罗格列酮和曲格列酮对人喉癌Hep-2细胞影响的实验研究

发布时间：2018-06-18 10:15

本文选题：喉癌 + 罗格列酮　；参考：《郑州大学》2010年博士论文

【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(P0.05)。 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(P0.01),S期细胞比例逐渐降低(P0.01),而G2/M期细胞比例也呈逐渐降低趋势(P0.01)；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(P0.01)。 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(P0.01)。 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(P0.01)。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(P0.01)。 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(P0.01)。而HIF-1αmRNA表达水平在各浓度组均无明显改变(P0.05)。 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(P0.01)；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(P0.05)。 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(P0.01)。 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(P0.01)。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(P0.05)。 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(P0.01)。 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(P0.05)。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。
[Abstract]:Cancer of the larynx is an epithelia in the Department of Otolaryngology, which is a malignant tumor. 11%-22%., which accounts for the malignant tumor of the otorhinolaryngology, has made great progress in the treatment of laryngology in recent decades, but it still needs to be improved in terms of survival and quality of life. With the continuous emergence of new chemotherapeutic drugs, the treatment of larynx cancer has also been transferred to the hand. The comprehensive treatment of surgery, radiotherapy and chemotherapy, therefore, it is of great significance to research and explore more and more effective chemotherapeutic drugs for laryngeal cancer.
Peroxisome poliferator-activited receptor (PPAR) is a nuclear transcription factor. It has a variety of biological effects and participates in a variety of physiological responses, especially in the storage and metabolism of fat. The present study shows that PPAR is divided into three subtypes (alpha, delta). The PPARy subtype is most closely related to tumor, so it is also the most extensive and in-depth study of tumor function. Many reports show that PPARy is expressed in a variety of tumor tissues. When PPARy agonists are used, the proliferation and invasion of tumor cells can be inhibited, angiogenesis and apoptosis can be induced. The malignant biological behavior of the tumor cells is changed. Therefore, recent advances in the study of PPARy as the target of drugs have become one of the hotspots in the research and development of antitumor drugs.
PPARy agonists can be divided into two major categories: natural agonists and synthetic agonists. Synthetic PPARy agonists are the most representative of the thiazolidane two ketone (thiazolidinedione compounds, TZDs), and are clinically applied to the treatment of diabetes. In recent years, a large number of studies have shown that TZDs can inhibit the proliferation of many tumor cells and inhibit the tumor. The formation of neovascularization, block cell cycle and induce apoptosis, and TZDs as the main therapeutic drug for diabetes is widely used for many years because of its good curative effect and less side effects. Therefore, the inhibitory effect of TZDs on tumor proliferation is more meaningful. The research shows that the mechanism of TZDs is very complex, and even some scholars have proposed TZDs. It is possible to play an anti-tumor role by activating other pathways outside PPAR gamma, but the anti-tumor effect of TZDs is beyond doubt.
In order to investigate the effect of TZDs on laryngeal cancer Hep-2 cells, this study first used TZDs to represent Rosiglitazone (RGZ) and Zugle (Troglitazone, TRG) in Hep-2 cells in vitro. MTT, flow cytometry, Real-time PCR, Western and various molecular biology techniques were observed in vitro. The effect of cell proliferation, apoptosis and hypoxia induced HIF-1 alpha and VEGF and its mechanism are discussed. On the basis of in vitro experiments, we observe and observe the effects of RGZ and TRG on the subcutaneous transplanted tumor of human larynx Hep-2 cells in nude mice and the toxic reaction of RGZ and TRG in nude mice bearing tumor, and develop new ideas and methods for the chemotherapy of larynx cancer. In the future, it provides the theoretical basis and experimental basis for the prevention and control of laryngeal cancer with PPAR gamma as a target. It provides valuable experimental basis for the development of TZDs and clinical application. This study is divided into the following three chapters:
Chapter one: the effects of RGZ and TRG on the proliferation and apoptosis of human laryngeal carcinoma Hep-2 cells and its mechanism.
1. MTT colorimetric assay was used to detect the change of Hep-2 cell proliferation activity after 12,24,36,48,60,72h treated by 12.5,25,50100 mol/L RGZ or TRG.
2. flow cytometry was used to detect the apoptosis rate and cell cycle distribution of Hep-2 cells after 0,24,48,72h treated with 50 mol/L RGZ or TRG.
3. Real-time PCR and Western methods were used to detect the changes in COX-2 and p65 mRNA and protein and VEGFmRNA levels in Hep-2 cells after the action of 50 mol/L RGZ or TRG on 0,24,48,72h.
4. ELISA method was used to detect the change of VEGF protein level in Hep-2 cell culture supernatant after 25,50100 24h mol/L or RGZ treatment.
Result
The proliferation activity of cells was significantly inhibited after 1.RGZ or TRG treatment, and the inhibition rate increased in a concentration dependent and time-dependent manner (P0.05).
The results of 2. flow cytometry showed that after the action of 50 mol/L RGZ or TRG, the proportion of cells in G0/G1 phase increased gradually (P0.01), the proportion of cells in S phase decreased gradually (P0.01), and the proportion of G2/M phase cells decreased gradually (P0.01). As RGZ or TRG work extended, the apoptosis rate was gradually increased in time dependent. There were significant differences between different time groups (P0.01).
The results of 3. Real-time PCR and Western showed that the COX-2 and p65 mRNA and protein and VEGF expressions in Hep-2 cells after the action of 50 mol/L RGZ or TRG were time dependent, and there was a significant difference between the different time groups.
