体变细长瘦弱,透明度下降,并有不同程度的死亡细胞。而PM_(2.5)暴露后,BEAS-2B细胞形态的改变是细胞之间空隙加大,生长密度降低,漂浮细胞增多。随着PM_(2.5)浓度的增加,晃动培养液,漂浮细胞增多,并且可以观察到细胞中存在颗粒状物质。(3)BEAS-2B细胞经CSC处理24h后,在AnnexinⅤ-FITC/PI散点图上可见随着CSC浓度的增加,位于右下和右上象限的早凋和晚凋细胞逐渐增多,且与对照组相比,BEAS-2B细胞的凋亡率显著高于对照组;而BEAS-2B细胞经PM_(2.5)处理24h后,在AnnexinⅤ-FITC/PI散点图上可见随着PM_(2.5)浓度的增加,位于左上和右上象限的细胞膜破损和晚凋细胞逐渐增多,且与对照组相比,BEAS-2B细胞的凋亡率显著高于对照组。(4)BEAS-2B细胞在CSC的刺激下,各处理组EC-SOD、MDA、8-OHd G水平显著高于对照组,而GSH水平显著低于对照组,结果表明CSC可通过氧化应激方式对BEAS-2B细胞造成毒性损伤;而BEAS-2B细胞在PM_(2.5)的刺激下,中、低剂量组的EC-SOD、MDA、8-OHd G水平升高缓慢,但在高剂量组则极显著高于对照组,而GSH水平显著低于对照组,结果表明PM_(2.5)可导致BEAS-2B细胞发生氧化应激反应并不明显。(5)BEAS-2B细胞在CSC的刺激下,各处理组TNF-α,IL-1β,IL-6,IL-8水平显著高于对照组,结果显示CSC可导致细胞发生炎症反应;而BEAS-2B细胞在PM_(2.5)的刺激下,各处理组TNF-α,IL-1β,IL-6,IL-8水平极显著高于对照组,结果显示PM_(2.5)可导致细胞免疫炎症水平升高,释放大量炎性介质,从而造成细胞损伤。3、CSC和PM_(2.5)引起BEAS-2B细胞凋亡的机制(1)CSC蛋白芯片及Western blotting实验结果显示BEAS-2B细胞相关凋亡蛋白TNF-R1、p21、IGF-1R、PI3K、AKT表达水平显著升高,Fas L表达水平显著降低,由此我们推测IGF-1R/PI3K/AKT通路可能是引发CSC暴露BEAS-2B细胞发生恶性转化并死亡的作用机制。(2)PM_(2.5)凋亡抗体芯片、RT-PCR和Western blotting实验结果均显示BEAS-2B细胞在PM_(2.5)的刺激下,相关凋亡蛋白TNF-R1、Fas、HTRA、BID、Caspase8表达水平显著升高,IGF-2、TNF-R2表达水平显著降低。4.PM_(2.5)引起BEAS-2B细胞炎症反应的机制PM_(2.5)芯片结果显示炎症因子IL-8、IL-13、TNF-α、GM-CSF、IL-2、IL-17、TGF-b1、IP-10、I-309、IL-4、EOTAXIN、IL-3、EOTAXIN-2表达升高,炎症因子TIMP-2、IL-10表达降低。利用KEGG数据库和ELISA方法验证结果显示IL-4、IL-13、Eotaxin、TNF-α、IL-3等相关炎症因子的表达升高激活哮喘相关通路最终导致BEAS-2B细胞发生炎症反应引起支气管损伤。结论1、CSC和PM_(2.5)在所包含的物质成分及各成分含量上均存在差异。在多环芳烃类物质方面:CSC中主要检出13种多环芳烃类物质,含量占前三位的分别是:蒽、苯并蒽、苯并k荧蒽,而PM_(2.5)中主要检测出11种多环芳烃类物质,含量占前三位的分别是:苯并b荧蒽、苯并k荧蒽、屈。在重金属类物质方面:CSC中主要检出5种重金属类物质,含量占前三位的分别是:Zn、Cu、Cr,而PM_(2.5)中主要检出8种重金属类物质,含量占前三位的分别是:Zn、Pb、Mn。2、CSC和PM_(2.5)对BEAS-2B细胞的毒性效应存在差异。CSC对BEAS-2B细胞的半数抑制浓度(IC50)约为80μg/ml,PM_(2.5)对BEAS-2B细胞的半数抑制浓度(IC50)约为165μg/ml,可见CSC对BEAS-2B细胞的毒性大于PM_(2.5);CSC导致BEAS-2B细胞变狭长瘦弱,PM_(2.5)则可导致细胞回缩变形并且可在细胞中观察到颗粒状物质,可见CSC导致BEAS-2B发生形态学变化与PM_(2.5)存在差异;CSC和PM_(2.5)均可诱导BEAS-2B细胞的氧化应激和炎症反应,而CSC致BEAS-2B细胞发生氧化应激反应水平显著高于PM_(2.5),PM_(2.5)则主要是通过炎症反应作用于BEAS-2B细胞。3、CSC和PM_(2.5)诱导BEAS-2B细胞凋亡机制不同。CSC可以通过TNF-R1、p21、IGF-1R的表达升高引发BEAS-2B细胞凋亡,并通过促进IGF-1R表达升高激活下游PI3K及AKT蛋白导致BEAS-2B细胞的恶性转化及凋亡。而PM_(2.5)则通过TNF-R1、Fas、BID、Caspase8、HTRA的表达升高来影响BEAS-2B细胞凋亡。4、PM_(2.5)可以通过IL-4、IL-13、Eotaxin、TNF-α、IL-3等相关炎症因子的表达升高激活哮喘相关通路最终导致BEAS-2B细胞发生炎症反应引起支气管损伤。
[Abstract]:Objective to cultivate human bronchial epithelial cells by in vitro (BEAS-2B), to observe and compare the two kinds of air particles of cigarette smoke condensate (cigarette smoke condensate, CSC) and atmospheric particulates in Tianjin city (fine particulate matter, PM_ (2.5)) toxic effect on BEAS-2B cells, reveals the toxic mechanism, provide scientific basis for the prevention and control of two kinds of typical air particles caused by respiratory