【作者】 张勇；

【导师】 娄卫华；

【作者基本信息】 郑州大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 喉癌是耳鼻咽喉科原发于上皮的恶性肿瘤,占耳鼻咽喉科恶性肿瘤的11%-22%。尽管近几十年来喉癌的治疗方法取得了较大的进展,但在生存率和患者生活质量方面仍然需要提高。随着新的化疗药物的不断出现,喉癌的治疗也过渡到了手术,放疗和化疗的综合性治疗,因此研究和探索喉癌更多的更有效的化疗药物有着深远的意义。过氧化酶增殖体激活受体(peroxisome poliferator-activited receptor, PPAR)是一种细胞核转录因子,具有多种生物学效应并参与多种机体生理反应,尤其是在脂肪的贮存和代谢中发挥重要的无可比拟的作用。目前的研究表明PPAR分为三种亚型(α,δ,γ),其中PPARy亚型与肿瘤关系最为密切,因此在肿瘤的功能研究的也得最为广泛和深入。很多报道都显示PPARy在多种肿瘤组织中表达,当应用PPARy激动剂作用后就可以引起肿瘤细胞增殖和侵袭能力的抑制、血管形成减少及诱导凋亡等肿瘤细胞的恶性生物学行为改变。因此,近年来以PPARy为靶点的药物研究进展迅速,已成为当前抗肿瘤药物研究开发的热点之一PPARy激动剂可分为天然激动剂和合成激动剂两大类。人工合成PPARy激动剂则以噻唑烷二酮类(thiazolidinedione compounds, TZDs)最为代表,在临床上应用于糖尿病的治疗。近几年的大量研究表明TZDs可以有效地抑制多种肿瘤细胞的增殖,抑制肿瘤新生血管的形成,阻滞细胞周期的进行,诱导凋亡,况且TZDs作为糖尿病主要治疗药物因其疗效好,副作用少在临床上广泛应用多年,所以TZDs对肿瘤的增殖抑制作用显得更为有意义。研究表明TZDs的抑瘤机制十分复杂,甚至有学者提出TZDs有可通过激活PPARγ外的其它途径来发挥抗肿瘤的作用,但是TZDs的抗肿瘤作用却是毋庸质疑的。为了深入探讨TZDs对喉癌Hep-2细胞的影响,本研究首先应用TZDs代表药物罗格列酮(Rosiglitazone, RGZ)和曲格列酮(Troglitazone, TRG)在体外作用于Hep-2细胞,采用MTT、流式细胞术、Real-time PCR、western和ELISA多种分子生物学技术体外观察了TZDs对Hep-2细胞增殖、凋亡和缺氧诱导的HIF-1α和VEGF的影响并探讨其发生机制；接着在体外实验的基础上,我们研究观察RGZ和TRG对人喉癌Hep-2细胞裸鼠皮下移植瘤影响及机制及RGZ和TRG在荷瘤裸鼠的毒性反应,为喉癌化学治疗拓展新的思路和方法,为将来以PPARγ为靶点的喉癌防治提供理论基础及实验依据,为TZDs的开发、临床应用提供有价值的实验依据。本研究分为以下三章：第一章RGZ和TRG对人喉癌Hep-2细胞增殖、凋亡的影响及机制研究方法1.采用MTT比色法测定经12.5、25、50、100μmol/L RGZ或TRG分别作用12、24、36、48、60、72h后的Hep-2细胞增殖活性的改变。2.采用流式细胞术分别检测经50μmol/L RGZ或TRG作用0、24、48、72h后的Hep-2细胞凋亡率和细胞周期分布。3.采用Real-time PCR和western方法分别检测经50μmol/L RGZ或TRG作用0、24、48、72h后Hep-2细胞中COX-2和p65 mRNA和蛋白及VEGFmRNA水平的变化。4.采用ELISA方法分别检测经25、50、100μmol/L RGZ或TRG作用24h后Hep-2细胞培养上清液中VEGF蛋白水平的变化。结果1.RGZ或TRG作用后导致细胞的增殖活性明显受到抑制,其抑制率呈浓度依赖性和时间依赖性增加(P<0.05)。2.流式细胞术结果显示50μmol/L RGZ或TRG作用24、48、72h后,G0/G1期细胞比例呈时间依赖性逐渐升高(P<0.01),S期细胞比例逐渐降低(P<0.01),而G2/M期细胞比例也呈逐渐降低趋势(P<0.01)；随着RGZ或TRG作用时间延长,细胞凋亡率呈时间依赖性逐渐增加,不同时间组间存在显著性差异(P<0.01)。3. Real-time PCR和western结果显示经50μmol/L RGZ或TRG作用24、48、72h后Hep-2细胞中COX-2和p65 mRNA和蛋白及VEGF mRNA表达水平呈时间依赖性的下降,不同时间组间有显著性差异(P<0.01)。4. ELISA结果显示经25、50、100μmol/L RGZ或TRG作用24h后Hep-2细胞培养上清液中VEGF蛋白水平呈时间依赖性的下降,不同时间组间有显著性差异(P<0.01)。第二章RGZ和TRG对缺氧诱导的Hep-2细胞HIF-1α和VEGF表达的影响研究方法1采用MTT法检测缺氧环境下经12.5、25、50、100μmol/LRGZ或TRG分别作用12、24、36、48、60、72h后Hep-2细胞增殖活性的改变。2.缺氧环境下经25、50、100μmol/L RGZ或TRG作用Hep-2细胞24h后,采用Real-time PCR和ELISA法分别检测细胞中VEGF mRNA和培养上清液蛋白水平的变化。3.采用Real-time PCR和western检测RGZ或TRG作用Hep-2细胞16h后Hep-2细胞中HIF-1αmRNA和蛋白水平的变化。4.采用western方法检测缺氧环境下经25、50、100μmol/L RGZ或TRG作用6h后Hep-2细胞中p-AKT/AKT, p-ERK1/2/ERK1/2, p-STAT3/STAT3蛋白水平的变化。5.采用transwell小室侵袭实验检测50μmol/LRGZ或TRG在缺氧和常氧环境下对Hep-2细胞侵袭能力的影响。6.采用明胶酶谱法分析50μmol/LRGZ或TRG在缺氧和常氧环境下对Hep-2细胞MMP-2活性的影响。结果1.MTT结果表明缺氧环境下RGZ和TRG作用Hep-2细胞后细胞的增殖活性受到明显抑制,且比常氧环境下对细胞的抑制作用更为明显,细胞抑制率具有时间依赖性和剂量依赖性(P<0.01)。2. Real-time PCR和ELISA结果显示RGZ和TRG呈浓度依赖性抑制缺氧诱导的培养上清液中VEGF蛋白和细胞内mRNA的水平,各组间差异有统计学意义(P<0.01)。而HIF-1αmRNA表达水平在各浓度组均无明显改变(P>0.05)。3. western结果表明RGZ和TRG呈浓度依赖性抑制缺氧诱导的Hep-2细胞HIF-1α、p-AKT、p-ERK1/2和p-STAT3蛋白的表达,各组间差异有统计学意义(P<0.01)；而AKT、ERK1/2和STAT3蛋白表达水平在各浓度组均无明显改变,各组间差异无统计学意义(P>0.05)。4. Transwell小室侵袭实验结果表明缺氧和常氧环境下RGZ和TRG都可以明显的抑制Hep-2细胞的侵袭,各组间差异有统计学意义(P<0.01)。5.明胶酶谱法分析结果表明缺氧和常氧环境下RGZ和TRG均可以抑制Hep-2细胞MMP-2活性,各组间差异有统计学意义(P<0.01)。