OSAS模式IH對腫瘤轉(zhuǎn)移相關(guān)蛋白表達的影響
發(fā)布時間：2018-07-13 13:08
【摘要】：睡眠呼吸暫停綜合征(SAS)最早報道于上個世紀(jì)五十年代,后隨著其發(fā)病率逐年上升,引起了越來越廣泛的重視。根據(jù)多導(dǎo)睡眠監(jiān)測結(jié)果,SAS可以分為阻塞性、中樞性、混合性三種類型,三者發(fā)病機制及臨床表現(xiàn)有一定的差異。流行病學(xué)顯示,在成年人群中以阻塞性最多見。OSAS發(fā)病機制復(fù)雜,主要與解剖因素、神經(jīng)-體液因素有關(guān),解剖因素主要指患者上氣道解剖結(jié)構(gòu)存在不同程度的狹窄,這種狹窄存在于患者靜止?fàn)顟B(tài)時,并且于睡眠狀態(tài)下狹窄程度加劇;神經(jīng)-體液因素主要包括患者各種內(nèi)分泌紊亂如甲狀腺功能減退等。OSAS導(dǎo)致的主要病理生理學(xué)特征是夜間反復(fù)的間歇低氧(IH)與持續(xù)的高碳酸血癥。OSAS危害巨大,其導(dǎo)致的危害不僅局限與疾病本身,更多的是在于在其病理生理學(xué)上導(dǎo)致的全身多個系統(tǒng)并發(fā)癥。OSAS常見的并發(fā)癥包括心血管系統(tǒng)、呼吸系統(tǒng)、內(nèi)分泌系統(tǒng)等在內(nèi)多個系統(tǒng)疾病。近幾年的研究發(fā)現(xiàn),OSAS患者合并腫瘤機率、病死率不同程度的提高,且致腫瘤病情進展迅速。對此,有學(xué)者推測OSAS導(dǎo)致的IH可能是促進腫瘤發(fā)生發(fā)展、腫瘤細胞增殖的重要危險因素之一。腫瘤發(fā)生發(fā)展過程中伴隨著不同程度的上皮間質(zhì)化(EMT)、氧化應(yīng)激、炎癥反應(yīng)等事件,OSAS模式IH能否對腫瘤發(fā)生發(fā)展過程中的這些參與事件產(chǎn)生影響,其中詳細機制并不十分清楚。本研究以人肺癌A549細胞與小鼠黑色素瘤B16F10細胞為研究對象,模擬間歇低氧環(huán)境,建立腫瘤細胞-間歇低氧模型,以研究OSAS模式IH對腫瘤細胞相關(guān)蛋白表達及生物學(xué)行為的影響,并探討其中機制,以希望能夠為防治腫瘤提供新的思路和措施。目的1.探索OSAS模式IH對人肺癌A549細胞HIF-1α與YAP及P-YAP蛋白表達的影響。2.探索OSAS模式IH對小鼠黑色素瘤B16F10細胞NF-κB、NRF2及MMP-9蛋白表達的影響。方法分別以人肺癌A549細胞與小鼠黑色素瘤B16F10細胞為對象,進行正常細胞培養(yǎng)后,進行IH環(huán)境培養(yǎng)艙暴露。IH環(huán)境培養(yǎng)艙的環(huán)境設(shè)置為低氧5mins、復(fù)氧5mins,并根據(jù)需要設(shè)置低氧-復(fù)氧循環(huán)。艙內(nèi)最低氧狀態(tài)時氧氣濃度控制在5%左右,復(fù)氧時艙內(nèi)氧氣濃度控制在21%左右。暴露時長分別設(shè)置為1h、3h、6h、據(jù)此分為IH1組(IH1)、IH3組(IH3)、IH6組(IH6),并采用置于正常細胞培養(yǎng)箱內(nèi)培養(yǎng)的細胞作為對照組N組(N)。采用Western-blot法檢測人肺癌A549細胞乏氧誘導(dǎo)因子-1α(HIF-1α)、YAP、P-YAP蛋白表達水平,q RT-PCR法檢測HIF-1α、YAP m RNA的表達水平。