本文關(guān)鍵詞：HIF-1α與BCL-2的交互作用對(duì)非小細(xì)胞肺癌細(xì)胞放射敏感性的影響　出處：《安徽醫(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： 放射 非小細(xì)胞肺癌 放射敏感性 乏氧誘導(dǎo)因子-1α B細(xì)胞淋巴瘤-2

【摘要】：目的:我國(guó)肺癌的發(fā)病率和死亡率均居惡性腫瘤的首位,并且隨著老齡化社會(huì)的發(fā)展,肺癌對(duì)人們健康的影響日益突出。大多數(shù)非小細(xì)胞肺癌(non-small cell lung cancer,NSCLC)患者被確診時(shí)已經(jīng)處于中晚期,而中晚期肺癌病人的5年生存率不足2%。放療是治療NSCLC的重要手段之一,然而由于腫瘤內(nèi)部存在著乏氧細(xì)胞,會(huì)對(duì)常規(guī)放療產(chǎn)生抵抗作用。乏氧誘導(dǎo)因子-1α(hypoxia inducible factor-1α,HIF-1α)是一種重要的轉(zhuǎn)錄因子,與腫瘤的惡性表型密切相關(guān),可調(diào)節(jié)下游多種靶基因,使細(xì)胞適應(yīng)乏氧環(huán)境,并降低腫瘤細(xì)胞的放射敏感性。B細(xì)胞淋巴瘤-2基因(B-cell lymphoma-2,BCL-2)作為一種重要的抗凋亡基因在多種腫瘤組織內(nèi)部存在著高表達(dá),可以促使腫瘤細(xì)胞在不利的條件下存活下來。在放療中BCL-2通過抑制腫瘤細(xì)胞的凋亡,使腫瘤細(xì)胞對(duì)放療產(chǎn)生抵抗作用。本研究以人NSCLC細(xì)胞—H1299細(xì)胞為試驗(yàn)?zāi)Ｐ?觀察受照射細(xì)胞中的HIF-1α表達(dá)是否影響B(tài)CL-2的表達(dá)水平,以及HIF-1α/BCL-2信號(hào)對(duì)NSCLC細(xì)胞放射敏感性的調(diào)節(jié)作用。方法:本研究采用2種帶有高表達(dá)HIF-1α質(zhì)粒的NSCLC細(xì)胞(H1299細(xì)胞)作為實(shí)驗(yàn)對(duì)象,其中帶有突變型HIF-1α質(zhì)粒的H1299細(xì)胞可在常氧下高表達(dá)HIF-1α(簡(jiǎn)稱為H1299/M-HIF-1α細(xì)胞),帶有野生型HIF-1α質(zhì)粒的H1299細(xì)胞在乏氧狀態(tài)下高表達(dá)HIF-1α(簡(jiǎn)稱為H1299/W-HIF-1α細(xì)胞),同時(shí)以帶有空白質(zhì)粒的H1299細(xì)胞(簡(jiǎn)稱為H1299/F細(xì)胞)和親本H1299細(xì)胞作為對(duì)照。首先采用Western Blot方法檢測(cè)四種細(xì)胞在常氧、乏氧、HIF-1α靶向抑制劑—3-(5'-羥甲基-2'-呋喃基)-1-苯甲基吲唑(3-[50-hydroxymethyl-20furyl]-1-benzyl in dazole,YC-1)和BCL-2靶向抑制劑—2-乙基-6氨基-4-溴(1-氰-2-乙氧-2表乙)-4-色原烯-3酸(2-Amino-6-bromo-α-cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acidethylester,ha14-1)預(yù)處理情況下的hif-1α和bcl-2蛋白的表達(dá)情況,并在相同預(yù)處理?xiàng)l件下對(duì)4種細(xì)胞進(jìn)行x線照射,照射劑量點(diǎn)分別設(shè)定為0、1、2、3、5和7gy。采用克隆形成實(shí)驗(yàn)觀察細(xì)胞的存活情況,并通過單擊多靶模型(multi-target-singlehittingmodel)計(jì)算不同細(xì)胞的d0值進(jìn)行比較,通過細(xì)胞增殖實(shí)驗(yàn)計(jì)算細(xì)胞群體倍增時(shí)間(populationdoublingtime,pdt),觀察細(xì)胞在不同處理?xiàng)l件下的增殖變化,并進(jìn)一步利用westernblot檢測(cè)受照射細(xì)胞中hif-1α和bcl-2蛋白的表達(dá)變化。結(jié)果:westernblot實(shí)驗(yàn)顯示,常氧條件下h1299/m-hif-1α細(xì)胞高表達(dá)hif-1α和bcl-2蛋白。采用氯化鈷(cobaltchloride,cocl2)進(jìn)行化學(xué)乏氧處理后,h1299/w-hif-1α、h1299/f和h1299細(xì)胞均高表達(dá)hif-1α和bcl-2蛋白,以h1299/w-hif-1α最為明顯,當(dāng)采用yc-1預(yù)處理后,4種細(xì)胞的hif-1α水平均明顯降低,伴隨bcl-2的表達(dá)水平下降。進(jìn)而采用ha14-1處理后,可顯著下調(diào)4種細(xì)胞中bcl-2的表達(dá)。細(xì)胞照射后的westernblot結(jié)果顯示,在常氧情況下,x線照射可明顯誘導(dǎo)4種細(xì)胞hif-1α和bcl-2的表達(dá),而經(jīng)乏氧和yc-1預(yù)處理的4種細(xì)胞hif-1α和bcl-2水平在中、低劑量的x線照射后并未出現(xiàn)顯著變化,在高劑量照射后2種蛋白的表達(dá)水平則明顯降低,采用ha14-1預(yù)處理后,4種受照射細(xì)胞中的hif-1α和bcl-2水平均明顯降低�？