第三章RGZ和TRG对人喉癌Hep-2细胞裸鼠移植瘤的影响研究方法1.建立人喉癌裸鼠移植瘤模型后随机分为对照组、RGZ实验组和TRG实验组,每组6只,治疗组瘤内注射50μmoL/L的RGZ和TRG,每3天注射一次；瘤内注射0.5%二甲基亚砜。共治疗4周。2.每d观察裸鼠的饮食、活动等生理状况。每周测量瘤体大小及裸鼠体质量,绘制肿瘤生长变化曲线。待4周后治疗结束时处死小鼠,剥瘤称重,计算质量抑瘤率。HE染色后光学显微镜下观察脑、心、肝、肺、脾和肾等重要脏器变化。3.采用免疫组化技术检测各组裸鼠移植瘤CD34抗原表达情况,并对染色结果进行统计分析计算MVD值。4.采用Real-time PCR和western技术检测各组移植瘤COX-2、p65、HIF-1α和VEGF mRNA及蛋白表达情况。结果1.与对照组相比,RGZ和TRG能明显抑制裸鼠移植瘤的生长,对照组和实验组裸鼠瘤体积、瘤质量组间比较差异具有统计学意义(P<0.05)。2.治疗过程中,裸鼠未见明显不良反应。治疗结束时各组心、肝、肺、脾、肾等重要脏器无明显器质性改变。3.与对照组相比,实验组肿瘤组织中MVD值明显减少,三组间MVD值相比差异具有统计学意义(P<0.01)。4. Real-time PCR和western结果表明RGZ和TRG能抑制裸鼠移植瘤COX-2、p65和VEGF mRNA和蛋白及HIF-1α蛋白的表达,组间比较差异具有统计学意义(P<0.05)。结论1.RGZ和TRG对Hep-2细胞具有浓度和时间依赖性增殖抑制作用,阻滞Hep-2细胞于G0/G1期,诱导细胞凋亡,其机制与抑制COX-2和p65蛋白和mRNA及VEGF mRNA的表达,下调VEGF蛋白的分泌有关。2.缺氧环境下RGZ或TRG依然对Hep-2细胞增殖有明显的抑制作用,能抑制VEGFmRNA的表达和培养上清液VEGF蛋白的分泌,下调缺氧诱导的HIF-1α蛋白水平；其机制与抑制AKT、ERK1/2和STAT3蛋白磷酸化及通过蛋白酶体通路促进HIF-1α蛋白的降解密切相关；能抑制常氧和缺氧环境下Hep-2细胞的侵袭,其机制与降低MMP-2活性密切相关。3.RGZ和TRG能抑制喉癌裸鼠移植瘤生长,降低肿瘤组织MVD值,下调喉癌裸鼠移植瘤COX-2、p65、HIF-1α和VEGF表达。且对机体无明显毒、副作用。更多还原

【Abstract】 Laryngeal cancer is otorhinolaryngology primary malignant tumor in the epithelium, accounting for 11%-22% of otorhinolaryngology malignant tumors. Although in recent decades the treatment of laryngeal cancer has made considerable progress, but the survival rate and improvement in the quality of life of patients still need to improve. With the new chemotherapy drugs emerging, the treatment of laryngeal cancer is also transit to the comprehensive treatment consisted of surgery, radiotherapy and chemotherapy, so the research and exploration of laryngeal more and more effective chemotherapy with far-reaching significance.Peroxisome proliferator-activated receptor (peroxisome poliferator-activited receptor, PPAR) is a nuclear transcription factor with multiple biological effects involved in a variety of physiological responses of the body, especially in the fat storage and metabolism play an important unparalleled role. The current studies have shown that PPAR is divided into three subtypes (α,δ,γ), in which PPARy subtypes and tumor most closely, so the study on it functions in the tumor development also is the most extensive and in-depth. a lot of reports have shown that PPARy expression in various tumor tissues, when applied with PPARy agonists will result in reducing the malignant biological behavior of tumor cells such as inhibition of tumor cell proliferation and of invasion, angiogenesis and induction of apoptosis etc. Therefore in recent years PPARy as a target with the rapid progress in drug research, which has become is one of hot research and development of antitumor drugs.PPARy agonists can be divided into natural agonists and synthetic agonists two categories. Synthetic PPARy agonists thiazolidinedione compounds, (TZDs) is most representative, which have the applyzed clinical treatment of diabetes. In recent years, a large number of studies have shown that TZDs can effectively inhibit a variety of tumor cell proliferation, inhibit tumor angiogenesis, block cell cycle progresses, induce