采用Western-blot法檢測小鼠黑色素瘤B16F10細胞氧化應(yīng)激核轉(zhuǎn)錄因子相關(guān)因子-2(NRF2)、炎癥相關(guān)核轉(zhuǎn)錄因子NF-κB、IκBα、基質(zhì)金屬蛋白酶-9(MMP-9)的表達。結(jié)果1.HIF-1α和YAP及P-YAP表達Western blot結(jié)果顯示H1、IH3、IH6組HIF-1α蛋白表達較N組均出現(xiàn)增高,且有大體上隨著IH處理時間的增長,而表達逐步增高的趨勢。IH1、IH3、IH6組YAP蛋白表達較N組均出現(xiàn)增高,且其增高的趨勢與IH處理時間呈時間依賴關(guān)系。IH1、IH3、IH6組P-YAP蛋白表達較N組均增降低,且降低趨勢與IH處理的時間亦有時間依賴性。q RT-PCR結(jié)果顯示IH1組(2.91±0.04)、IH3組(5.69±0.17)HIF-1αm RNA的相對表達量較N組(1.01±0.05)均顯著增高(P0.05),IH6組(9.18±0.59)HIF-1αm RNA的相對表達量較N組極顯著增高(P0.01),并且發(fā)現(xiàn)細胞中HIF-1αm RNA的相對表達量與IH處理的時間呈依賴關(guān)系。IH1組(2.50±0.18)、IH3組(4.07±0.25)YAP m RNA的相對表達量較N組(1.00±0.01)均顯著增高(P0.05),IH6組(9.18±0.58)YAP m RNA的相對表達量較N組有極顯著增高(P0.01),并且發(fā)現(xiàn)細胞中YAP m RNA的相對表達量與IH處理的時間呈依賴關(guān)系(P0.05)。2.NRF2、NF-κB P65、IκBα、MMP-9蛋白表達與N組相比,IH1組、IH3組、IH6組NRF2蛋白的表達出現(xiàn)了顯著的升高趨勢。與N組相比,IH1組、IH3組、IH6組NFκB P65蛋白的表達出現(xiàn)了顯著的升高趨勢。與N組相比,CH組、IH組IκBα蛋白的表達均出現(xiàn)了顯著的降低趨勢。且并且IH暴露時IκBα蛋白的降解程度要高于CH暴露時。與N組相比,IH組MMP-9蛋白的表達出現(xiàn)了顯著的增高。結(jié)論1.OSAS模式IH可以促進人肺癌A549細胞內(nèi)HIF-1α、YAP高表達,P-YAP蛋白低表達;并且促進了HIF-1αm RNA表達上升、YAP m RNA表達上升。提示Hippo-YAP信號通路失調(diào)可能是OSAS患者合并腫瘤患者風(fēng)險增加、病情進展加快的原因之一。2.OSAS模式IH可以促進小鼠黑色素瘤B16F10細胞內(nèi)NRF2高表達,提示OSAS合并腫瘤患者,病情進展較快可能與OSAS導(dǎo)致的氧化應(yīng)激事件有關(guān)3.OSAS模式IH可以促進小鼠黑色素瘤B16F10細胞內(nèi)NF-κb p65高表達,還可以促進Lewis小鼠肺癌細胞中IκBα蛋白低表達。提示炎癥反應(yīng)在OSAS患者合并腫瘤風(fēng)險增加過程中發(fā)揮了重要作用。4.腫瘤細胞-低氧模型中發(fā)現(xiàn)Lewis小鼠腺癌細胞內(nèi)MMP-9蛋白表達升高,提示間歇低氧可以促進腫瘤浸潤轉(zhuǎn)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R766;R730.5
本文編號：2119481
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R766;R730.5
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