HIF-1α和BCL2在涎腺腺样囊性癌组织中的表达以及对细胞放射敏感性的影响

发布时间：2018-05-21 09:16

本文选题：乏氧诱导因子-1α + B淋巴细胞因子2　；参考：《安徽医科大学》2017年硕士论文

【摘要】：第一部分HIF-1α/BCL-2在SACC中的表达以及与临床病理特征的关系目的:乏氧诱导因子-1α(Hypoxia inducible factor-1α,HIF-1α)在很多癌症患者的预后及治疗的反应性方面扮演着重要角色,本研究观察HIF-1α及B淋巴细胞因子-2(B-cell lymphoma-2,BCL-2)在涎腺腺样囊性癌(Salivary gland adenoid cystic carcinoma,SACC)中的表达情况,并分析其与临床病理特征间的相互关系。方法:收集来自安徽医科大学第一附属医院的56例SACC患者的手术切除蜡块,通过免疫组织化学染色,观察HIF-1α和BCL-2的表达。然后,通过半定量免疫反应性积分(Semiquantitatively immunoreactive scoer,IRS)的方法分析SACC中HIF-1α和BCL-2表达水平。结果:在36例(64.3%)中存在HIF-1α的表达(IRS≥3),且表达水平于TNM分期紧密相关(P=0.03),同时,HIF-1α的表达水平于SACC的组织来源也具有统计学关联(P=0.004)。在55例(98.2%)中发现有BCL-2的表达,其中44例(78.6%)呈强阳性。统计发现,HIF-1α的IRS于BCL-2具有相关性(β=0.236,P=0.013),并且HIF-1α于TNM分期的相关性依赖于BCL-2的高表达(Z=2.43,P=0.015)。结论:通过检测HIF-1α的蛋白表达将有助于SACC患者的预后评价。第二部分HIF-1α对SACC细胞放射敏感性的影响目的:涎腺腺样囊性癌(salivary ade noid cystic carcinoma,SACC)是唾液腺上皮源性的恶性肿瘤。术后复发和远处转移是引起SACC患者死亡的主要原因,如何有效地控制肿瘤的复发与转移,对于提高患者的生存率至关重要。以往的临床研究认为SACC细胞具有较强的放射抵抗性,因此,对SACC放射敏感性的研究意义重大。HIF-1是一种在实体肿瘤组织中常见的转录因子,由α和β亚基组成的异源二聚体。通常情况下HIF-1β表达稳定,HIF-1α受到氧浓度的调节,抑制HIF-1α的表达可以有效限制肿瘤的生长,同时,HIF-1α的表达水平升高可使多种肿瘤细胞对放疗产生抵抗作用。BCL-2为一种抗凋亡基因,是肿瘤细胞放射敏感性的重要靶分子。研究发现在肾脏等恶性肿瘤中,HIF-1α与BCL-2具有明显的相关性,并与患者的愈后不良有关。本研究的目的是构建高表达HIF-1α的SACC细胞,观察SACC中是否存在HIF-1α/BCL-2信号通路,以及HIF-1α在SACC中的表达对肿瘤细胞放射敏感性的影响。方法:用高表达HIF-1α的重组质粒和EGFP载体对ACC-2细胞进行转染获得的常氧下高表达HIF-1α的ACC-2/M-HIF-1α细胞、乏氧下高表达HIF-1α的ACC-2/W-HIF-1α及对照载体细胞ACC-2/F。采用RT-PCR和Western Blot的方法检测细胞中HIF-1α的蛋白及m RNA的表达水平,通过在不同处理下对4中细胞进行不同剂量的X线照射后观察细胞内HIF-1α和BCL-2蛋白表达水平并通过增殖实验观察细胞的增殖能力。在常氧环境中,对4种细胞进行0、2、3、5Gy的X线照射,采用克隆形成的方法观察和计算各细胞的存活分数。结果:成功转染并筛选出高表达HIF-1α的SACC细胞,并经RT-PCR检测结果显示,ACC-2/M-HIF-1α和ACC-2/W-HIF-1α细胞中HIF-1α的m RNA表达水平明显升高(P0.05)。采用Co Cl2化学模拟乏氧后经Western Blot检测结果显示,4种细胞中HIF-1α及BCL-2表达水平均有上升,而经YC-1显著降低。然后,进行了不同预处理并经不同剂量X线照射后,通过Western Blot检测结果显示,常氧条件X线照射下,细胞内HIF-1α和BCL-2的表达水平升高,而在乏氧高剂量照射后会有降低,YC-1处理后的结果类似。通过细胞增殖实验,结果显示ACC-2/MHIF-1α的倍增时间(Td)明显高于其他3种细胞,4种细胞均表现出受照射倍增时间延长的现象。通过集落形成实验可以观察到,ACC-2/M-HIF-1α的D0值高于其他3种细胞。结论:本研究结果表明,细胞内HIF-1α的高表达可以增加SACC细胞的放射抵抗性,并且HIF-1α和BCL-2的相关性同样存在于SACC中。
[Abstract]:The first part of the expression of HIF-1 alpha /BCL-2 in SACC and the relationship with the clinicopathological features: hypoxic inducible factor -1 alpha (Hypoxia inducible factor-1 alpha, HIF-1 a) plays an important role in the prognosis and reactivity of many cancer patients. This study observed HIF-1 alpha and B lymphocyte factor -2 (B-cell). The expression of Salivary gland adenoid cystic carcinoma (SACC) and its relationship with the clinicopathological features were analyzed. Methods: 56 cases of SACC patients from the First Affiliated Hospital of Medical University Of Anhui were collected and the surgical excised wax blocks were collected, and the expression of HIF-1 A and BCL-2 was observed by immunohistochemical staining. Then, the expression level of HIF-1 alpha and BCL-2 in SACC was analyzed by Semiquantitatively immunoreactive scoer (IRS). Results: the expression of HIF-1 alpha (IRS > 3) was found in 36 cases (64.3%), and the expression level was closely related to TNM stages (P=0.03), and the expression level of HIF-1 alpha was also the source of tissue. There was a statistical correlation (P=0.004). The expression of BCL-2 was found in 55 cases (98.2%), of which 44 cases (78.6%) were strongly positive. It was found that the IRS of HIF-1 alpha was associated with BCL-2 (beta =0.236, P=0.013), and the correlation of HIF-1 a in TNM staging depended on the high expression of BCL-2 (Z=2.43, P=0.015). The prognostic evaluation of SACC patients. Second part HIF-1 alpha affects the radiosensitivity of SACC cells. Objective: salivary adenoid cystic