寺⌒纬蓪�(shí)驗(yàn)結(jié)果顯示,常氧條件下,m細(xì)胞的d0值(d0(m)=4.375gy)明顯高于其他3種細(xì)胞(分別為d0(w)=3.845gy;d0(f)=3.311gy;d0(h1299)=3.021gy);乏氧下m和w細(xì)胞的存活曲線和d0值(d0(m)=4.787gy;d0(w)=4.679gy)明顯高于其他2種細(xì)胞(d0(f)=3.877gy;d0(h1299)=4.070gy),并且4種細(xì)胞的氧增比(oxygenenhancementratios,oers)分別為:oer(m)=1.094,oer(w)=1.217,oer(f)=1.171,oer(h1299)=1.347;yc-1預(yù)處理后,4種細(xì)胞的增敏比(sensitivityenhancementratios,sers)分別為:ser(m)=1.225,ser(w)=1.128,ser(f)=1.146,ser(h1299)=1.457;ha14-1預(yù)處理后,4種細(xì)胞的ser值為:ser(m)=1.4,ser(w)=1.828,ser(f)=1.335,ser(h1299)=1.221。在細(xì)胞增殖實(shí)驗(yàn)中,常氧條件下,受照射細(xì)胞的pdt會(huì)隨劑量加大逐漸增加,而在乏氧狀態(tài)下,受照射細(xì)胞的pdt并沒有明顯的增加,yc-1預(yù)處理后,低劑量照射并未明顯增加乏氧細(xì)胞的PDT,HA14-1預(yù)處理則顯著延長(zhǎng)受照射細(xì)胞的PDT,但是增加程度低于常氧受照射細(xì)胞。結(jié)論:當(dāng)NSCLC細(xì)胞處于乏氧狀態(tài)下,HIF-1α表達(dá)水平升高,對(duì)X線照射產(chǎn)生抵抗作用,其作用機(jī)制與HIF-1α/BCL-2信號(hào)通路的活化有關(guān),同時(shí)在有氧狀態(tài)下,低LET射線可誘導(dǎo)HIF-1α的表達(dá),進(jìn)而導(dǎo)致HIF-1α/BCL-2信號(hào)活化,從而引起細(xì)胞的抗性作用。因此阻斷HIF-1α/BCL-2信號(hào)可以提高NSCLC細(xì)胞的放射敏感性。
[Abstract]:Objective: in China, the incidence and mortality of lung cancer in malignant tumor, and with the development of aging society, the impact on people's health, lung cancer has become increasingly prominent. The majority of non small cell lung cancer (non-small cell lung cancer, NSCLC) patients were diagnosed at an advanced stage, and in patients with advanced lung cancer 5 years the survival rate of less than 2%. radiotherapy is one of the important means of treatment of NSCLC, however, due to the tumor exists inside the hypoxic cells are resistant to conventional radiotherapy. The role of hypoxia inducible factor alpha -1 (hypoxia inducible factor-1 HIF-1 alpha, alpha) is an important transcription factor, and is closely related to the malignant phenotype of tumor, adjustable a variety of downstream target genes, make the cells adapt to hypoxia environment, and reduce the radiation sensitivity of tumor cells to.B cell lymphoma -2 gene (B-cell lymphoma-2 BCL-2) is one of the most important anti apoptotic genes in a variety of There is a high expression of tumor tissue, can promote tumor cell survival under adverse conditions. In the radiotherapy of BCL-2 by inhibiting the apoptosis of tumor cells, the tumor cells resistant to radiotherapy. In this study, human NSCLC cells H1299 cells as experimental model, to observe the expression will affect the expression level of BCL-2 irradiation cells in the HIF-1 