DEN诱导大鼠肝癌模型的代谢组学研究
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摘要
第一部分DEN诱导大鼠肝癌模型的代谢组学研究
[研究目的]
肝细胞性肝癌(简称肝癌,Hepatocellular Carcinoma, HCC)是我国最常见的恶性肿瘤之一,目前的研究认为：肝炎病毒感染及其他致癌因素是肝癌发生的主要外因。同时,肝癌和其它恶性肿瘤一样,其发生发展是由体内多基因多蛋白参与、多步骤协同的复杂过程。寻找肝癌发生、发展过程中的相关代谢产物有助于研究肝癌发生及发展的机制,从而为肝癌的预防、诊断及治疗提供理论基础。
组学概念的提出为研究者提供了一个整体研究疾病的新思路,基因组学、转录组学、蛋白组学等已经不为大家所陌生,作为系统生物学最下游的代谢组学,是一门新的科学,主要研究生物体对病理生理刺激或基因修饰所产生代谢物的质和量的动态变化规律。这种整体的研究模式可全面了解代谢物质在疾病发生、发展过程中的变化规律,对发病机制研究以及疾病的诊断、治疗和疗效评估具有重要意义。因此,我们采用该理论,结合现代生物质谱技术,对DEN诱导大鼠肝炎—肝硬化—肝癌模型以及慢性肝炎、肝硬化和肝癌病人进行代谢组学研究。
本研究拟首先通过DEN诱导建立大鼠肝炎—肝硬化—肝癌动物模型,观察在不同发展阶段血浆代谢小分子的变化情况,从中找出不同发展阶段大鼠血浆中潜在的代谢物分子标签；然后对模型肝脏组织进行代谢组研究,并与疾病的发展阶段进行关联；最后将这些代谢物分子标签在人血浆中进行验证,观察其对肝癌的预测能力。
[研究方法]
1.建立DEN诱导大鼠肝炎—肝硬化—肝癌模型；
2.模型不同阶段大鼠血浆的代谢组检测；
3.基于大鼠血浆代谢组的预测模型建立；
4.潜在大鼠血浆代谢产物分子标志物的筛选及鉴定；
5.模型不同阶段大鼠肝脏组织的代谢组检测；
6.基于大鼠肝脏组织代谢组的预测模型建立；
7.潜在血浆代谢产物分子标志物在肝炎、肝硬化、肝癌病人血浆中的验证及筛选。
[结果]
1.DEN诱导的大鼠肝癌模型可以很好的模拟人肝炎—肝硬化—肝癌的发展进程；
2.大鼠血浆的代谢组检测发现相对于NC组而言,DEN组大鼠在炎症期、硬化期及肝癌期,分别有382个、427个以及445个代谢物离子有显著性差异,其中又分别有253个、330个以及383个代谢物离子升高；
3.基于大鼠血浆代谢组数据建立的PLS-DA分析模型,计算出5个主成份,前两个主成份可以分别表示致癌过程及年龄增长的代谢物变化；
4.基于肝癌阶段与非肝癌阶段代谢组数据建立的OPLS模型,筛选出对模型贡献最大的50个变量,其中一半与HCC正相关,另一半与非HCC正相关；
5.用这50个代谢产物另外建立PLS-DA模型,计算出两个主成份,模型能够很好地区分炎症期、硬化期和肝癌期三个阶段；
6.对50个代谢产物进行鉴定,发现包括氨基酸、胆汁酸、磷脂、游离脂肪酸、神经鞘氨醇以及肉碱等；
7.基于大鼠肝脏组织的代谢组检测发现相对于NC组而言,DEN组大鼠在炎症期、硬化期及肝癌期,分别有326个、337个以及631个代谢物离子有显著性差异,在肝癌和肿瘤邻近组织中也发现有578个代谢产物存在差异,瘤旁组织与肝硬化组织更为接近,只有146个代谢产物不同；
8.基于大鼠肝脏组织代谢组数据建立的PLS-DA模型,计算出三个主成份,模型能够很好地区分炎症期、硬化期和肝癌期三个阶段；
9.基于大鼠肝癌组织和肿瘤邻近组织代谢组数据建立的OPLS模型,发现牛磺酸结合胆汁酸是最重要的代谢产物；
10.人血浆的代谢组与上述50个不同代谢产物相比,共有29个可以配对上；
11.用这29个代谢产物所建立的PLS-DA模型,能够很好地区分肝炎、肝硬化及肝细胞癌病人；
