鹅去氧胆酸衍生物HS-1200抑制大鼠原发性肝癌及HepG2裸鼠肝癌移植瘤的研究

发布时间：2018-05-08 06:19

本文选题：原发性肝癌 + DEN　；参考：《山东大学》2017年博士论文

【摘要】：研究背景及目的:原发性肝癌是最常见的恶性肿瘤之一,是全球癌症死亡的主要原因。其发病率高,预后差,仅2012年,全球就约有782500例新的原发性肝癌病例发生,同时约有745500例肝癌死亡病例发生。肝细胞癌(hepatocellular carcinoma,HCC)占原发性肝癌患者的多数。由于原发性肝细胞癌具有高转移率和高复发率,尽管近年的诊断和治疗方法取得了相当大的的进步,但死亡率仍然很高。目前用于预防肝癌药物的数量是很有限的,而且很多的药物仍然仅处于临床试验阶段,所以当务之急是研究和发现新的肝癌药物和方法,希望我们的研究能为肝癌预防和治疗提供新的思路。目前亲水性胆汁酸的抗癌作用日益得到人们的关注。熊去氧胆酸(ursodeoxycholic acid,UDCA)属于亲水性胆汁酸,很多证据表明它可以抑制体外培养的肝癌细胞株增殖,降低大鼠肝癌模型的肝癌发生率,它还可以降低丙型肝炎患者罹患肝癌的风险,并改善原发性胆汁性肝硬化病人的预后。鹅去氧胆酸(chenodeoxycholic acid,CDCA)是知的最丰富的初级胆汁酸,它可以减少膳食胆固醇吸收,并使胆固醇结石溶解,其衍生物HS-1200是亲水性胆汁酸的新成员。现已报道鹅去氧胆酸衍生物HS-1200 {N-[(3 α,5 β,7 α)-3,7-二羟基-24-氧代胆留烷-24-基)β-丙氨酸苄酯}在数种人类癌症中显示其抗癌活性,HS-1200可以在人前列腺癌细胞、骨肉瘤细胞、乳腺癌细胞中诱导癌细胞凋亡并抑制癌细胞增殖,而且可以诱导人肝癌细胞株BEL7402、HepG2凋亡。然而,HS-1200对生物体内肝癌是否有潜在的抗癌作用尚未阐明。为此,我们建立了两种动物肝癌模型,分别通过口服和腹腔注射两种给药途径来验证HS-1200对生物体内肝癌的作用。第一部分鹅去氧胆酸衍生物HS-1200抑制二乙基亚硝胺诱导的大鼠原发性肝癌的实验研究研究目的:虽然肝癌发展的分子机制仍然未全部为人所知,但DNA氧化损伤被认为是参与人类肝癌发生和进展的重要原因。氧化应激由于过度生产活性氧(reactive oxygen species,ROS)等自由基被认为引起遗传不稳定性,从而导致致癌作用。8-羟基脱氧鸟苷(8-OHdG)是一种被人们广泛接受的R0S触发氧化损伤DNA的损伤标记物,同时也被确定为慢性丙型肝炎病毒感染者进一步发展为肝细胞癌的危险因素。MTH1(mutT homologl)基因是肝细胞DNA修复基因,编码的MTH1蛋白通过抑制8-OHdG错并入DNA链而有利于氧化诱导的DNA损伤的修复。肿瘤组织中MTH1的上调表达可以消除肿瘤细胞中过量的8-OHdG,从而参与DNA损伤修复过程,使得肿瘤细胞继续分裂与增殖,维持肿瘤细胞的生存。现在已经有报道在一些人类癌症如肾癌、乳腺癌、结肠直肠癌中MTH1的表达增加。另外,也有报道证实与相邻的非癌组织相比,MTH1在肝癌组织中明显增加。然而,其在肝癌发生中的确切作用还不是很清楚。我们试图研究HS-1200抑制肝癌发生的潜在能力,并探讨MTH1参与肝癌发生的作用。鉴于此,我们建立了通过腹腔注射二乙基亚硝胺(diethylnitrosamine,DEN)诱导产生大鼠原发性肝癌的动物模型,我们以不同剂量的HS-1200干预此模型,来观察HS-1200对于模型大鼠肝功能和肝癌发生的作用,并研究大鼠肝组织MTH1mRNA的水平,从而探讨HS-1200的抗肿瘤活性。研究方法:实验共需145只雄性Wistar鼠,其中50只大鼠用于确定HS-1200潜在的肝毒性和肾毒性,20只用于通过腹腔内注射DEN进行DEN诱导的原发性肝癌的实验性造模。在进行了 HS-1200的安全性无毒评估和大鼠肝癌模型成功建模之后,其它75只大鼠进行如下实验:大鼠随机分为5组:对照组(N = 15),HS-1200组(N = 15),HCC(肝细胞癌)组(N = 15),HCC+低剂量 HS-1200 组(N = 15)和 HCC+高剂量HS-1200(N = 15)组。后三组按照实验造模条件腹腔内注射DEN建立大鼠肝癌模型,同时后四组通过每日口服灌胃法对大鼠进行不同剂量HS-1200的药物干预。20周后,处死大鼠,称大鼠体重,取其肝脏称重,计算肝重/体重比(肝系数)及结节计数,并进行病理学检验。大鼠心脏采血用ELISA法检测血清中AFP、ALT、AST的水平。取各组大鼠的肝组织,分别提取RNA,应用实时荧光定量RT-PCR法检测各组大鼠肝组织中MTH1mRNA的表达情况。