本文選題：低氧　切入點(diǎn)：K562細(xì)胞　出處：《浙江大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：低氧是實(shí)體瘤發(fā)生發(fā)展過(guò)程中存在的環(huán)境條件之一,是腫瘤發(fā)生惡性轉(zhuǎn)化甚至轉(zhuǎn)移的啟動(dòng)因子。同時(shí),缺氧誘導(dǎo)因子(Hypoxia-inducible factor, HIF)與腫瘤的生長(zhǎng)和發(fā)展密切相關(guān),是近年來(lái)國(guó)內(nèi)外抗腫瘤研究的一個(gè)重要的治療靶點(diǎn)。3-(4-溴苯基)-2-(乙砜基)-6-甲基喹喔啉-1,4-二氧化物(Q39)是由替拉扎明(tirapazamine,TPZ)結(jié)構(gòu)修飾得到的具有自主知識(shí)產(chǎn)權(quán)的全新化合物,可在低氧條件下誘導(dǎo)腫瘤細(xì)胞發(fā)生凋亡,因此對(duì)其低氧抗腫瘤作用進(jìn)行研究,將有助于了解Q39乃至TPZ類似物發(fā)揮低氧誘導(dǎo)腫瘤凋亡的機(jī)制,為相關(guān)低氧抗腫瘤藥物的研究和開(kāi)發(fā)提供科學(xué)依據(jù)。本研究探索了Q39在低氧條件下誘導(dǎo)人白血病K562細(xì)胞和人肝癌Bel-7402細(xì)胞凋亡的作用機(jī)制。 方法：1)MTT法測(cè)定Q39對(duì)人白血病K562細(xì)胞和肝癌Bel-7402細(xì)胞的增殖抑制作用,計(jì)算其半數(shù)抑制濃度(IC5o)。2)采用DAPI染色法觀察K562細(xì)胞的凋亡情況。3)采用PI染色結(jié)合流式細(xì)胞術(shù)測(cè)定K562細(xì)胞和Bel-7402細(xì)胞的凋亡率。4)JC-1染色法觀察Q39對(duì)K562細(xì)胞線粒體膜電位(△Ψm)的影響。5)采用Western blotting法檢測(cè)低氧條件下細(xì)胞內(nèi)procaspase-3、cleaved caspase-3、PARP、 Bax、Bcl-2、p-ERK、p-JNK、p-p38、VEGF和HIF-1α等蛋白表達(dá)的變化。6)采用免疫熒光法觀察HIF-1α經(jīng)Q39作用后的入核情況。 結(jié)果： 一、Q39在低氧條件下誘導(dǎo)人白血病K562細(xì)胞的凋亡的作用機(jī)制研究：1)Q39在低氧條件(3%O2)下能夠明顯抑制人白血病K562細(xì)胞的增殖,其IC50為(0.21±0.05)μmol/L。2)經(jīng)DAPI染色證實(shí),Q39作用6h后開(kāi)始誘導(dǎo)K562細(xì)胞發(fā)生凋亡,后期細(xì)胞體積縮小,并出現(xiàn)凋亡小體。3)流式細(xì)胞術(shù)結(jié)果顯示：20%02環(huán)境中K562細(xì)胞與Q39共孵育0、12、24和48h的凋亡率分別為5.5%、5.1%、4.6%和24.4%,而3%02環(huán)境中相應(yīng)的誘導(dǎo)凋亡率分別達(dá)到了4.1%、34.5%、42.5和60.1%,表現(xiàn)出明顯的時(shí)效依賴性及低氧選擇性。4)JC-1染色實(shí)驗(yàn)結(jié)果顯示：孵育0、6、12、24和48h后Q39使K562細(xì)胞的△中m呈明顯下降趨勢(shì),并且具有時(shí)效依賴關(guān)系,提示Q39誘導(dǎo)K562的凋亡可能是通過(guò)線粒體信號(hào)轉(zhuǎn)導(dǎo)通路實(shí)現(xiàn)。5) Western-blotting結(jié)果顯示：Q39在低氧條件下降低K562細(xì)胞的HIF-1α、VEGF、procaspase-3和Bcl-2蛋白表達(dá),顯著增加Bax和cleaved caspase-3蛋白表達(dá),并且促使PARP裂解。Q39顯著下調(diào)了p-ERK和p-p38的表達(dá)水平,同時(shí)增加了p-JNK的表達(dá),兩者的調(diào)節(jié)具有時(shí)效依賴性。 二、Q39在低氧條件下誘導(dǎo)人肝癌Bel-7402細(xì)胞凋亡的作用機(jī)制研究：1)Q39在低氧條件下抑制人肝癌Bel-7402細(xì)胞增殖,其ICso為(3.87±0.56) μmol/L。2)Q39在低氧條件下誘導(dǎo)Bel-7402細(xì)胞凋亡,5、10、20μmol/L的Q39引起的凋亡率分別為(27.1±4.8)%、(50.2±5.6)%和(62.5±6.2)%,其誘導(dǎo)凋亡作用呈濃度依賴性增加。3)隨著低氧作用時(shí)間的延長(zhǎng),ERKl/2,JN和p38總蛋白水平變化不明顯；而各自的磷酸化水平卻呈現(xiàn)良好的時(shí)效依賴性,說(shuō)明低氧條件激活MAPK通路表達(dá)。4)Q39在低氧條件下抑制人肝癌細(xì)胞Bel-7402的ERK1/2的磷酸化,但并不影響p38和JNK的磷酸化,說(shuō)明Q39在低氧條件下發(fā)揮抗腫瘤活性時(shí)可能主要影響ERKl/2的活性。5)免疫熒光法證實(shí)在3%氧壓環(huán)境下Q39作用16小時(shí)后能明顯阻斷低氧誘導(dǎo)的HIF-1α入核過(guò)程,影響其細(xì)胞核內(nèi)外分布,減少具備活性的形成HIF-1α;同時(shí)Western blot法檢測(cè)Q39(0-20μmol/L)作用后HIF-1α蛋白表達(dá),結(jié)果發(fā)現(xiàn)Q39明顯降低低氧激活的HIF-1α蛋白表達(dá),說(shuō)明Q39可能通過(guò)抑制ERK1/2磷酸化影響HIF-1α的轉(zhuǎn)位,最終導(dǎo)致HIF-1α蛋白表達(dá)水平降低,從而引起細(xì)胞的凋亡。 