12.二元logistic回归进行计算,方程式显示如下(1,2)：5个代谢产物进入最后的肝癌预测方程。这些产物包括：二氢(神经)鞘氨醇(Measured mass:301.2980,X1),棕榈酰肉碱(Measured mass:399.3351,X2),一种胆汁酸衍生物(Measured mass:356.2711,X3),溶血磷脂酰胆碱(LPE(22：5),Measured mass:569.3475,X4),以及溶血磷脂酰乙醇胺(LPC(16：0),Measuredmass:453.2856,X5),5个代谢产物组合后的ROS曲线下面积为0.951；
13.5个代谢产物在肝癌病人血浆相对浓度与肝炎病人及肝硬化病人相比,除了胆汁酸衍生物在HCC与肝硬化之间差异没有统计学意义,均有显著差异。
[结论]
本研究采用液相色谱—质谱联用分析技术(LC-MS),对DEN诱导的大鼠肝癌动物模型血浆及肝脏组织进行代谢组学研究,筛选出区分肝癌与非肝癌的50个代谢产物,与肝炎、肝硬化及肝癌病人的血浆代谢组数据比较,最终寻找到5个潜在代谢标志物：二氢(神经)鞘氨醇,棕榈酰肉碱,一种胆汁酸衍生物,溶血磷脂酰胆碱,以及溶血磷脂酰乙醇胺。研究结果为寻找肝癌分子标志物、肝癌代谢相关通路研究及肝癌的诊断提供了新的思路与方法。
第二部分Hedgehog信号通路在肝内胆管癌发生发展中的作用
[研究目的]
肝内胆管癌(Intrahepatic Cholangiocarcinoma,ICC),又称胆管细胞癌或周围型胆管癌,是来自肝内二级分支以下胆管树上皮的肿瘤。在世界范围内,肝内胆管癌是仅次于肝细胞癌的第二大肝脏恶性肿瘤,占原发性肝脏恶性肿瘤的10%-20%,其发病率近年来呈上升趋势。肝内胆管癌具有发生隐匿、恶性程度高、发展迅速、手术切除率低、复发转移早、临床预后差等特点,严重影响病人的生活质量及生存率。以往对这类非肝细胞起源的肝脏恶性肿瘤的发生与发展过程缺乏系统的了解,研究水平也相对滞后。因此针对临床肝内胆管癌诊治中的难点进行深入研究,尤其是研究其发生、发展以及复发、转移的机制,以寻找新的有效的肝内胆管癌特异性治疗方法十分必要。
Hedgehog(Hh)基因是编码一系列分泌蛋白的基因家族,1980年首先在果蝇中被发现。Hh信号转导通路最早被发现对胚胎发育过程中的细胞分化和增殖起重要作用。近年来通过对其信号转导途径深入研究,发现Hh通路的异常在人类肿瘤的形成中也有极其重要的作用。HH基因在不同组织中过度表达以及靶基因突变,均可导致细胞特异性的组织增生,参与肿瘤的发生和发展。Hh通路与肿瘤的关系最早在Glioma中被证实,其与脑部神经瘤的形成联系密切,后又发现Hh信号异常与基底细胞癌、肺癌、前列腺癌、口腔癌和消化道肿瘤等的形成均有重要关系。
Hh信号通路在肝脏发育、细胞分化以及肿瘤发生上均起重要作用,本实验拟分析Hh信号通路相关分子在肝内胆管癌组织及肝内胆管癌细胞系中的表达,体内外调节Hh信号通路观察其对肝内胆管癌细胞系生物学特性的影响,并分析Hh信号通路下游转录分子Gli1、Gli2表达水平与肝内胆管癌病人临床病理资料及预后的相关性。以上工作有助全面揭示肝内胆管癌发生、发展的分子机制,深化对Hh信号通路的认识,为寻找肝内胆管癌新治疗靶点提供线索和思路。
[研究方法]
1. Real-time PCR检测正常肝内胆管上皮组织与对肝内胆管细胞癌组织中Hh通路信号分子的mRNA表达水平；
2.免疫组织化学检测正常肝内胆管上皮组织与对肝内胆管细胞癌组织中Hh通路信号分子的蛋白表达水平；
3.体外用小分子化合物改变Hh信号通路的活性,观察其对肝内胆管癌细胞系生物学特性的影响；
4.体内用小分子化合物改变Hh信号通路的活性,观察其对肝内胆管癌细胞系生物学特性的影响；
5.Hh通路信号分子与肝内胆管癌病人临床病理资料及预后资料的统计分析。
[结果]
1.Hh信号通路在肝内胆管癌组织中异常激活：Hh信号通路配体shh和ihh分别有87.8%和78.0%表达升高,受体ptchl有87.8%表达升高,下游分子smo有85.4%表达升高,下游转录分子gli1、gli2及gli3分别有65.9%、87.8%及36.6%表达升高,下游靶分子hhip、bcl2和c-myc分别有17.1%、75.6%和82.9%表达升高；
2.Hh信号通路在肝内胆管癌细胞系QBC939、RBE及HCCC-9810中也异常激活；
