OSAS模式IH对肿瘤转移相关蛋白表达的影响

发布时间：2018-07-13 13:08

【摘要】：睡眠呼吸暂停综合征(SAS)最早报道于上个世纪五十年代,后随着其发病率逐年上升,引起了越来越广泛的重视。根据多导睡眠监测结果,SAS可以分为阻塞性、中枢性、混合性三种类型,三者发病机制及临床表现有一定的差异。流行病学显示,在成年人群中以阻塞性最多见。OSAS发病机制复杂,主要与解剖因素、神经-体液因素有关,解剖因素主要指患者上气道解剖结构存在不同程度的狭窄,这种狭窄存在于患者静止状态时,并且于睡眠状态下狭窄程度加剧;神经-体液因素主要包括患者各种内分泌紊乱如甲状腺功能减退等。OSAS导致的主要病理生理学特征是夜间反复的间歇低氧(IH)与持续的高碳酸血症。OSAS危害巨大,其导致的危害不仅局限与疾病本身,更多的是在于在其病理生理学上导致的全身多个系统并发症。OSAS常见的并发症包括心血管系统、呼吸系统、内分泌系统等在内多个系统疾病。近几年的研究发现,OSAS患者合并肿瘤机率、病死率不同程度的提高,且致肿瘤病情进展迅速。对此,有学者推测OSAS导致的IH可能是促进肿瘤发生发展、肿瘤细胞增殖的重要危险因素之一。肿瘤发生发展过程中伴随着不同程度的上皮间质化(EMT)、氧化应激、炎症反应等事件,OSAS模式IH能否对肿瘤发生发展过程中的这些参与事件产生影响,其中详细机制并不十分清楚。本研究以人肺癌A549细胞与小鼠黑色素瘤B16F10细胞为研究对象,模拟间歇低氧环境,建立肿瘤细胞-间歇低氧模型,以研究OSAS模式IH对肿瘤细胞相关蛋白表达及生物学行为的影响,并探讨其中机制,以希望能够为防治肿瘤提供新的思路和措施。目的1.探索OSAS模式IH对人肺癌A549细胞HIF-1α与YAP及P-YAP蛋白表达的影响。2.探索OSAS模式IH对小鼠黑色素瘤B16F10细胞NF-κB、NRF2及MMP-9蛋白表达的影响。方法分别以人肺癌A549细胞与小鼠黑色素瘤B16F10细胞为对象,进行正常细胞培养后,进行IH环境培养舱暴露。IH环境培养舱的环境设置为低氧5mins、复氧5mins,并根据需要设置低氧-复氧循环。舱内最低氧状态时氧气浓度控制在5%左右,复氧时舱内氧气浓度控制在21%左右。暴露时长分别设置为1h、3h、6h、据此分为IH1组(IH1)、IH3组(IH3)、IH6组(IH6),并采用置于正常细胞培养箱内培养的细胞作为对照组N组(N)。采用Western-blot法检测人肺癌A549细胞乏氧诱导因子-1α(HIF-1α)、YAP、P-YAP蛋白表达水平,q RT-PCR法检测HIF-1α、YAP m RNA的表达水平。采用Western-blot法检测小鼠黑色素瘤B16F10细胞氧化应激核转录因子相关因子-2(NRF2)、炎症相关核转录因子NF-κB、IκBα、基质金属蛋白酶-9(MMP-9)的表达。结果1.HIF-1α和YAP及P-YAP表达Western blot结果显示H1、IH3、IH6组HIF-1α蛋白表达较N组均出现增高,且有大体上随着IH处理时间的增长,而表达逐步增高的趋势。IH1、IH3、IH6组YAP蛋白表达较N组均出现增高,且其增高的趋势与IH处理时间呈时间依赖关系。IH1、IH3、IH6组P-YAP蛋白表达较N组均增降低,且降低趋势与IH处理的时间亦有时间依赖性。q RT-PCR结果显示IH1组(2.91±0.04)、IH3组(5.69±0.17)HIF-1αm RNA的相对表达量较N组(1.01±0.05)均显著增高(P0.05),IH6组(9.18±0.59)HIF-1αm RNA的相对表达量较N组极显著增高(P0.01),并且发现细胞中HIF-1αm RNA的相对表达量与IH处理的时间呈依赖关系。IH1组(2.50±0.18)、IH3组(4.07±0.25)YAP m RNA的相对表达量较N组(1.00±0.01)均显著增高(P0.05),IH6组(9.18±0.58)YAP m RNA的相对表达量较N组有极显著增高(P0.01),并且发现细胞中YAP m RNA的相对表达量与IH处理的时间呈依赖关系(P0.05)。2.NRF2、NF-κB P65、IκBα、MMP-9蛋白表达与N组相比,IH1组、IH3组、IH6组NRF2蛋白的表达出现了显著的升高趋势。与N组相比,IH1组、IH3组、IH6组NFκB P65蛋白的表达出现了显著的升高趋势。与N组相比,CH组、IH组IκBα蛋白的表达均出现了显著的降低趋势。且并且IH暴露时IκBα蛋白的降解程度要高于CH暴露时。与N组相比,IH组MMP-9蛋白的表达出现了显著的增高。结论1.OSAS模式IH可以促进人肺癌A549细胞内HIF-1α、YAP高表达,P-YAP蛋白低表达;并且促进了HIF-1αm RNA表达上升、YAP m RNA表达上升。提示Hippo-YAP信号通路失调可能是OSAS患者合并肿瘤患者风险增加、病情进展加快的原因之一。2.OSAS模式IH可以促进小鼠黑色素瘤B16F10细胞内NRF2高表达,提示OSAS合并肿瘤患者,病情进展较快可能与OSAS导致的氧化应激事件有关3.OSAS模式IH可以促进小鼠黑色素瘤B16F10细胞内NF-κb p65高表达,还可以促进Lewis小鼠肺癌细胞中IκBα蛋白低表达。提示炎症反应在OSAS患者合并肿瘤风险增加过程中发挥了重要作用。4.肿瘤细胞-低氧模型中发现Lewis小鼠腺癌细胞内MMP-9蛋白表达升高,提示间歇低氧可以促进肿瘤浸润转移。
[Abstract]:Sleep apnea syndrome (SAS) is first reported in the 50s of last century, and with its incidence increasing year by year, it has attracted more and more attention. According to the results of polysomnography, SAS can be divided into three types of obstructive, central and mixed, the three disease mechanism and clinical manifestations are different. Epidemiology shows that The most complicated pathogenesis of.OSAS in adult population is complex, mainly related to anatomical factors and nerve fluid factors. Anatomical factors mainly mean that there are different degrees of stenosis in the upper airway anatomy of the patients. This stenosis exists in the static state of the patient, and the degree of stenosis is aggravated in the sleep state; the main factors of the nerve and body fluid are the main factors. The main pathophysiological features of.OSAS, including a variety of endocrine disorders such as hypothyroidism, are that repeated nocturnal intermittent hypoxic (IH) and persistent hypercapnia,.OSAS, are not only limited to the disease itself, but also in its pathophysiological system. The common complications of.OSAS include the cardiovascular system, the respiratory system, the endocrine system and many other systemic diseases. In recent years, the study found that the OSAS patients were combined with the tumor probability, the mortality rate was improved to different degrees, and the tumor was progressing rapidly. Some scholars suggested that the OSAS induced IH may be to promote the development of tumor. One of the most important risk factors for tumor cell proliferation. The development process of tumor is accompanied by different degrees of epithelial interstitial (EMT), oxidative stress, inflammation and other