本文關(guān)鍵詞：煙草煙霧冷凝物和大氣細(xì)顆粒物對BEAS-2B細(xì)胞的毒性及機(jī)制研究　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 煙草煙霧冷凝物 細(xì)顆粒物 人支氣管上皮細(xì)胞 毒作用機(jī)制

【摘要】：目的利用體外培養(yǎng)人支氣管上皮細(xì)胞(BEAS-2B),觀察并比較兩種空氣顆粒物煙草煙霧冷凝物(cigarette smoke condensate,CSC)和天津市大氣細(xì)顆粒物(fine particulate matter,PM_(2.5))對BEAS-2B細(xì)胞的毒性效應(yīng),揭示相關(guān)毒作用機(jī)制,為預(yù)防和控制兩種典型空氣顆粒物所致呼吸系統(tǒng)疾病提供科學(xué)基礎(chǔ)。方法1.兩種顆粒物染毒液的制備和處理:CSC:選用3R4F參比卷煙為樣品。按GB/T5606.1-2004的規(guī)定取樣,捕集煙草煙霧冷凝物并調(diào)整濃度得10mg/ml的CSC樣品母液,-80℃保存?zhèn)溆谩M_(2.5):利用嶗山2030型中流量智能TSP采樣器的第三級采集細(xì)顆粒物,隨后進(jìn)行超聲洗脫處理,制成細(xì)顆粒物干粉,配成終濃度為100mg/ml的細(xì)顆粒物母液,-80℃保存?zhèn)溆谩?.利用CCK-8法檢測不同濃度顆粒物對BEAS-2B細(xì)胞存活率的影響,明確CSC和PM_(2.5)的半數(shù)抑制濃度并確定后續(xù)實驗的干預(yù)劑量;利用倒置顯微鏡觀察兩種顆粒物對BEAS-2B細(xì)胞形態(tài)的影響;采用Annexin V-FITC/PI雙染試劑盒,利用流式細(xì)胞儀分析兩種顆粒物對細(xì)胞凋亡的影響。3.利用酶聯(lián)免疫(ELISA)法檢測顆粒物暴露后BEAS-2B細(xì)胞裂解液中MDA含量、GSH、8-OHd G表達(dá)水平以及細(xì)胞培養(yǎng)上清中SOD水平,明確顆粒物暴露導(dǎo)致的細(xì)胞氧化應(yīng)激反應(yīng);利用酶聯(lián)免疫(ELISA)法檢測顆粒物暴露后BEAS-2B細(xì)胞培養(yǎng)液中IL-1β、TNF-α、IL-6和IL-8的含量,明確顆粒物暴露導(dǎo)致的炎癥反應(yīng)。4.利用定制的凋亡抗體芯片Ray Bio?Human Apoptosis Antibody Array G1高通量篩選CSC和PM_(2.5)誘導(dǎo)BEAS-2B細(xì)胞凋亡時涉及的凋亡蛋白,并利用RT-PCR和Western blotting方法對差異的凋亡蛋白及其所在通路蛋白進(jìn)行驗證。5.利用定制的炎癥抗體芯片Ray Bio?Human Inflammation Antibody Array G Series III篩選PM_(2.5)暴露導(dǎo)致BEAS-2B細(xì)胞中炎癥因子表達(dá)的變化,并利用ELISA方法對差異炎癥因子及其所在通路進(jìn)行驗證。結(jié)果1、CSC和PM_(2.5)的成分分析。CSC中共檢測出13種多環(huán)芳烴類有機(jī)物,含量由多到少依次為:蒽(18.99%)、苯并蒽(11.57%)、苯并k熒蒽(8.52%)、苯并b熒蒽(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%)、熒蒽(3.23%)、芴(2.13%)、苯并芘(0.81%);另外檢出5種重金屬類物質(zhì),含量由多到少依次為:Zn(8.86%)、Cu(2.65%)、Cd(0.44%)、Mn(0.22%)、Hg(0.09%)。