3.在体外用Hh信号通路特异性抑制剂KAAD-Cyclopamine能够抑制QBC939、RBE和HCCC-9810的增殖,且其抑制程度与KAAD-Cyclopamine呈剂量依赖性；
4.在体内Cyclopamine能够明显抑制QBC939细胞种植肿瘤的大小；
5.肝内胆管癌组织中Hh信号通路下游转录分子Gli1蛋白高表达的病人更易发生肝内转移,预后更差；
6.肝内胆管癌组织中Hh信号通路下游转录分子Gli2蛋白高表达的病人更易发生肝内转移和血管侵犯,预后更差；
7.单因素分析发现发现血清CA19-9>37units/ml、最大肿瘤直径>5cm、伴有肝内转移、淋巴结转移、血管侵犯、T3/4、TNMⅢ/Ⅳ期和Gli1、Gli2蛋白在肿瘤组织中高表达的肝内胆管癌病人总体生存率更低；
8.多因素分析发现最大肿瘤直径、肝内转移、淋巴结转移和Gli1的表达高低与肝内胆管癌病人的预后密切相关,是独立的预后因素。
[结论]本研究从人肝内胆管癌组织标本和体外培养的肝内胆管癌细胞系两个方面,检测发现Hh信号通路在其中异常激活；用Hh信号通路特异性抑制剂在体内外改变通路活性,均能够明显抑制肝内胆管癌细胞系的增殖能力；肝内胆管癌组织中Hh信号通路下游转录分子Gli1的表达高低与肝内转移及病人预后密切相关,Gli2的表达高低与肝内转移、血管侵犯、T分期及病人预后密切相关；Gli1的表达高低是肝内胆管癌的独立预后因素。本研究可加深人们对肝内胆管癌发生发展及其调控机理的认识,为研发肝内胆管癌靶向治疗新策略提供靶点。
Part I Metabonomic investigations of DEN induced hepatocellular carcinoma in SD rats
Hepatocellular carcinoma (HCC) is a predominant malignancy in the liver. It has become the third leading cause of death worldwide according to the estimation of WTO. HCC has a high incidence level in the East Asia countries. Cirrhosis is considered as the major risk factor for liver carcinogenesis. Hepatitis B and C virus infection, aflatoxin B1 exposure and alcohol drinking are also the commonly risk factors for HCC. In China, HBV infection is the most prevalent cause of HCC and account for more than 80% cases. The HBV related cirrhosis is also accounted for 2.7% of the HCC incidence in East Asia. Although the mechanism of carcinogenesis of HBV is still not very clear, the chronic necroinflammation with subsequent fibrosis and hepatocyte proliferation are generally considered as the major risk factors. CT and MRI are the major tools for the diagnosis, and AFP is a commonly used biomarker in the clinic. The diagnosis of HCC still is a great challenge for the clinic.