apoptosis. Moreover, TZDs as a diabetes treatment drug, because of its good curative effect, less side-effects widely used in clinical practice for many years, so TZDs inhibit the proliferation of the tumor is much more meaningful. Studies have shown that the inhibitory mechanism of TZDs is very complicated, and even some academics have suggested that TZDs anti-tumor effect were through activate PPARy and independent PPARy ways, but the anti-tumor effect of TZDs is needless to be questioned.In ordor to study the impact of TZDs on the laryngeal Hep-2 cells, TZDs on behalf of drug rosiglitazone (RGZ) and troglitazone (TRG) first in vitro on the Hep-2 cells, using MTT, flow cytometry, Real-time PCR, ELISA, western, etc, molecular biology techniques in vitro to observed the impact of TZDs on Hep-2 cell proliferation, apoptosis and hypoxia-induced HIF-la and VEGF and to explore the impact mechanism; Then, based on the observation of our vitro experiments, we observed the impact of the RGZ and TRG on human laryngeal cancer Hep-2 cells nude mice transplanted tumor and explored it mechanism, and the toxicity of RGZ and TRG in this process. For take PPARy as a target for prevention and treatment of laryngeal cancer provide a theoretical basis and experimental evidence, for TZDs development, clinical application provide valuable experimental evidence. This study is divided into the following three parts:The first chapter:the impact of RGZ and TRG on human laryngeal cancer Hep-2 cell proliferation, apoptosis and its mechanismMethods1. The proliferative activity of the Hep-2 cells treated with RGZ or TRG at 12.5、25、50、100μmol/L for 12,24,36,48,60 or 72h was respectively analyzed by MTT assay.2. The apoptosis rate and cell cycle distribution of the Hep-2 cells treated with 50μmol/L RGZ or TRG for 0,24,48 or 72h were respectively analyzed by FCM.3. The expression of COX-2 and p65 mRNA and protein, VEGF mRNA in the Hep-2 cells treated with 50μmol/L RGZ or TRG for 0,24,48 or 72h were respectively analyzed by Real-time PCR and western.4. The VEGF protein levels in Hep-2 cell culture supernatant after treated with 25,50,100μmol/L RGZ or TRG for 24h were detected by ELISA.Results1.The proliferative activity of the cells treated with RGZ or TRG was inhibited obviously. The inhibitory rate of cell growth in dose-dependent and time-dependent manner (P<0.05)2. FCM results showed that after treated with 50μmol/L RGZ or TRG for 0h、 24、48、72h, the proportion of the cells in G0/G1 stage increased in a time dependent manner(P<0.01); that cells in S stage decreased (P<0.01); that cells in G2/M stage also decreased(P>0.05). the apoptosis rate of the cells increased gradually with the prolonging action time of RGZ or TRG, There were significant differences among different time groups(P<0.01).3. Real-time PCR and western results showed that the expression of COX-2、p65 and VEGF mRNA and protein in Hep-2 cells decreased in a time dependent manner after treat with 50μmol/L RGZ or TRG for Oh、24、48、72h, There