HIF-1α与BCL-2的交互作用对非小细胞肺癌细胞放射敏感性的影响

发布时间：2018-01-05 15:05

  本文关键词：HIF-1α与BCL-2的交互作用对非小细胞肺癌细胞放射敏感性的影响　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 放射 非小细胞肺癌 放射敏感性 乏氧诱导因子-1α B细胞淋巴瘤-2

【摘要】：目的:我国肺癌的发病率和死亡率均居恶性肿瘤的首位,并且随着老龄化社会的发展,肺癌对人们健康的影响日益突出。大多数非小细胞肺癌(non-small cell lung cancer,NSCLC)患者被确诊时已经处于中晚期,而中晚期肺癌病人的5年生存率不足2%。放疗是治疗NSCLC的重要手段之一,然而由于肿瘤内部存在着乏氧细胞,会对常规放疗产生抵抗作用。乏氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)是一种重要的转录因子,与肿瘤的恶性表型密切相关,可调节下游多种靶基因,使细胞适应乏氧环境,并降低肿瘤细胞的放射敏感性。B细胞淋巴瘤-2基因(B-cell lymphoma-2,BCL-2)作为一种重要的抗凋亡基因在多种肿瘤组织内部存在着高表达,可以促使肿瘤细胞在不利的条件下存活下来。在放疗中BCL-2通过抑制肿瘤细胞的凋亡,使肿瘤细胞对放疗产生抵抗作用。本研究以人NSCLC细胞—H1299细胞为试验模型,观察受照射细胞中的HIF-1α表达是否影响BCL-2的表达水平,以及HIF-1α/BCL-2信号对NSCLC细胞放射敏感性的调节作用。方法:本研究采用2种带有高表达HIF-1α质粒的NSCLC细胞(H1299细胞)作为实验对象,其中带有突变型HIF-1α质粒的H1299细胞可在常氧下高表达HIF-1α(简称为H1299/M-HIF-1α细胞),带有野生型HIF-1α质粒的H1299细胞在乏氧状态下高表达HIF-1α(简称为H1299/W-HIF-1α细胞),同时以带有空白质粒的H1299细胞(简称为H1299/F细胞)和亲本H1299细胞作为对照。首先采用Western Blot方法检测四种细胞在常氧、乏氧、HIF-1α靶向抑制剂—3-(5'-羟甲基-2'-呋喃基)-1-苯甲基吲唑(3-[50-hydroxymethyl-20furyl]-1-benzyl in dazole,YC-1)和BCL-2靶向抑制剂—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)预处理情况下的hif-1α和bcl-2蛋白的表达情况,并在相同预处理条件下对4种细胞进行x线照射,照射剂量点分别设定为0、1、2、3、5和7gy。采用克隆形成实验观察细胞的存活情况,并通过单击多靶模型(multi-target-singlehittingmodel)计算不同细胞的d0值进行比较,通过细胞增殖实验计算细胞群体倍增时间(populationdoublingtime,pdt),观察细胞在不同处理条件下的增殖变化,并进一步利用westernblot检测受照射细胞中hif-1α和bcl-2蛋白的表达变化。结果:westernblot实验显示,常氧条件下h1299/m-hif-1α细胞高表达hif-1α和bcl-2蛋白。采用氯化钴(cobaltchloride,cocl2)进行化学乏氧处理后,h1299/w-hif-1α、h1299/f和h1299细胞均高表达hif-1α和bcl-2蛋白,以h1299/w-hif-1α最为明显,当采用yc-1预处理后,4种细胞的hif-1α水平均明显降低,伴随bcl-2的表达水平下降。进而采用ha14-1处理后,可显著下调4种细胞中bcl-2的表达。细胞照射后的westernblot结果显示,在常氧情况下,x线照射可明显诱导4种细胞hif-1α和bcl-2的表达,而经乏氧和yc-1预处理的4种细胞hif-1α和bcl-2水平在中、低剂量的x线照射后并未出现显著变化,在高剂量照射后2种蛋白的表达水平则明显降低,采用ha14-1预处理后,4种受照射细胞中的hif-1α和bcl-2水平均明显降低。克隆形成实验结果显示,常氧条件下,m细胞的d0值(d0(m)=4.375gy)明显高于其他3种细胞(分别为d0(w)=3.845gy;d0(f)=3.311gy;d0(h1299)=3.021gy);乏氧下m和w细胞的存活曲线和d0值(d0(m)=4.787gy;d0(w)=4.679gy)明显高于其他2种细胞(d0(f)=3.877gy;d0(h1299)=4.070gy),并且4种细胞的氧增比(oxygenenhancementratios,oers)分别为:oer(m)=1.094,oer(w)=1.217,oer(f)=1.171,oer(h1299)=1.347;yc-1预处理后,4种细胞的增敏比(sensitivityenhancementratios,sers)分别为:ser(m)=1.225,ser(w)=1.128,ser(f)=1.146,ser(h1299)=1.457;ha14-1预处理后,4种细胞的ser值为:ser(m)=1.4,ser(w)=1.828,ser(f)=1.335,ser(h1299)=1.221。在细胞增殖实验中,常氧条件下,受照射细胞的pdt会随剂量加大逐渐增加,而在乏氧状态下,受照射细胞的pdt并没有明显的增加,yc-1预处理后,低剂量照射并未明显增加乏氧细胞的PDT,HA14-1预处理则显著延长受照射细胞的PDT,但是增加程度低于常氧受照射细胞。结论:当NSCLC细胞处于乏氧状态下,HIF-1α表达水平升高,对X线照射产生抵抗作用,其作用机制与HIF-1α/BCL-2信号通路的活化有关,同时在有氧状态下,低LET射线可诱导HIF-1α的表达,进而导致HIF-1α/BCL-2信号活化,从而引起细胞的抗性作用。因此阻断HIF-1α/BCL-2信号可以提高NSCLC细胞的放射敏感性。
[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 绉嶇粏鑳瀐if-1伪鍜宐cl-2鐨勮〃杈，

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