Sprague-Dawley (SD) rats exposed to diethylnitrosamine (DEN) are suitable for the study of the hepatocarcinogenesis. As an environmental carcinogen, DEN can induce HCC by alkylate DNA molecules, and can be converted into highly reactive molecules by P450-dependent mono-oxygenase. Since the DEN induced hepatocarcinogenesis progresses from inflammation to cirrhosis, finally to HCC, it is similar to progress of HBV infection disease. This animal model is widely used in the study of the HCC, either for the mechanism of carcinogenesis or the seeking of diagnostic biomarkers.
To obtain a systemic understanding and find potential biomarkers of the hepatocarcinogenesis, diethylnitrosamine (DEN) induced rat model of HCC was recruited in this study. Liquid chromatography coupled to time of flight mass spectrometry system was used for data acquisition. As a result,50 differential metabolites between HCC and non-HCC plasma were selected from rat models,29 of which could be found from the patients. A further filtration obtained 5 metabolites which could be considered as potential biomarker candidates for HCC, including dihydrosphingosine, Lysophosphatidyl ethanolamine (LPE,16:0), Palmityl-L-carnitine, Lysophosphatidyl choline (LPC,22:5), and a bile acid derivative. Considering the influences of ageing, LPE (16:0) could be a better one among the group. The metabolic profiling of rat tissue also indicated the latent mechanism of the up-regulation of LPE. The current study represented a suitable template for the searching and validation of potential biomarkers from plasma metabonome.
Part II Activation of hedgehog signaling in intrahepatic cholangiocarcinoma
Intrahepatic cholangiocarcinoma (ICC) is a rare malignant tumor which arises from the epithelial cells of intrahepatic bile ducts (beyond the second order bile ducts). The incidence of ICC is reported to be about 10% of primary liver cancers and its lethality is also on the top of primary liver cancers. Chronic cholangitis, HCV/HBV infection, choledochal cysts etal are significantly associated with ICC. ICC has the worst prognosis of any tumor arising in the liver; its 5-year survival is poor, and accompanied by a high recurrence rate. The overall 5-year survival rate ranges 15%-20%. The development and progression of ICC is a complicated process involving multiple genes and transforming steps. But the mechanisms underlying the development of ICC remain unclear. Therefore, searching for new ICC associated molecules may broaden our view to the progress of ICC.
Activation of the hedgehog (Hh) pathway is shown to be involved in several types of gastrointestinal cancers. Here, we provide evidence to indicate that hedgehog signaling activation occurs frequently in ICC. We detect mRNA expression of Hh molecules in 41 ICC tissues and 8 normal intrahepatic bile duct epithelia. Expression of shh and ptchl are both elevated in about 87.8% of ICC. Consistent with this, hedgehog target genes Gli1 and Gli2 are elevated in 65.9% and 87.8% of the tumors, respectively, suggesting that the hedgehog pathway is frequently activated in ICC. We found QBC939, RBE and HCCC-9810 cells with detectable hedgehog target genes. Specific inhibition of hedgehog signaling in these three cell lines by smoothened (SMO) antagonist, KAAD-cyclopamine, or with Shh neutralizing antibodies decreases expression of hedgehog target genes, inhibits cell growth and results in apoptosis. Our data suggested that abnormal Hh signaling activation plays important roles in the development and progression of ICC.
We found that a high expression of nuclear Gill was correlated with positive intrahepatic metastasis (P<0.05) and a high expression of nuclear Gil2 was correlated with positive intrahepatic metastasis and venous invasion. The survival curve of the group with high Gill or Gli2 nuclear expression was significantly lower than that of the group with low Gill or Gli2 nuclear expression (P<0.005). According to the multivariate analyses, high Glil expression can be considered an independent prognostic factor whose occurrence indicates a worse prognosis. We conclude that an elevated Gli1 expression adds an independent parameter that can be used when determining the prognosis of patients with ICC. Inhibition of the Hh pathway molecules might be a valid therapeutic strategy for ICC.
