本文關(guān)鍵詞：P38促分裂素原活化蛋白激酶在重鉻酸鉀誘導(dǎo)A549細(xì)胞凋亡中的作用　出處：《鄭州大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

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【摘要】：鉻(Cr)作為一種常見的化學(xué)元素,化合價(jià)態(tài)為-2到+6,其中以+3和+6價(jià)態(tài)最為常見。三價(jià)鉻在有效劑量下是無毒的,是人和動(dòng)物必需的營(yíng)養(yǎng)素。六價(jià)鉻是一種劇毒劑,具有致癌性、致突變性、致畸性、免疫毒性、生殖毒性、神經(jīng)毒性,是一種重金屬環(huán)境污染物。 六價(jià)鉻廣泛存在于香煙煙霧、汽車尾氣和生活環(huán)境等,主要用于電鍍行業(yè)、制革行業(yè)、化學(xué)工業(yè)、染料行業(yè)、冶金行業(yè),還有不銹鋼焊接和木材加工行業(yè)等。六價(jià)鉻能夠被細(xì)胞內(nèi)的還原系統(tǒng)還原,進(jìn)而產(chǎn)生大量的低價(jià)態(tài)鉻離子,低價(jià)態(tài)鉻離子容易與DNA結(jié)合產(chǎn)生DNA加合物；鉻被還原過程中,會(huì)產(chǎn)生大量的活性氧,然后誘導(dǎo)細(xì)胞的氧化應(yīng)激反應(yīng),進(jìn)而導(dǎo)致細(xì)胞DNA直接損傷以及細(xì)胞修復(fù)系統(tǒng)的遠(yuǎn)期損傷,同時(shí)活性氧對(duì)細(xì)胞的刺激產(chǎn)生一系列的信號(hào)傳導(dǎo)級(jí)聯(lián)反應(yīng)。有實(shí)驗(yàn)證明六價(jià)鉻能夠影響細(xì)胞凋亡信號(hào)傳導(dǎo)通路。 P38MAPK能夠被各種生長(zhǎng)因子、炎性細(xì)胞因子以及各種物理化學(xué)刺激因素激活,激活后的P38激酶能夠調(diào)節(jié)細(xì)胞的生長(zhǎng)、分化、凋亡,進(jìn)而影響其下游通路。半胱氨酸的天冬氨酸蛋白水解酶-3在細(xì)胞凋亡中起著重要作用,在Caspase蛋白酶級(jí)聯(lián)切割過程中起核心因子作用。近年來P38-MAPK和Caspase-3是介導(dǎo)細(xì)胞增殖和凋亡等熱點(diǎn)問題的重要因子。 目的： 基于上述分析,該研究通過建立體外細(xì)胞模型,培養(yǎng)人肺癌上皮細(xì)胞(A549細(xì)胞),然后使其暴露于不同濃度的重鉻酸鉀染毒液中,觀察各分組凋亡率水平,并且從蛋白表達(dá)水平上分析重鉻酸鉀對(duì)A549細(xì)胞激活的磷酸化P38、總P38和Caspase-3等相關(guān)凋亡蛋白的表達(dá)水平,進(jìn)而探討重鉻酸鉀誘導(dǎo)A549細(xì)胞凋亡機(jī)制,為進(jìn)一步了解重鉻酸鉀誘導(dǎo)肺癌機(jī)制提供理論依據(jù)。 方法： 1.首先建立A549細(xì)胞的體外細(xì)胞模型。 2.利用MTT比色法來測(cè)定0μmol/L、2.5μmol/L、5μmol/L、10μmol/L的重鉻酸鉀對(duì)A549細(xì)胞生長(zhǎng)的影響,并且確定合適的染毒濃度。 3.用流式細(xì)胞儀檢測(cè)重鉻酸鉀對(duì)A549細(xì)胞凋亡的影響,觀察加入P38抑制劑后染毒組細(xì)胞凋亡率和抑制劑組細(xì)胞凋亡率的表達(dá)水平。 4. Western blot法檢測(cè)重鉻酸鉀染毒后P-P38、P38和Caspase-3的蛋白表達(dá)水平。 5.統(tǒng)計(jì)分析。運(yùn)用SPSS12.0統(tǒng)計(jì)軟件,數(shù)據(jù)用x±s表示,采用t檢驗(yàn)、單因素方差分析(analysis of variance,ANOVA)對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析(檢驗(yàn)水準(zhǔn)a=0.05)。 結(jié)果： 1.MTT結(jié)果：染毒濃度為40μmol/L時(shí),A549細(xì)胞的增殖抑制率為76.25%。根據(jù)細(xì)胞染毒后的細(xì)胞活性不同,確定了IC50=7.6μmol/L,選用染毒濃度為0μmol/L、2.5μmol/L、5μmol/L、10μmol/L進(jìn)行下一步實(shí)驗(yàn)。 2.流式細(xì)胞儀檢測(cè)細(xì)胞凋亡結(jié)果為：當(dāng)重鉻酸鉀染毒濃度是0μmol/L的時(shí)候,正常染毒組A組和加抑制劑染毒組B組相比升高,差異無統(tǒng)計(jì)學(xué)意義(P0.05)；當(dāng)重鉻酸鉀染毒濃度是2.5μmol/L、5μmol/L、10μmol/L的時(shí)候,正常染毒組A組和加抑制劑染毒組B組相比下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05) 3. Westblot實(shí)驗(yàn)檢測(cè)蛋白表達(dá)：重鉻酸鉀正常染毒組的P-P38蛋白和Caspase-3蛋白相對(duì)表達(dá)量分別比起加抑制劑重鉻酸鉀組的P-P38蛋白和Caspase-3蛋白相對(duì)表達(dá)量升高,差異有統(tǒng)計(jì)學(xué)意義(P0.001)；各分組細(xì)胞的P38總蛋白在不同刺激下差異沒有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論： 重鉻酸鉀能誘導(dǎo)A549細(xì)胞凋亡,呈劑量反應(yīng)關(guān)系；P38信號(hào)傳導(dǎo)通路在重鉻酸鉀致A549細(xì)胞凋亡中起促進(jìn)作用；SB203580能抑制P38信號(hào)傳導(dǎo)通路,對(duì)A549細(xì)胞凋亡其保護(hù)作用。