carcinoma (salivary ade noid cystic carcinoma, SACC) is a malignant tumor of salivary gland epithelial origin. Postoperative recurrence and distant metastasis are the main causes of the death and death of SACC patients. How to effectively control the recurrence of the tumor Hair and metastasis are essential to improve the survival rate of patients. Previous clinical studies suggest that SACC cells have strong radiosensitivity. Therefore, the study of SACC radiosensitivity is significant.HIF-1 is a common transcription factor in solid tumor tissue, a heterogenous two polymer consisting of alpha and beta subunits. Generally, HIF-1 beta The expression of HIF-1 A is regulated by the concentration of oxygen, and the inhibition of the expression of HIF-1 a can effectively restrict the growth of the tumor. At the same time, the increase of the expression level of HIF-1 a can cause a variety of tumor cells to resist radiotherapy..BCL-2 is an anti apoptotic gene, and it is an important target for the radiation sensitivity of tumor cells. The purpose of this study is to construct a SACC cell with high expression of HIF-1 alpha and to observe the existence of HIF-1 alpha /BCL-2 signaling pathway in SACC and the effect of HIF-1 alpha expression in SACC on the radiosensitivity of tumor cells in SACC. Methods: the recombination of the expression of HIF-1 a in SACC on the radiosensitivity of the tumor cells. Method: the recombination of the high expression of HIF-1 alpha in the HIF-1. ACC-2/M-HIF-1 alpha cells with high expression of HIF-1 a under normal oxygen were obtained by transfection of plasmids and EGFP vectors to ACC-2 cells, ACC-2/W-HIF-1 a with high expression of HIF-1 alpha in hypoxia and ACC-2/F. using RT-PCR and Western Blot to detect the protein of HIF-1 alpha in cells and the expression level of M proteins in the cells by different treatments to 4. After the cells were irradiated with different doses, the expression of HIF-1 alpha and BCL-2 protein was observed and the proliferation ability of the cells was observed through the proliferation experiment. In the atmospheric environment, 4 kinds of cells were irradiated with 0,2,3,5Gy, and the survival fraction of each cell was observed and calculated by the method of clone formation. The expression of SACC cells of HIF-1 alpha and the results of RT-PCR detection showed that the m RNA expression level of HIF-1 alpha in ACC-2/M-HIF-1 A and ACC-2/W-HIF-1 alpha cells increased significantly (P0.05). After Co Cl2 chemical analogue of hypoxia, the results showed that the average of the 4 cells was increased, and then decreased significantly. After different pretreatments and irradiated by different doses of X-ray, the results of Western Blot detection showed that the expression level of HIF-1 alpha and BCL-2 increased in the cells under the X-ray irradiation of normal oxygen, but decreased after high dose of hypoxia, and the result after YC-1 treatment was similar. The results of cell proliferation showed that the multiplication of ACC-2/MHIF-1 alpha was multiplied by the results of cell proliferation. The time (Td) was significantly higher than that of the other 3 cells, and the 4 cells showed prolonged exposure to exposure. The D0 value of ACC-2/M-HIF-1 alpha was higher than that of the other 3 cells. Conclusion: the results of this study suggest that the high expression of HIF-1 alpha in cells can increase the radiation resistance of SACC cells, and HIF-1 alpha and B The correlation of CL-2 also exists in SACC.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8

【相似文献】