【作者】 苗战军；

【导师】 张凤瑞； 彭利；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:肝细胞癌（hepatocellular carcinoma,HCC）是我国最常见的恶性肿瘤之一,其发病率高且难以治愈,病程进展迅速,易侵袭、转移、复发率高。随着医疗技术和医学科学研究的发展,对肝细胞癌的诊断及治疗技术已经有了很大的提高。但是关于肝细胞癌发生发展的确切机制还不清楚,目前仍然没有有效的预防措施,因此深入地研究肝细胞癌的发病机理以及探讨新的预防及治疗措施具有极其重要的临床意义。过氧化物酶体增殖物激活受体（Peroxisome proliferator -activated receptors,PPARs）是甾体激素受体超家族的新成员,PPARs共有3个亚型即PPARα、PPARβ、PPARγ,它们在结构、功能及组织分布上均有差异。PPARs的生物学功能复杂,参与调节脂质代谢与糖代谢、脂肪细胞分化和能量平衡、炎症反应、动脉粥样硬化及肿瘤细胞的分化与凋亡等生理和病理过程。近年来的许多研究表明PPARγ在多种肿瘤细胞中均有表达,通过多种途径抑制细胞增殖、诱导细胞凋亡及分化、抑制血管生成和降低肿瘤侵袭能力等。PPARγ的配体包括天然型配体和合成型配体两类。前者由实验证实从前列腺素D2衍生出的一系列前列腺素代谢产物是PPARγ的天然配体,其中15-脱氧前列腺J2（15d-PGJ2）与PPARγ的结合作用最强。合成型配体主要是胰岛素增敏剂噻唑烷二酮类化合物（ Thiazolidinediones ,TZDs）,包括罗格列酮（Rosiglitazone,Rosi）、匹格列酮（pioglitazone）、曲格列酮（troglitazone）等,临床上主要用于Ⅱ型糖尿病的治疗[1]。国外的一些实验证明PPARγ激动剂可以有效地预防肿瘤的发生,有望在肿瘤的化学预防中发挥重要作用[2 ]。罗格列酮是噻唑烷二酮类药物中生物利用度最高药效最强的一种药物,且其毒副作用相对较小。我们以前的试验结果表明罗格列酮可以显著抑制肝癌SMMC-7721细胞的生长并且诱导凋亡。本试验通过研究罗格列酮对肝癌细胞SMMC-7721中VEGF、AKT₁/2及P-AKT₁表达的影响,探讨PPARγ抑制肝癌SMMC-7721细胞生长的可能作用机制。方法: 1 SMMC-7721细胞常规培养,取培养48小时的细胞加入罗格列酮分0.1、1、10、100μmol/L4个浓度组,以及选取加入10μmol/L药物的细胞取24h、48h、72h三个作用时间,采用Western Blot法进行检测,观察罗格列酮的对SMMC-7721细胞中VEGF基因表达的影响来探讨罗格列酮对肝细胞癌血管生成影响的可能作用机制。2 SMMC-7721细胞常规培养,取培养48小时的细胞加入罗格列酮分1、10、100μmol/L3个浓度组,以及选取加入10μmol/L药物的细胞取0.5h、1h、2h、6h四个作用时间,采用Western Blot法进行检测,观察罗格列酮对SMMC-7721细胞中AKT_1/2、P-AKT₁基因表达的影响来探讨罗格列酮对肝细胞癌的抑制作用是否与PI3K/AKT信号转导通路有关。应用单因素方差分析统计光密度研究有无量效及时效关系。结果: 1罗格列酮可以显著降低SMMC-7721细胞株中VEGF蛋白的表达,并呈显著的时间依赖性和剂量依赖性变化（P<0.05）。2罗格列酮对SMMC-7721细胞株中AKT_1/2、P-AKT₁蛋白的表达无显著影响,表现在PVDF膜上显示条带亮度及宽度无明显变化（P>0.05）。结论:1罗格列酮可以显著降低SMMC-7721细胞株中VEGF蛋白的表达,并且呈显著的时间依赖性和剂量依赖性（P<0.05）。说明罗格列酮可通过降低肝细胞癌中VEGF的表达来抑制肿瘤血管的生长。2罗格列酮对SMMC-7721细胞AKT_1/2、P-AKT₁蛋白的表达无显著影响,也无浓度及时间依赖性（P>0.05）。表明罗格列酮可能不是通过直接抑制PI3K/AKT信号转导通路来抑制SMMC-7721细胞中VEGF的表达。更多还原

【Abstract】 Objective: The hepatocellular carcinoma is one of the clinically common malignant tumors,which has high mortality and difficult to cure. It progresses fast,invade、transfer and recur easily. Along with the development of medical technology and medical research,have already had a great exaltation to the diagnosis and the treatment technique of HCC. But at present the certain mechanism of HCC genesis is not clear,there is no effective protective measure yet. Thus it’s greatly significant in clinic to research the etiopathogenesis of HCC and investigate new protective measure.Peroxisome proliferator activated receptor is a new member of the NHR super family. PPARs subfamily has defined as PPARα, PPARβand PPARγ. They differ in their structure, function and tissue distribution specificity. Several lines of evidence indicate that PPARγplays an important role in regulating adipocyte and glucose metabolism、adipocyte differentiation、energy balance、inflammatory reaction、artherosclerosis,inducing differentiation and apoptosis of tumor cell. Recent data showed that ligands for PPARγoverexpressed in many tumor cell, through