were significant differences among different time groups(P<0.01).4. The ELISA results showed that the VEGF protein levels in Hep-2 cell culture supernatant decreased in a dose dependent manner after treated with 25,50,100μmol/L RGZ or TRG for 24h. There were significant differences among different time groups(P<0.01).The second chapter:the RGZ and TRG inhibit hypoxia induced HIF-1αand VEGF Expression in Hep-2 cellsMethods1. The proliferative activity of the Hep-2 cells treated with RGZ and TRG at 12.5、25、50、100μmol/L for 12,24,36,48,60,72h under hypoxia was analyzed respectively by MTT assay.2. The level of hypoxia induced VEGF protein in the supernatants of cultured Hep-2 cells and VEGF mRNA treated with 50μmol/L RGZ or TRG at 25、50、100μmol/L were respectively analyzed by ELISA and Real-time PCR.3 The expression of HIF-1αprotein and mRNA in the Hep-2 cells treated with RGZ or TRG at 25、50、100μmol/L under hypoxia respectively were analyzed by western and Real-time PCR.4. The expression of HIF-1α, p-AKT/AKT, p-ERK1/2/ERK1/2, p-STAT3/STAT3 protein in the Hep-2 cells treated with RGZ or TRG at 25、50、100μmol/L under hypoxia were respectively analyzed by western.5. The impact of RGZ or TRG on invasion of Hep-2 cells under normoxia and hypoxia were detected by transwell.6. The impact of RGZ or TRG on MMP-2 activity in Hep-2 cells under normoxia and hypoxia were detected by zymography.Results1. MTT results showed that the proliferative activity of the cells treated with RGZ or TRG was inhibited under hypoxia, this effect even more obviously than that under normoxia. The inhibitory rate of cell growth in dose and time dependent manner(P<0.01).2. Real-time PCR and ELISA results showed that the level of secretive VEGF protein in the supernatants of cultured Hep-2 cells and mRNA in cells decreased in a time dependent manner after treated with RGZ or TRG at 25、50、100μmol/L, There were significant differences among different groups(P<0.01). while HIF-la mRNA have not obvious changes between different time groups (P<0.01).3. Western results showed that the expression of HIF-1α、p-AKT、p-ERK1/2 and p-STAT3 protein in Hep-2 cells decreased in a time dependent manner after treated with RGZ or TRG at 25、50、100μmol/L under hypoxia, There were significant differences among different groups(P<0.01); while AKT、ERK1/2 and STAT3 protein have not obvious changes among different groups (P<0.01).4. transwell invasive studies results showed that RGZ or TRG can inhibit Hep-2 cells invasion, There were significant differences among different groups(P<0.01).5. Zymography results showed that RGZ or TRG can inhibit Hep-2 cell MMP-2 activity under normoxia and hypoxia. There were significant differences among different time groups(P<0.01).The third chapter:the impact of RGZ and the TRG on human laryngeal cancer Hep-2 cell nude mice xenograftsMethods1. After establishment of human laryngeal carcinoma xenografts in nude mice were randomly divided into control group, RGZ experimental group and TRG