[研究目的]
肝细胞性肝癌(简称肝癌,Hepatocellular Carcinoma, HCC)是我国最常见的恶性肿瘤之一,目前的研究认为：肝炎病毒感染及其他致癌因素是肝癌发生的主要外因。同时,肝癌和其它恶性肿瘤一样,其发生发展是由体内多基因多蛋白参与、多步骤协同的复杂过程。寻找肝癌发生、发展过程中的相关代谢产物有助于研究肝癌发生及发展的机制,从而为肝癌的预防、诊断及治疗提供理论基础。
组学概念的提出为研究者提供了一个整体研究疾病的新思路,基因组学、转录组学、蛋白组学等已经不为大家所陌生,作为系统生物学最下游的代谢组学,是一门新的科学,主要研究生物体对病理生理刺激或基因修饰所产生代谢物的质和量的动态变化规律。这种整体的研究模式可全面了解代谢物质在疾病发生、发展过程中的变化规律,对发病机制研究以及疾病的诊断、治疗和疗效评估具有重要意义。因此,我们采用该理论,结合现代生物质谱技术,对DEN诱导大鼠肝炎—肝硬化—肝癌模型以及慢性肝炎、肝硬化和肝癌病人进行代谢组学研究。
本研究拟首先通过DEN诱导建立大鼠肝炎—肝硬化—肝癌动物模型,观察在不同发展阶段血浆代谢小分子的变化情况,从中找出不同发展阶段大鼠血浆中潜在的代谢物分子标签；然后对模型肝脏组织进行代谢组研究,并与疾病的发展阶段进行关联；最后将这些代谢物分子标签在人血浆中进行验证,观察其对肝癌的预测能力。
[研究方法]
1.建立DEN诱导大鼠肝炎—肝硬化—肝癌模型；
2.模型不同阶段大鼠血浆的代谢组检测；
3.基于大鼠血浆代谢组的预测模型建立；
4.潜在大鼠血浆代谢产物分子标志物的筛选及鉴定；
5.模型不同阶段大鼠肝脏组织的代谢组检测；
6.基于大鼠肝脏组织代谢组的预测模型建立；
7.潜在血浆代谢产物分子标志物在肝炎、肝硬化、肝癌病人血浆中的验证及筛选。
[结果]
1.DEN诱导的大鼠肝癌模型可以很好的模拟人肝炎—肝硬化—肝癌的发展进程；
2.大鼠血浆的代谢组检测发现相对于NC组而言,DEN组大鼠在炎症期、硬化期及肝癌期,分别有382个、427个以及445个代谢物离子有显著性差异,其中又分别有253个、330个以及383个代谢物离子升高；
3.基于大鼠血浆代谢组数据建立的PLS-DA分析模型,计算出5个主成份,前两个主成份可以分别表示致癌过程及年龄增长的代谢物变化；
4.基于肝癌阶段与非肝癌阶段代谢组数据建立的OPLS模型,筛选出对模型贡献最大的50个变量,其中一半与HCC正相关,另一半与非HCC正相关；
5.用这50个代谢产物另外建立PLS-DA模型,计算出两个主成份,模型能够很好地区分炎症期、硬化期和肝癌期三个阶段；
6.对50个代谢产物进行鉴定,发现包括氨基酸、胆汁酸、磷脂、游离脂肪酸、神经鞘氨醇以及肉碱等；
7.基于大鼠肝脏组织的代谢组检测发现相对于NC组而言,DEN组大鼠在炎症期、硬化期及肝癌期,分别有326个、337个以及631个代谢物离子有显著性差异,在肝癌和肿瘤邻近组织中也发现有578个代谢产物存在差异,瘤旁组织与肝硬化组织更为接近,只有146个代谢产物不同；
8.基于大鼠肝脏组织代谢组数据建立的PLS-DA模型,计算出三个主成份,模型能够很好地区分炎症期、硬化期和肝癌期三个阶段；
9.基于大鼠肝癌组织和肿瘤邻近组织代谢组数据建立的OPLS模型,发现牛磺酸结合胆汁酸是最重要的代谢产物；
10.人血浆的代谢组与上述50个不同代谢产物相比,共有29个可以配对上；
11.用这29个代谢产物所建立的PLS-DA模型,能够很好地区分肝炎、肝硬化及肝细胞癌病人；
12.二元logistic回归进行计算,方程式显示如下(1,2)：5个代谢产物进入最后的肝癌预测方程。这些产物包括：二氢(神经)鞘氨醇(Measured mass:301.2980,X1),棕榈酰肉碱(Measured mass:399.3351,X2),一种胆汁酸衍生物(Measured mass:356.2711,X3),溶血磷脂酰胆碱(LPE(22：5),Measured mass:569.3475,X4),以及溶血磷脂酰乙醇胺(LPC(16：0),Measuredmass:453.2856,X5),5个代谢产物组合后的ROS曲线下面积为0.951；