結(jié)論：Q39在低氧條件下對(duì)K562細(xì)胞和Bel-7402細(xì)胞具有較好的抑制作用,并能通過(guò)降低HIF-1α蛋白表達(dá)和調(diào)節(jié)其他凋亡相關(guān)蛋白表達(dá)誘導(dǎo)細(xì)胞凋亡。Q39誘導(dǎo)K562細(xì)胞凋亡可能是通過(guò)激活依賴于caspase級(jí)聯(lián)反應(yīng)的線粒體通路和低氧相關(guān)的HIF-1信號(hào)傳導(dǎo)通路實(shí)現(xiàn)的。Q39誘導(dǎo)Bel-7402細(xì)胞凋亡可能通過(guò)調(diào)控ERKl/2信號(hào)通路,阻斷HIF-1α入核過(guò)程,促進(jìn)HIF-1α蛋白降解,減少HIF-1α蛋白合成。上述實(shí)驗(yàn)結(jié)果將有利于我們發(fā)現(xiàn)低氧條件下新的抗腫瘤藥物作用靶點(diǎn),對(duì)阻斷和抑制HIF-1α介導(dǎo)的低氧腫瘤生長(zhǎng)和惡性化提供新的思路。
[Abstract]:Objective: hypoxia is one of the process of tumor occurrence and development of environmental conditions, is the malignant transformation of tumor metastasis and promoter. At the same time, hypoxia inducible factor (Hypoxia-inducible, factor, HIF) is closely associated with tumor growth and development, is an important therapeutic target of.3- in recent years at home and abroad of anti-tumor research (4- Bromophenyl) -2- (ethyl sulfone) -6- methyl quinoxaline -1,4- dioxide (Q39) is composed of tirapazamine (tirapazamine, TPZ) modified structure obtained with independent intellectual property rights of the new compounds can induce tumor cell apoptosis under hypoxic conditions, so the research on the anti-tumor effect of hypoxia, will help to understand Q39 and TPZ analogues play mechanism of hypoxia induced apoptosis of the tumor cells, and provide a scientific basis for the research and development related to hypoxia antitumor drugs. This study explored Q39 in hypoxia induced The mechanism of apoptosis of human leukemia K562 cells and human hepatocellular carcinoma Bel-7402 cells.
Methods: 1) the determination of inhibitory effect of Q39 on human leukemia cells K562 and human hepatoma Bel-7402 cells proliferation by MTT method, to calculate the half inhibitory concentration (IC5o).2) was observed by DAPI staining and the apoptosis of K562 cells.3) using PI staining combined with K562 cells and Bel-7402 cells apoptosis were measured by flow cytometry. The rate of.4 JC-1) staining method to observe the effect of Q39 on mitochondrial membrane potential of K562 cells (lpli m) the effect of.5 procaspase-3 cells in hypoxic conditions) were detected by Western blotting cleaved Caspase-3, PARP, Bax, Bcl-2, p-ERK, p-JNK, p-p38, VEGF and HIF-1 expression of.6 alpha protein) immunofluorescence was used to observe HIF-1 alpha after the action of Q39 into the nucleus.
Result錛，

本文編號(hào)：1565187