experimental group, n=6, the treatment group for each 3d to the local injection of RGZ and TRG on tumor tissue at 50μmoL/L; control group was injected with 0.5% DMSO. Were treated 4w.2. Nude mice were observed every day for diet, activity and other physiological conditions. Each tumor size and nude body mass were measured every week, drown tumor growth curve. when the mice were killed at the end of treatment after 4W treatment, stripped and weighted tumor, calculated the quality of tumor inhibition rate. the changes of brain, heart, liver, lung, spleen and kidney and other important organs was observed under optical microscope after HE staining.3. The expression of CD34 antigen of in each nude mice xenografts group was detected by immunohistochemistry method, and calculated staining results for MVD values.4. The expression of COX-2、p65、VEGF and HIF-1αmRNA and protein in each nude mice xenografts group was detected by Real-time PCR and western.Results 1. Compared with the control group, RGZ and TRG inhibited tumor growth, the control group and experimental group of nude mice tumor volume, tumor mass were significantly difference, there were statistically significant difference among three groups (P<0.05).2. No significant adverse effects in nude mice was found during the course of treatment. There were no significant organic changes of heart, liver, lung, spleen, kidneys and other important organs in each group at the end of treatment.3. Compared with the control group, experimental group MVD in tumor tissue was significantly reduced, the difference of MVD values among the three groups were statistically significant (P<0.01).4. Real-time and western PCR results showed that VEGF、COX-2 and NF-κB mRNA and protein and HIF-1αprotein expression was significantly lower in the RGZ and TRG experimental group tumor xenografts in nude mice than that in the control group, the difference between the three groups was statistically significant(P<0.05).Conclusion1. The RGZ and the TRG inhibited Hep-2 cells proliferation in a concentration and time-dependent manner; block Hep-2 cells in the G0/G1 phase and induce apoptosis, its mechanism were related to inhibition of COX-2 and p65 protein and mRNA and VEGF mRNA expression, and reduced VEGF protein secretion.2. The RGZ and the TRG inhibited Hep-2 cells proliferation, inhibited VEGF expression in a dose-dependent manner under hypoxia, inhibited HIF-la protein expression in a dose-dependent manner under hypoxia. Its mechanism were related to inhibiting AKT, ERK1/2 and STAT3 protein phosphorylation and promoting the degradation of HIF-1αprotein through the proteasome pathway, inhibited the invasion of Hep-2 cells under normoxia and hypoxia, its mechanism were relate to reduced activity of the MMP-2.3. The RGZ and the TRG can inhibit laryngeal carcinoma xenografts growth, reduce the value of tumor MVD, down-regulateCOX-2, p65, HIF-1αand VEGF expression in Hep-2 cell xenografts in nude mice. the body had no significant toxic side effects.更多还原