13.5个代谢产物在肝癌病人血浆相对浓度与肝炎病人及肝硬化病人相比,除了胆汁酸衍生物在HCC与肝硬化之间差异没有统计学意义,均有显著差异。
[结论]
本研究采用液相色谱—质谱联用分析技术(LC-MS),对DEN诱导的大鼠肝癌动物模型血浆及肝脏组织进行代谢组学研究,筛选出区分肝癌与非肝癌的50个代谢产物,与肝炎、肝硬化及肝癌病人的血浆代谢组数据比较,最终寻找到5个潜在代谢标志物：二氢(神经)鞘氨醇,棕榈酰肉碱,一种胆汁酸衍生物,溶血磷脂酰胆碱,以及溶血磷脂酰乙醇胺。研究结果为寻找肝癌分子标志物、肝癌代谢相关通路研究及肝癌的诊断提供了新的思路与方法。
第二部分Hedgehog信号通路在肝内胆管癌发生发展中的作用
[研究目的]
肝内胆管癌(Intrahepatic Cholangiocarcinoma,ICC),又称胆管细胞癌或周围型胆管癌,是来自肝内二级分支以下胆管树上皮的肿瘤。在世界范围内,肝内胆管癌是仅次于肝细胞癌的第二大肝脏恶性肿瘤,占原发性肝脏恶性肿瘤的10%-20%,其发病率近年来呈上升趋势。肝内胆管癌具有发生隐匿、恶性程度高、发展迅速、手术切除率低、复发转移早、临床预后差等特点,严重影响病人的生活质量及生存率。以往对这类非肝细胞起源的肝脏恶性肿瘤的发生与发展过程缺乏系统的了解,研究水平也相对滞后。因此针对临床肝内胆管癌诊治中的难点进行深入研究,尤其是研究其发生、发展以及复发、转移的机制,以寻找新的有效的肝内胆管癌特异性治疗方法十分必要。
Hedgehog(Hh)基因是编码一系列分泌蛋白的基因家族,1980年首先在果蝇中被发现。Hh信号转导通路最早被发现对胚胎发育过程中的细胞分化和增殖起重要作用。近年来通过对其信号转导途径深入研究,发现Hh通路的异常在人类肿瘤的形成中也有极其重要的作用。HH基因在不同组织中过度表达以及靶基因突变,均可导致细胞特异性的组织增生,参与肿瘤的发生和发展。Hh通路与肿瘤的关系最早在Glioma中被证实,其与脑部神经瘤的形成联系密切,后又发现Hh信号异常与基底细胞癌、肺癌、前列腺癌、口腔癌和消化道肿瘤等的形成均有重要关系。
Hh信号通路在肝脏发育、细胞分化以及肿瘤发生上均起重要作用,本实验拟分析Hh信号通路相关分子在肝内胆管癌组织及肝内胆管癌细胞系中的表达,体内外调节Hh信号通路观察其对肝内胆管癌细胞系生物学特性的影响,并分析Hh信号通路下游转录分子Gli1、Gli2表达水平与肝内胆管癌病人临床病理资料及预后的相关性。以上工作有助全面揭示肝内胆管癌发生、发展的分子机制,深化对Hh信号通路的认识,为寻找肝内胆管癌新治疗靶点提供线索和思路。
[研究方法]
1. Real-time PCR检测正常肝内胆管上皮组织与对肝内胆管细胞癌组织中Hh通路信号分子的mRNA表达水平；
2.免疫组织化学检测正常肝内胆管上皮组织与对肝内胆管细胞癌组织中Hh通路信号分子的蛋白表达水平；
3.体外用小分子化合物改变Hh信号通路的活性,观察其对肝内胆管癌细胞系生物学特性的影响；
4.体内用小分子化合物改变Hh信号通路的活性,观察其对肝内胆管癌细胞系生物学特性的影响；
5.Hh通路信号分子与肝内胆管癌病人临床病理资料及预后资料的统计分析。
[结果]
1.Hh信号通路在肝内胆管癌组织中异常激活：Hh信号通路配体shh和ihh分别有87.8%和78.0%表达升高,受体ptchl有87.8%表达升高,下游分子smo有85.4%表达升高,下游转录分子gli1、gli2及gli3分别有65.9%、87.8%及36.6%表达升高,下游靶分子hhip、bcl2和c-myc分别有17.1%、75.6%和82.9%表达升高；
2.Hh信号通路在肝内胆管癌细胞系QBC939、RBE及HCCC-9810中也异常激活；