PM_(2.5)中共檢測出11種多環(huán)芳烴類有機(jī)物,含量由多到少依次為:苯并b熒蒽(6.50%)、苯并k熒蒽(5.62%)、屈(4.71%)、苯并芘(4.53%)、苯并傒(4..05%)、苯并蒽(3.92%)、熒蒽(3.87%)、茚并芘(3.21%)、芘(2.82%)、菲(0.69%)、蒽(0.26%);另外檢出8種重金屬類物質(zhì),含量由多到少依次為: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)對BEAS-2B細(xì)胞的毒性效應(yīng)。(1)根據(jù)CCK-8實驗結(jié)果得出CSC對BEAS-2B細(xì)胞的半數(shù)抑制濃度(IC50)約為80μg/ml,即設(shè)置CSC染毒劑量為(0μg/ml、10μg/ml、20μg/ml、40μg/ml);PM_(2.5)對BEAS-2B細(xì)胞的半數(shù)抑制濃度(IC50)約為165μg/ml,因此設(shè)置PM_(2.5)染毒劑量為(0μg/ml、17.5μg/ml、35μg/ml、70μg/ml)。(2)BEAS-2B細(xì)胞經(jīng)CSC暴露24h,可見細(xì)胞發(fā)生形態(tài)學(xué)改變。與對照組相比,細(xì)胞胞漿疏松,觸角變狹長,彼此連接減少,細(xì)胞整體變細(xì)長瘦弱,透明度下降,并有不同程度的死亡細(xì)胞。而PM_(2.5)暴露后,BEAS-2B細(xì)胞形態(tài)的改變是細(xì)胞之間空隙加大,生長密度降低,漂浮細(xì)胞增多。隨著PM_(2.5)濃度的增加,晃動培養(yǎng)液,漂浮細(xì)胞增多,并且可以觀察到細(xì)胞中存在顆粒狀物質(zhì)。(3)BEAS-2B細(xì)胞經(jīng)CSC處理24h后,在AnnexinⅤ-FITC/PI散點圖上可見隨著CSC濃度的增加,位于右下和右上象限的早凋和晚凋細(xì)胞逐漸增多,且與對照組相比,BEAS-2B細(xì)胞的凋亡率顯著高于對照組;而BEAS-2B細(xì)胞經(jīng)PM_(2.5)處理24h后,在AnnexinⅤ-FITC/PI散點圖上可見隨著PM_(2.5)濃度的增加,位于左上和右上象限的細(xì)胞膜破損和晚凋細(xì)胞逐漸增多,且與對照組相比,BEAS-2B細(xì)胞的凋亡率顯著高于對照組。(4)BEAS-2B細(xì)胞在CSC的刺激下,各處理組EC-SOD、MDA、8-OHd G水平顯著高于對照組,而GSH水平顯著低于對照組,結(jié)果表明CSC可通過氧化應(yīng)激方式對BEAS-2B細(xì)胞造成毒性損傷;而BEAS-2B細(xì)胞在PM_(2.5)的刺激下,中、低劑量組的EC-SOD、MDA、8-OHd G水平升高緩慢,但在高劑量組則極顯著高于對照組,而GSH水平顯著低于對照組,結(jié)果表明PM_(2.5)可導(dǎo)致BEAS-2B細(xì)胞發(fā)生氧化應(yīng)激反應(yīng)并不明顯。(5)BEAS-2B細(xì)胞在CSC的刺激下,各處理組TNF-α,IL-1β,IL-6,IL-8水平顯著高于對照組,結(jié)果顯示CSC可導(dǎo)致細(xì)胞發(fā)生炎癥反應(yīng);而BEAS-2B細(xì)胞在PM_(2.5)的刺激下,各處理組TNF-α,IL-1β,IL-6,IL-8水平極顯著高于對照組,結(jié)果顯示PM_(2.5)可導(dǎo)致細(xì)胞免疫炎癥水平升高,釋放大量炎性介質(zhì),從而造成細(xì)胞損傷。3、CSC和PM_(2.5)引起B(yǎng)EAS-2B細(xì)胞凋亡的機(jī)制(1)CSC蛋白芯片及Western blotting實驗結(jié)果顯示BEAS-2B細(xì)胞相關(guān)凋亡蛋白TNF-R1、p21、IGF-1R、PI3K、AKT表達(dá)水平顯著升高,Fas L表達(dá)水平顯著降低,由此我們推測IGF-1R/PI3K/AKT通路可能是引發(fā)CSC暴露BEAS-2B細(xì)胞發(fā)生惡性轉(zhuǎn)化并死亡的作用機(jī)制。