The results of 4. ELISA showed that the level of VEGF protein in the supernatant of Hep-2 cells cultured by 25,50100 mol/L RGZ or TRG was time dependent, and there was a significant difference between different time groups (P0.01).
The second chapter is about the effect of RGZ and TRG on the expression of HIF-1 and VEGF in hypoxia induced Hep-2 cells.
Method
1 MTT method was used to detect the change of Hep-2 cell proliferation activity after 12,24,36,48,60,72h treatment by 12.5,25,50100 or mol/LRGZ or TRG in hypoxia environment.
The changes of VEGF mRNA in cells and protein levels in culture supernatant were detected by Real-time PCR and ELISA method in 2. mol/L RGZ or TRG Hep-2 cells 24h under anoxic environment.
3. Real-time PCR and Western were used to detect the changes of HIF-1 mRNA mRNA and protein levels in Hep-2 cells after RGZ or TRG acted on 16h cells.
4. the changes in the level of p-AKT/AKT, p-ERK1/2/ERK1/2, p-STAT3/STAT3 protein in Hep-2 cells after 25,50100 micron mol/L RGZ or TRG were detected by western method.
5. Transwell cell invasion assay was used to detect the effect of 50 mol/LRGZ or TRG on the invasion of Hep-2 cells under hypoxia and normoxic environment.
6. gelatin zymography was used to analyze the effects of 50 mol/LRGZ or TRG on MMP-2 activity in Hep-2 cells under hypoxia and normoxic environment.
Result
1.MTT results showed that the proliferation activity of RGZ and TRG cells in Hep-2 cells under anoxic environment was significantly inhibited, and the inhibitory effect on cells was more obvious than that under the normal oxygen environment. The cell inhibition rate was time dependent and dose dependent (P0.01).
2. Real-time PCR and ELISA results showed that RGZ and TRG showed a concentration dependent inhibition of the level of VEGF protein and intracellular mRNA in the culture supernatant induced by hypoxia (P0.01), but the expression level of HIF-1 alpha mRNA was not significantly changed in the concentration groups (P0.05).
The results of 3. Western showed that RGZ and TRG showed a concentration dependent inhibition of the expression of HIF-1 alpha, p-AKT, p-ERK1/2 and p-STAT3 protein in Hep-2 cells induced by hypoxia (P0.01), but there was no significant change in the expression of AKT, ERK1/2 and STAT3 protein in each group. There was no significant difference between the groups.
The results of 4. Transwell cell invasion test showed that both RGZ and TRG could significantly inhibit the invasion of Hep-2 cells in hypoxia and normoxic environment, and there were significant differences between each group (P0.01).
5. the results of gelatin zymography showed that RGZ and TRG could inhibit MMP-2 activity in Hep-2 cells under hypoxia and normoxic environment, and the difference was statistically significant among all groups (P0.01).
The third chapter is about the influence of RGZ and TRG on human laryngeal carcinoma Hep-2 cell xenografts in nude mice.
1. the nude mice model of human larynx cancer was randomly divided into control group, RGZ experimental group and TRG experimental group, 6 rats in each group. The treatment group was injected with 50 mu moL/L RGZ and TRG, once every 3 days, and 0.5% two methyl sulfoxide was injected into the tumor for 4 weeks.
2. every d observed the diet, activity and other physiological conditions of nude mice. The tumor size and mass of nude mice were measured weekly and the tumor growth curve was plotted. After 4 weeks of treatment, the mice were killed and the tumor was weighed and the tumor suppressor rate was calculated by.HE staining. The changes of the brain, heart, liver, lung, spleen and kidney were observed under the optical microscope.
3. immunohistochemical staining was used to detect the expression of CD34 antigen in nude mice xenografts, and the MVD values were analyzed by statistical analysis.
4. Real-time PCR and Western were used to detect the expression of COX-2, p65, HIF-1 alpha and VEGF mRNA and protein in each group.
Result
1. compared with the control group, RGZ and TRG could obviously inhibit the growth of xenografts in nude mice, and there was a significant difference between the tumor volume and the mass group of the control group and the experimental group (P0.05).
2. during treatment, no obvious adverse reactions were observed in nude mice. No significant organic changes were found in heart, liver, lung, spleen, kidney and other important organs at the end of treatment.
3. compared with the control group, the MVD value in the tumor tissue of the experimental group was significantly reduced, and the difference of MVD between the three groups was statistically significant (P0.01).
The results of 4. Real-time PCR and Western showed that RGZ and TRG could inhibit the expression of COX-2, p65 and VEGF mRNA and protein and HIF-1 alpha protein in nude mice, and there was a statistically significant difference between groups (P0.05).
conclusion
1.RGZ and TRG have inhibitory effect on Hep-2 cells in concentration and time dependent proliferation, blocking Hep-2 cells in G0/G1 stage and inducing apoptosis. The mechanism is related to the inhibition of COX-2 and p65 protein and the expression of mRNA and VEGF mRNA, and down regulation of the secretion of VEGF protein.
In 2. anoxic environment, RGZ or TRG still inhibited the proliferation of Hep-2 cells, inhibited the expression of VEGFmRNA and the secretion of VEGF protein in the culture supernatant, and lowered the level of HIF-1 alpha protein induced by hypoxia; its mechanism was closely related to the inhibition of the phosphorylation of AKT, ERK1/2 and STAT3 protein and the degradation of HIF-1 alpha protein through the egg white enzyme pathway. It can inhibit the invasion of Hep-2 cells in normoxic and hypoxic environment, and its mechanism is closely related to the reduction of MMP-2 activity.
3.RGZ and TRG can inhibit the growth of transplanted tumor in nude mice, reduce the MVD value of tumor tissue, and down regulate the expression of COX-2, p65, HIF-1 A and VEGF in the transplanted tumor of the larynx, and have no toxic and side effects on the body.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.65

【参考文献】