[Abstract]:Chromium (Cr) as a common chemical element, the valence of -2 to +6, in which +3 and +6 are the most common valence. Trivalent chromium is non-toxic in the effective dose, are essential nutrients for human and animal. Six chromium is a toxic agent, has carcinogenicity, mutagenicity, teratogenicity, immunotoxicity, reproductive toxicity, neurotoxicity, is a kind of heavy metal pollutants.
Six chromium exists widely in cigarette smoke, car exhaust and living environment, mainly used in electroplating industry, leather industry, chemical industry, dyestuff industry, metallurgical industry, and the welding of stainless steel and wood processing industries. Six chromium can restore the system to restore the cells, which produce large amounts of low valence chromium ions, low price chromium ions easily combined with DNA to produce DNA adducts; chromium reduction process, will produce large amounts of reactive oxygen species, and then induced cellular oxidative stress response, causing long-term damage cell DNA damage and cell repair system, and stimulate the active oxygen to the cells produce a series of signal transduction cascade reaction. Experiments have proved that six chromium can affect apoptosis signaling pathways.
P38MAPK can be a variety of growth factors, activation of inflammatory cytokines and various physical and chemical stimuli, after activation of P38 kinase can regulate cell growth, differentiation, apoptosis, thereby affecting the downstream pathway. Cysteine aspartic acid protease -3 in cell apoptosis plays an important role, played a key role in the Caspase factor protease cascade in the cutting process. In recent years, P38-MAPK and Caspase-3 is an important factor mediated cell proliferation and apoptosis and other hot issues.
Objective:
Based on the above analysis, this research established a cell model in vitro, cultured human lung epithelial cells (A549 cells), which is then exposed to potassium dichromate exposure to different concentrations of liquid, observe the apoptosis rate of each group, and from the protein expression of phosphorylated P38 potassium dichromate on A549 cell activation level, the expression level of P38 and total Caspase-3 protein related to apoptosis, and to explore the mechanism of A549 cell apoptosis induced by potassium dichromate, in order to further understand the mechanism of lung cancer induced by potassium dichromate and provide a theoretical basis.
Method錛，

本文編號(hào)：1423183