(2)PM_(2.5)凋亡抗體芯片、RT-PCR和Western blotting實驗結(jié)果均顯示BEAS-2B細(xì)胞在PM_(2.5)的刺激下,相關(guān)凋亡蛋白TNF-R1、Fas、HTRA、BID、Caspase8表達(dá)水平顯著升高,IGF-2、TNF-R2表達(dá)水平顯著降低。4.PM_(2.5)引起B(yǎng)EAS-2B細(xì)胞炎癥反應(yīng)的機(jī)制PM_(2.5)芯片結(jié)果顯示炎癥因子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表達(dá)升高,炎癥因子TIMP-2、IL-10表達(dá)降低。利用KEGG數(shù)據(jù)庫和ELISA方法驗證結(jié)果顯示IL-4、IL-13、Eotaxin、TNF-α、IL-3等相關(guān)炎癥因子的表達(dá)升高激活哮喘相關(guān)通路最終導(dǎo)致BEAS-2B細(xì)胞發(fā)生炎癥反應(yīng)引起支氣管損傷。結(jié)論1、CSC和PM_(2.5)在所包含的物質(zhì)成分及各成分含量上均存在差異。在多環(huán)芳烴類物質(zhì)方面:CSC中主要檢出13種多環(huán)芳烴類物質(zhì),含量占前三位的分別是:蒽、苯并蒽、苯并k熒蒽,而PM_(2.5)中主要檢測出11種多環(huán)芳烴類物質(zhì),含量占前三位的分別是:苯并b熒蒽、苯并k熒蒽、屈。在重金屬類物質(zhì)方面:CSC中主要檢出5種重金屬類物質(zhì),含量占前三位的分別是:Zn、Cu、Cr,而PM_(2.5)中主要檢出8種重金屬類物質(zhì),含量占前三位的分別是:Zn、Pb、Mn。2、CSC和PM_(2.5)對BEAS-2B細(xì)胞的毒性效應(yīng)存在差異。CSC對BEAS-2B細(xì)胞的半數(shù)抑制濃度(IC50)約為80μg/ml,PM_(2.5)對BEAS-2B細(xì)胞的半數(shù)抑制濃度(IC50)約為165μg/ml,可見CSC對BEAS-2B細(xì)胞的毒性大于PM_(2.5);CSC導(dǎo)致BEAS-2B細(xì)胞變狹長瘦弱,PM_(2.5)則可導(dǎo)致細(xì)胞回縮變形并且可在細(xì)胞中觀察到顆粒狀物質(zhì),可見CSC導(dǎo)致BEAS-2B發(fā)生形態(tài)學(xué)變化與PM_(2.5)存在差異;CSC和PM_(2.5)均可誘導(dǎo)BEAS-2B細(xì)胞的氧化應(yīng)激和炎癥反應(yīng),而CSC致BEAS-2B細(xì)胞發(fā)生氧化應(yīng)激反應(yīng)水平顯著高于PM_(2.5),PM_(2.5)則主要是通過炎癥反應(yīng)作用于BEAS-2B細(xì)胞。3、CSC和PM_(2.5)誘導(dǎo)BEAS-2B細(xì)胞凋亡機(jī)制不同。CSC可以通過TNF-R1、p21、IGF-1R的表達(dá)升高引發(fā)BEAS-2B細(xì)胞凋亡,并通過促進(jìn)IGF-1R表達(dá)升高激活下游PI3K及AKT蛋白導(dǎo)致BEAS-2B細(xì)胞的惡性轉(zhuǎn)化及凋亡。而PM_(2.5)則通過TNF-R1、Fas、BID、Caspase8、HTRA的表達(dá)升高來影響B(tài)EAS-2B細(xì)胞凋亡。4、PM_(2.5)可以通過IL-4、IL-13、Eotaxin、TNF-α、IL-3等相關(guān)炎癥因子的表達(dá)升高激活哮喘相關(guān)通路最終導(dǎo)致BEAS-2B細(xì)胞發(fā)生炎癥反應(yīng)引起支氣管損傷。
[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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