events. Whether the OSAS model IH can affect these events in the process of tumor development, the detailed mechanism is not very clear. This study uses human lung cancer A549 thin. Cell and mouse melanoma B16F10 cells are used to simulate intermittent hypoxic environment and establish a tumor cell intermission hypoxia model to study the effect of OSAS model IH on the expression of tumor cell related proteins and biological behavior, and to explore the mechanism in order to provide new ideas and measures for the prevention and control of tumor. Objective 1. to explore the OSAS model. The effect of IH on the expression of HIF-1 alpha, YAP and P-YAP protein in human lung cancer A549 cells.2. to explore the effect of OSAS mode IH on NF- kappa B, NRF2 and protein expression in mouse melanoma B16F10 cells. The environment of IH environment culture cabin is set to low oxygen 5mins, reoxygenate 5mins, and set low oxygen reoxygenation cycle according to the need. The oxygen concentration is controlled at about 5% in the lowest oxygen state of the cabin, and the oxygen concentration in the cabin is controlled at about 21% when reoxygenation. The exposure time is set to 1H, 3h, 6h respectively, according to this, IH1 group (IH1), IH3 group (IH3), IH6 group (IH6), and put in The cells cultured in the normal cell culture box were used as the control group N group (N). The Western-blot method was used to detect the hypoxia inducible factor -1 alpha (HIF-1 alpha), the expression level of YAP, P-YAP protein in human lung cancer A549 cells. The Q RT-PCR method was used to detect the HIF-1 alpha and the expression level of YAP. -2 (NRF2), the expression of NF- kappa B, I kappa B alpha, and matrix metalloproteinase -9 (MMP-9) in inflammation related nuclear transcription factors. The expression of YAP protein in group IH6 was higher than that in group N, and the trend of YAP protein increased in time dependent relationship with IH treatment time.IH1, IH3, IH6 group, P-YAP protein expression was lower than that of N group, and the decreasing trend and IH processing time also had time dependent.Q results showed (2.91 + 0.04), and the relative table of (5.69 + 0.17) Compared with group N (1.01 + 0.05), the relative expression of HIF-1 alpha m RNA in IH6 group (9.18 + 0.59) was significantly higher than that in N group (P0.01), and the relative expression of HIF-1 alpha m RNA was found to be dependent on the duration of IH treatment (2.50 + 0.18), and the relative expression of 4.07 + 0.25 (4.07 + 0.25) was higher than that of the group (1 + 0.01). The relative expression of the IH6 group (9.18 + 0.58) YAP m RNA was significantly higher than that of the N group (P0.01), and the relative expression of YAP m RNA was found to be dependent on the IH processing time (P0.05). The expression of NF kappa B P65 protein in IH1, IH3 and IH6 groups increased significantly compared with the N group. Compared with the N group, the expression of I kappa B alpha protein in the CH group and IH group showed a significant decreasing trend. Conclusion 1.OSAS model IH can promote the HIF-1 alpha, YAP high expression and low expression of P-YAP protein in human lung cancer A549 cells, and promote the RNA expression of HIF-1 alpha m and YAP m RNA expression rise. S model IH can promote the high expression of NRF2 in mouse melanoma B16F10 cells, suggesting that OSAS is associated with tumor patients. The progression of the disease is faster and the 3.OSAS mode IH, which is associated with OSAS induced oxidative stress events, can promote the high expression of NF- kappa B p65 in murine melanoma B16F10 cells. It is suggested that the inflammatory reaction plays an important role in the process of increasing the risk of cancer in OSAS patients. The increase of MMP-9 protein expression in the adenocarcinoma cells of Lewis mice is found in the.4. tumor cell hypoxic model, suggesting that intermittent hypoxia can promote tumor invasion and metastasis.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R766;R730.5

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