diseases. The preparation and processing method of 1. two particle exposure: CSC: 3R4F liquid used for sampling reference cigarette samples. According to the provisions of GB/T5606.1-2004, collecting tobacco smoke condensate and adjust the concentration of CSC sample liquor 10mg/ml, -80 stored at the standby.PM_ (2.5): the Laoshan third type 2030 traffic intelligent TSP sampler collection of fine particles, followed by ultrasonic extraction treatment, dry powder made of fine particles, with a final concentration of 100mg/ml fine particles Particle -80 mother liquor, stored at the standby.2. to detect different concentrations of particles by the method of CCK-8 on BEAS-2B cell survival, clear CSC and PM_ (2.5) the IC50 and determine the intervention dose in the follow-up experiment; using inverted microscope was used to observe the effects of two kinds of particles on the morphology of BEAS-2B cells by Annexin V-FITC/PI; staining kit, using the analysis of two kinds of particles influence on apoptosis of.3. was detected by flow cytometry (ELISA) GSH content of MDA, BEAS-2B in cell lysate was detected after exposure to particulate matter, 8-OHd G expression level of SOD in the supernatant and cell culture, clear particulate matter exposure to oxidative stress leads to cell; using enzyme-linked immunosorbent assay (ELISA) in liquid IL-1 beta, BEAS-2B cell culture method was used to detect the particles after exposure to TNF- alpha, IL-6 and IL-8 content, clear particulate matter exposure to inflammatory reaction caused by.4. using customized. Die Ray Bio Human Apoptosis antibody chip? Antibody Array G1 CSC and PM_ high throughput screening (2.5) apoptosis proteins involved in apoptosis of BEAS-2B cells and the apoptosis protein of different pathway and their proteins were validated using.5. chip Ray Bio system inflammation antibody by RT-PCR and Western blotting Human Inflammation Antibody Array G? Series III PM_ (2.5) screening exposure leads to changes in the expression of inflammatory cytokines in BEAS-2B cells, and to verify the differences of inflammatory factors and their pathways by using ELISA method. The results of 1, CSC and PM_ (2.5) analysis of the components of.CSC were detected in 13 kinds of polycyclic aromatic hydrocarbons, content from more to less as follows: (18.99%) anthracene, benzo (11.57%) anthracene, benzo K fluoranthene (8.52%), benzo B fluoranthene (8.29%), and (8.17%), phenanthrene (6.97%), benzene and Xi (6.83%), Indeno pyrene (5.13%), pyrene (3.57%), benzanthracene (two 3.53%, fluoranthene (3.) 23%),鑺，

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