3.在体外用Hh信号通路特异性抑制剂KAAD-Cyclopamine能够抑制QBC939、RBE和HCCC-9810的增殖,且其抑制程度与KAAD-Cyclopamine呈剂量依赖性；
4.在体内Cyclopamine能够明显抑制QBC939细胞种植肿瘤的大小；
5.肝内胆管癌组织中Hh信号通路下游转录分子Gli1蛋白高表达的病人更易发生肝内转移,预后更差；
6.肝内胆管癌组织中Hh信号通路下游转录分子Gli2蛋白高表达的病人更易发生肝内转移和血管侵犯,预后更差；
7.单因素分析发现发现血清CA19-9>37units/ml、最大肿瘤直径>5cm、伴有肝内转移、淋巴结转移、血管侵犯、T3/4、TNMⅢ/Ⅳ期和Gli1、Gli2蛋白在肿瘤组织中高表达的肝内胆管癌病人总体生存率更低；
8.多因素分析发现最大肿瘤直径、肝内转移、淋巴结转移和Gli1的表达高低与肝内胆管癌病人的预后密切相关,是独立的预后因素。
[结论]本研究从人肝内胆管癌组织标本和体外培养的肝内胆管癌细胞系两个方面,检测发现Hh信号通路在其中异常激活；用Hh信号通路特异性抑制剂在体内外改变通路活性,均能够明显抑制肝内胆管癌细胞系的增殖能力；肝内胆管癌组织中Hh信号通路下游转录分子Gli1的表达高低与肝内转移及病人预后密切相关,Gli2的表达高低与肝内转移、血管侵犯、T分期及病人预后密切相关；Gli1的表达高低是肝内胆管癌的独立预后因素。本研究可加深人们对肝内胆管癌发生发展及其调控机理的认识,为研发肝内胆管癌靶向治疗新策略提供靶点。
Part I Metabonomic investigations of DEN induced hepatocellular carcinoma in SD rats
Hepatocellular carcinoma (HCC) is a predominant malignancy in the liver. It has become the third leading cause of death worldwide according to the estimation of WTO. HCC has a high incidence level in the East Asia countries. Cirrhosis is considered as the major risk factor for liver carcinogenesis. Hepatitis B and C virus infection, aflatoxin B1 exposure and alcohol drinking are also the commonly risk factors for HCC. In China, HBV infection is the most prevalent cause of HCC and account for more than 80% cases. The HBV related cirrhosis is also accounted for 2.7% of the HCC incidence in East Asia. Although the mechanism of carcinogenesis of HBV is still not very clear, the chronic necroinflammation with subsequent fibrosis and hepatocyte proliferation are generally considered as the major risk factors. CT and MRI are the major tools for the diagnosis, and AFP is a commonly used biomarker in the clinic. The diagnosis of HCC still is a great challenge for the clinic.
