本文選題：乏氧誘導(dǎo)因子-1α + B淋巴細(xì)胞因子2　； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分HIF-1α/BCL-2在SACC中的表達(dá)以及與臨床病理特征的關(guān)系目的:乏氧誘導(dǎo)因子-1α(Hypoxia inducible factor-1α,HIF-1α)在很多癌癥患者的預(yù)后及治療的反應(yīng)性方面扮演著重要角色,本研究觀察HIF-1α及B淋巴細(xì)胞因子-2(B-cell lymphoma-2,BCL-2)在涎腺腺樣囊性癌(Salivary gland adenoid cystic carcinoma,SACC)中的表達(dá)情況,并分析其與臨床病理特征間的相互關(guān)系。方法:收集來自安徽醫(yī)科大學(xué)第一附屬醫(yī)院的56例SACC患者的手術(shù)切除蠟塊,通過免疫組織化學(xué)染色,觀察HIF-1α和BCL-2的表達(dá)。然后,通過半定量免疫反應(yīng)性積分(Semiquantitatively immunoreactive scoer,IRS)的方法分析SACC中HIF-1α和BCL-2表達(dá)水平。結(jié)果:在36例(64.3%)中存在HIF-1α的表達(dá)(IRS≥3),且表達(dá)水平于TNM分期緊密相關(guān)(P=0.03),同時(shí),HIF-1α的表達(dá)水平于SACC的組織來源也具有統(tǒng)計(jì)學(xué)關(guān)聯(lián)(P=0.004)。在55例(98.2%)中發(fā)現(xiàn)有BCL-2的表達(dá),其中44例(78.6%)呈強(qiáng)陽性。統(tǒng)計(jì)發(fā)現(xiàn),HIF-1α的IRS于BCL-2具有相關(guān)性(β=0.236,P=0.013),并且HIF-1α于TNM分期的相關(guān)性依賴于BCL-2的高表達(dá)(Z=2.43,P=0.015)。結(jié)論:通過檢測(cè)HIF-1α的蛋白表達(dá)將有助于SACC患者的預(yù)后評(píng)價(jià)。第二部分HIF-1α對(duì)SACC細(xì)胞放射敏感性的影響目的:涎腺腺樣囊性癌(salivary ade noid cystic carcinoma,SACC)是唾液腺上皮源性的惡性腫瘤。術(shù)后復(fù)發(fā)和遠(yuǎn)處轉(zhuǎn)移是引起SACC患者死亡的主要原因,如何有效地控制腫瘤的復(fù)發(fā)與轉(zhuǎn)移,對(duì)于提高患者的生存率至關(guān)重要。以往的臨床研究認(rèn)為SACC細(xì)胞具有較強(qiáng)的放射抵抗性,因此,對(duì)SACC放射敏感性的研究意義重大。HIF-1是一種在實(shí)體腫瘤組織中常見的轉(zhuǎn)錄因子,由α和β亞基組成的異源二聚體。通常情況下HIF-1β表達(dá)穩(wěn)定,HIF-1α受到氧濃度的調(diào)節(jié),抑制HIF-1α的表達(dá)可以有效限制腫瘤的生長,同時(shí),HIF-1α的表達(dá)水平升高可使多種腫瘤細(xì)胞對(duì)放療產(chǎn)生抵抗作用。BCL-2為一種抗凋亡基因,是腫瘤細(xì)胞放射敏感性的重要靶分子。研究發(fā)現(xiàn)在腎臟等惡性腫瘤中,HIF-1α與BCL-2具有明顯的相關(guān)性,并與患者的愈后不良有關(guān)。本研究的目的是構(gòu)建高表達(dá)HIF-1α的SACC細(xì)胞,觀察SACC中是否存在HIF-1α/BCL-2信號(hào)通路,以及HIF-1α在SACC中的表達(dá)對(duì)腫瘤細(xì)胞放射敏感性的影響。方法:用高表達(dá)HIF-1α的重組質(zhì)粒和EGFP載體對(duì)ACC-2細(xì)胞進(jìn)行轉(zhuǎn)染獲得的常氧下高表達(dá)HIF-1α的ACC-2/M-HIF-1α細(xì)胞、乏氧下高表達(dá)HIF-1α的ACC-2/W-HIF-1α及對(duì)照載體細(xì)胞ACC-2/F。采用RT-PCR和Western Blot的方法檢測(cè)細(xì)胞中HIF-1α的蛋白及m RNA的表達(dá)水平,通過在不同處理下對(duì)4中細(xì)胞進(jìn)行不同劑量的X線照射后觀察細(xì)胞內(nèi)HIF-1α和BCL-2蛋白表達(dá)水平并通過增殖實(shí)驗(yàn)觀察細(xì)胞的增殖能力。在常氧環(huán)境中,對(duì)4種細(xì)胞進(jìn)行0、2、3、5Gy的X線照射,采用克隆形成的方法觀察和計(jì)算各細(xì)胞的存活分?jǐn)?shù)。結(jié)果:成功轉(zhuǎn)染并篩選出高表達(dá)HIF-1α的SACC細(xì)胞,并經(jīng)RT-PCR檢測(cè)結(jié)果顯示,ACC-2/M-HIF-1α和ACC-2/W-HIF-1α細(xì)胞中HIF-1α的m RNA表達(dá)水平明顯升高(P0.05)。采用Co Cl2化學(xué)模擬乏氧后經(jīng)Western Blot檢測(cè)結(jié)果顯示,4種細(xì)胞中HIF-1α及BCL-2表達(dá)水平均有上升,而經(jīng)YC-1顯著降低。然后,進(jìn)行了不同預(yù)處理并經(jīng)不同劑量X線照射后,通過Western Blot檢測(cè)結(jié)果顯示,常氧條件X線照射下,細(xì)胞內(nèi)HIF-1α和BCL-2的表達(dá)水平升高,而在乏氧高劑量照射后會(huì)有降低,YC-1處理后的結(jié)果類似。通過細(xì)胞增殖實(shí)驗(yàn),結(jié)果顯示ACC-2/MHIF-1α的倍增時(shí)間(Td)明顯高于其他3種細(xì)胞,4種細(xì)胞均表現(xiàn)出受照射倍增時(shí)間延長的現(xiàn)象。通過集落形成實(shí)驗(yàn)可以觀察到,ACC-2/M-HIF-1α的D0值高于其他3種細(xì)胞。結(jié)論:本研究結(jié)果表明,細(xì)胞內(nèi)HIF-1α的高表達(dá)可以增加SACC細(xì)胞的放射抵抗性,并且HIF-1α和BCL-2的相關(guān)性同樣存在于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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.8

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