various paths inhibited tumor cell proliferation,induced apoptosis and differentiation、inhibited angiogenesis and depressed invasion of tumor cell.PPARγcontains natural and synthetical ligands. The former by derivation from the prostaglandin D2 by the experiment confirmation of a series of prostaglandin metabolism is the PPARγof natural ligands,among them 15d-PGJ2 combines strongest to the PPARγ.The synthetical ligands mainly is the thiazolidinediones of euglycemic agent such as rosiglitazone, pioglitazone, troglitazone.This medicine mainly used for theⅡtype the treatment of the diabetes on the clinic. Some experiment of the abroad certificate the PPARγexcitomotory can prevent tumorous development availably,hoping can play an important on tumorous chemical prophylaxis.Rosiglitazone is the drug which is the highest bioavailability and drug action.Our previous experiment indicated rosiglitazone can significantly inhibit the growth of SMMC-7721 cell line .Our experiment further reveal the possible mechanism of action on the inhibition of hepatoma growth by investigating the effect of rosiglitazone（PPARγagent）on the genetic expression of VEGF、AKT_1/2 and P-AKT₁ of SMMC-7721 cell line.Methods: 1 We detected the protein expression of VEGF by Western Blot in four groups which were administered with rosiglitazone of various concentrations,0.1、1、10、100（μmol/L）respectively and the cell joined 10μmol/L of rosiglitazone,and on three action times（24h、48h、72h）in each group. 2 We determined the protein expression of AKT_1/2、P-AKT1 by Western Blot in three groups which were administered with rosiglitazone of various concentrations,1、10、100μmol/L respectively and the cell joined 10μmol/L of rosiglitazone,and on four action times （0.5h、1h、2h、6h） in each group. By analysis of variance for one factors （ANOVA）, optical density was analysised to investigate time-effect relationship and quantity-effect relationship.Results:1 Western-blot showed that rosiglitazone can decrease the expression of protein VEGF in human hepatoma cell line SMMC-7721 in a dose-dependence and time-dependent manner（ P<0.05）.2 Detect the expression of AKT_1/2、P-AKT₁ by Western Blot: No obvious difference of expression of AKT_1/2、P-AKT1were found（P>0.05）.Conclusion:1 Rosiglitazone can decrease the expression of protein VEGF in human hepatocellular carcinoma cell line SMMC-7721 in a dose-dependence and time-dependent manner（P<0.05）.The result suggested that rosiglitazone can inhibit the angiogenesis,cell proliferation and metastasis of HCC, It is suggested that rosiglitazone may be a potential molecular therapeutic target for Hepatocellular carcinoma.2 The result show that rosiglitazone was not significantly related with the expression of AKT_1/2、P-AKT₁（P>0.05）. It shows that rosiglitazone decreasing the expression of protein VEGF perhaps do not inhibit the PI3K/AKT signal conduction pathway.更多还原