發(fā)布時間：2018-01-14 10:25

  本文關鍵詞：P38促分裂素原活化蛋白激酶在重鉻酸鉀誘導A549細胞凋亡中的作用　出處：《鄭州大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： 重鉻酸鉀 A549細胞 P38MAPK Caspase-3 細胞凋亡

【摘要】：鉻(Cr)作為一種常見的化學元素,化合價態(tài)為-2到+6,其中以+3和+6價態(tài)最為常見。三價鉻在有效劑量下是無毒的,是人和動物必需的營養(yǎng)素。六價鉻是一種劇毒劑,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。六價鉻能夠被細胞內的還原系統(tǒng)還原,進而產(chǎn)生大量的低價態(tài)鉻離子,低價態(tài)鉻離子容易與DNA結合產(chǎn)生DNA加合物；鉻被還原過程中,會產(chǎn)生大量的活性氧,然后誘導細胞的氧化應激反應,進而導致細胞DNA直接損傷以及細胞修復系統(tǒng)的遠期損傷,同時活性氧對細胞的刺激產(chǎn)生一系列的信號傳導級聯(lián)反應。有實驗證明六價鉻能夠影響細胞凋亡信號傳導通路。 P38MAPK能夠被各種生長因子、炎性細胞因子以及各種物理化學刺激因素激活,激活后的P38激酶能夠調節(jié)細胞的生長、分化、凋亡,進而影響其下游通路。半胱氨酸的天冬氨酸蛋白水解酶-3在細胞凋亡中起著重要作用,在Caspase蛋白酶級聯(lián)切割過程中起核心因子作用。近年來P38-MAPK和Caspase-3是介導細胞增殖和凋亡等熱點問題的重要因子。 目的： 基于上述分析,該研究通過建立體外細胞模型,培養(yǎng)人肺癌上皮細胞(A549細胞),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,并且從蛋白表達水平上分析重鉻酸鉀對A549細胞激活的磷酸化P38、總P38和Caspase-3等相關凋亡蛋白的表達水平,進而探討重鉻酸鉀誘導A549細胞凋亡機制,為進一步了解重鉻酸鉀誘導肺癌機制提供理論依據(jù)。 方法： 1.首先建立A549細胞的體外細胞模型。 2.利用MTT比色法來測定0μmol/L、2.5μmol/L、5μmol/L、10μmol/L的重鉻酸鉀對A549細胞生長的影響,并且確定合適的染毒濃度。 3.用流式細胞儀檢測重鉻酸鉀對A549細胞凋亡的影響,觀察加入P38抑制劑后染毒組細胞凋亡率和抑制劑組細胞凋亡率的表達水平。 4. Western blot法檢測重鉻酸鉀染毒后P-P38、P38和Caspase-3的蛋白表達水平。 5.統(tǒng)計分析。運用SPSS12.0統(tǒng)計軟件,數(shù)據(jù)用x±s表示,采用t檢驗、單因素方差分析(analysis of variance,ANOVA)對數(shù)據(jù)進行統(tǒng)計分析(檢驗水準a=0.05)。 結果： 1.MTT結果：染毒濃度為40μmol/L時,A549細胞的增殖抑制率為76.25%。根據(jù)細胞染毒后的細胞活性不同,確定了IC50=7.6μmol/L,選用染毒濃度為0μmol/L、2.5μmol/L、5μmol/L、10μmol/L進行下一步實驗。 2.流式細胞儀檢測細胞凋亡結果為：當重鉻酸鉀染毒濃度是0μmol/L的時候,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)計學意義(P0.05)；當重鉻酸鉀染毒濃度是2.5μmol/L、5μmol/L、10μmol/L的時候,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)計學意義(P0.05) 3. Westblot實驗檢測蛋白表達：重鉻酸鉀正常染毒組的P-P38蛋白和Caspase-3蛋白相對表達量分別比起加抑制劑重鉻酸鉀組的P-P38蛋白和Caspase-3蛋白相對表達量升高,差異有統(tǒng)計學意義(P0.001)；各分組細胞的P38總蛋白在不同刺激下差異沒有統(tǒng)計學意義(P0.05)。 結論： 重鉻酸鉀能誘導A549細胞凋亡,呈劑量反應關系；P38信號傳導通路在重鉻酸鉀致A549細胞凋亡中起促進作用；SB203580能抑制P38信號傳導通路,對A549細胞凋亡其保護作用。
[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：1423183