Sprague-Dawley (SD) rats exposed to diethylnitrosamine (DEN) are suitable for the study of the hepatocarcinogenesis. As an environmental carcinogen, DEN can induce HCC by alkylate DNA molecules, and can be converted into highly reactive molecules by P450-dependent mono-oxygenase. Since the DEN induced hepatocarcinogenesis progresses from inflammation to cirrhosis, finally to HCC, it is similar to progress of HBV infection disease. This animal model is widely used in the study of the HCC, either for the mechanism of carcinogenesis or the seeking of diagnostic biomarkers.
To obtain a systemic understanding and find potential biomarkers of the hepatocarcinogenesis, diethylnitrosamine (DEN) induced rat model of HCC was recruited in this study. Liquid chromatography coupled to time of flight mass spectrometry system was used for data acquisition. As a result,50 differential metabolites between HCC and non-HCC plasma were selected from rat models,29 of which could be found from the patients. A further filtration obtained 5 metabolites which could be considered as potential biomarker candidates for HCC, including dihydrosphingosine, Lysophosphatidyl ethanolamine (LPE,16:0), Palmityl-L-carnitine, Lysophosphatidyl choline (LPC,22:5), and a bile acid derivative. Considering the influences of ageing, LPE (16:0) could be a better one among the group. The metabolic profiling of rat tissue also indicated the latent mechanism of the up-regulation of LPE. The current study represented a suitable template for the searching and validation of potential biomarkers from plasma metabonome.
Part II Activation of hedgehog signaling in intrahepatic cholangiocarcinoma
Intrahepatic cholangiocarcinoma (ICC) is a rare malignant tumor which arises from the epithelial cells of intrahepatic bile ducts (beyond the second order bile ducts). The incidence of ICC is reported to be about 10% of primary liver cancers and its lethality is also on the top of primary liver cancers. Chronic cholangitis, HCV/HBV infection, choledochal cysts etal are significantly associated with ICC. ICC has the worst prognosis of any tumor arising in the liver; its 5-year survival is poor, and accompanied by a high recurrence rate. The overall 5-year survival rate ranges 15%-20%. The development and progression of ICC is a complicated process involving multiple genes and transforming steps. But the mechanisms underlying the development of ICC remain unclear. Therefore, searching for new ICC associated molecules may broaden our view to the progress of ICC.
Activation of the hedgehog (Hh) pathway is shown to be involved in several types of gastrointestinal cancers. Here, we provide evidence to indicate that hedgehog signaling activation occurs frequently in ICC. We detect mRNA expression of Hh molecules in 41 ICC tissues and 8 normal intrahepatic bile duct epithelia. Expression of shh and ptchl are both elevated in about 87.8% of ICC. Consistent with this, hedgehog target genes Gli1 and Gli2 are elevated in 65.9% and 87.8% of the tumors, respectively, suggesting that the hedgehog pathway is frequently activated in ICC. We found QBC939, RBE and HCCC-9810 cells with detectable hedgehog target genes. Specific inhibition of hedgehog signaling in these three cell lines by smoothened (SMO) antagonist, KAAD-cyclopamine, or with Shh neutralizing antibodies decreases expression of hedgehog target genes, inhibits cell growth and results in apoptosis. Our data suggested that abnormal Hh signaling activation plays important roles in the development and progression of ICC.
We found that a high expression of nuclear Gill was correlated with positive intrahepatic metastasis (P<0.05) and a high expression of nuclear Gil2 was correlated with positive intrahepatic metastasis and venous invasion. The survival curve of the group with high Gill or Gli2 nuclear expression was significantly lower than that of the group with low Gill or Gli2 nuclear expression (P<0.005). According to the multivariate analyses, high Glil expression can be considered an independent prognostic factor whose occurrence indicates a worse prognosis. We conclude that an elevated Gli1 expression adds an independent parameter that can be used when determining the prognosis of patients with ICC. Inhibition of the Hh pathway molecules might be a valid therapeutic strategy for ICC.
引文
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