and HIF-1 alpha, alpha /BCL-2 signal on the radiosensitivity of NSCLC cell regulation. Methods: This study used 2 species with high HIF-1 expression plasmid of NSCLC cells (H1299 cells) as the experimental object, with the mutant HIF-1 plasmid in H1299 cells with high expression of HIF-1 alpha in normoxia (referred to as H1299/M-HIF-1 alpha cells), with wild type HIF-1 plasmid in H1299 cells under hypoxia and high expression of HIF-1 alpha (H1299/W-HIF-1 alpha cells), at the same time with blank plasmid H1299 Cells (H1299/F cells) and H1299 cells were used as control. Firstly using Western Blot method to detect four kinds of cells in normoxia, hypoxia, HIF-1 alpha inhibitor 3- (5'- hydroxymethyl -2'- furyl) -1- phenyl methyl indazole (3-[50-hydroxymethyl-20furyl]-1-benzyl in, dazole, YC-1) and BCL-2 inhibitor 2- -6 -4- (1- amino ethyl bromide cyanide -2- ethoxy -2 b) -4- chromene -3 acid (2-Amino-6-bromo- alpha -cyano-3- (ethoxycarbonyl) -4H-1-benzopyran-4-acetic acidethylester, HA14-1) expression of pretreatment conditions of HIF-1 alpha and bcl-2 protein, and X-ray irradiation on the 4 kinds of cells in the same pretreatment condition, radiation dose point set survival for 0,1,2,3,5 and 7gy. cells were observed by clone formation experiment, and by clicking on the multiple target model (multi-target-singlehittingmodel) to calculate d0 values were compared with cells, The cell proliferation assay cell population doubling time was calculated (populationdoublingtime, PDT), to observe the change of cell proliferation in the different treatment conditions, and further use of Westernblot to detect expression of HIF-1 alpha and bcl-2 protein in irradiated cells. Results: Westernblot assay showed that HIF-1 alpha and bcl-2 protein h1299/m-hif-1 alpha cell high expression under normoxic conditions using cobalt chloride (cobaltchloride, CoCl2) chemical hypoxia treatment, h1299/w-hif-1 alpha, h1299/f and H1299 cells had high expression of HIF-1 and bcl-2 protein, h1299/w-hif-1 alpha is most obvious, when using YC-1 after pretreatment, HIF-1 alpha level of 4 kinds of cells were significantly decreased with the decrease of bcl-2 expression level. Then the HA14-1 after treatment significantly reduced the expression of 4 kinds of cells in Bcl-2 cells after irradiation. The Westernblot results showed that under normoxia, X irradiation can induce 4 縐嶇粏鑳?yōu)hif-1偽鍜宐cl-2鐨勮〃杈，

本文編號(hào)：1383625