结果:1.20周末时HCC组、HCC+低剂量HS-1200组和HCC+高剂量HS-1200组均成功建立大鼠原发性肝癌模型,各组的成瘤率分别为93%(14/15)、73%(11/15)、53%(8/15)。对照组和HS-1200组大鼠的体重、肝重及肝脏系数无明显差异,HCC组、HCC+低剂量HS-1200组和HCC+高剂量HS-1200组大鼠的体重均明显低于对照组(P0.05);但与HCC组相比,动物接受低剂量或高剂量HS-1200治疗后显示其体重增加,肝脏重量和肝系数降低了(P0.05);且HCC+高剂量HS-1200组大鼠的体重较HCC+低剂量HS-1200组明显升高,肝重和肝脏系数明显降低(P0.05)。2.对照组和HS-1200组的大鼠肝脏均正常未见结节。HE肝脏病理形态学观察显示对照组和HS-1200组肝小叶结构正常,无变性和坏死现象;HCC组正常肝小叶组织结构消失,假小叶形成,肝细胞索排列紊乱,可见癌巢,肿瘤细胞异型性明显,可见大片状坏死;在应用HS-1200干预之后,癌巢形成和出血坏死减少了,肝脏病理有了明显改善,HCC+低剂量HS-1200组正常肝小叶结构消失,可见假小叶结构,偶见癌巢及出血坏死;HCC+高剂量HS-1200组肝脏病理改善更显著,仅偶见假小叶,无明显癌巢,出血坏死少见。3.与对照组相比,HCC组大鼠血清中ALT、AST和AFP的含量明显升高(P0.05与对照组比较),这意味着肝功能的恶化;而HS-1200干预之后,各项指标的升高得到了不同程度的逆转,较HCC组均有所降低(P0.05与HCC组比较);且HS-1200高剂量组各项指标的降低更明显。4.以正常对照组大鼠肝脏MTH1mRNA的表达水平1为参照,HCC组的相对表达量为16.23±0.74(P0.05)。应用HS-1200治疗显著逆转MTH1 mRNA的上调表达,高剂量HS-1200似乎在这方面有更高的功效(HCC+低剂量HS-1200组,9.48±0.46;HCC+高剂量 HS-1200 组,6.13±0.33;P0.05 与 HCC 组比较)。结论:1.HS-1200无明显肝、肾毒性,保证了此药物的安全性。DEN腹腔注射可以成功建立大鼠原发性肝癌模型。2.HS-1200可降低DEN诱导的大鼠肝脏瘤变率,并降低大鼠血清中AFP含量,说明HS-1200对大鼠原发性肝癌的发生具有抑制作用。3.HS-1200可降低大鼠血清中AST、ALT的水平,可降低大鼠肝脏中MTH1mRNA的表达,说明HS-1200对大鼠原发性肝癌发生的抑制作用与其改善大鼠肝功能抑制炎症、抑制氧化应激有关。第二部分鹅去氧胆酸衍生物HS-1200抑制HepG2裸鼠肝癌移植瘤的实验研究研究目的:为了进一步研究HS-1200对肝癌肿瘤血管生成及转移性肝癌的作用,我们通过瘤体接种BALB/C裸鼠,复制了人肝癌细胞株HepG2裸鼠皮下移植瘤模型,观察HS-1200干预对移植瘤组织结构、超微结构及新生血管生成的影响。以发现HS-1200对于肝癌肿瘤血管生成因子VEGF、bFGF表达及血管生成的作用,从而为HS-1200应用于临床肝癌及转移的治疗进一步积累实验依据。研究方法:人肝癌细胞株HepG2调整细胞密度为1.5×107/ml,将细胞悬液接种于裸鼠腋窝皮下。皮下成瘤至长径约10mm时,剥离瘤体,取离体瘤组织以每个大小2×2×2mm3接种于60余只裸鼠左侧腋窝皮下。待接种瘤体长至长径约10mm,将荷瘤裸鼠随机分对照组(N=20)、20mg/kg低剂量HS-1200组(N=20)、60 mg/kg高剂量HS-1200组(N=20)。后两组分别腹腔注射相对应的剂量的HS-1200,每天一次,对照组裸鼠以相同方式给予等量灭菌注射用水处理,共2周。之后处死裸鼠并剥离移植瘤,测量并称重移植瘤,计算瘤体积,计算低、高剂量HS-1200治疗组瘤重的抑瘤率。光镜和电镜观察各组移植瘤组织结构和超微结构,免疫组织化学法检测各组移植瘤VEGF、bFGF表达。结果:1.低、高剂量HS-1200组移植瘤体积、重量均明显小于对照组,抑瘤率分别为38.23%、47.05%。2.光镜观察,对照组见血管增生活跃,瘤细胞内癌巢分布,细胞呈明显异型性。HS-1200干预组移植瘤见血管数量稀少或缺如,癌巢明显减少,HS-1200高剂量组更明显。3.透射电镜观察,对照组呈现细胞形态怪异,核大畸形等恶性肿瘤特有的超微结构特点;低、高剂量HS-1200组移植瘤组织见不同时期凋亡细胞,凋亡小体存在,坏死瘤细胞及细胞崩解碎片可见,且HS-1200高剂量组更明显。4.免疫组化结果表明,低、高剂量HS-1200组移植瘤中VEGF、bFGF阳性表达比对照组明显下调(p0.05),且HS-1200高剂量组更明显。结论:1.HS-1200可抑制人肝癌细胞株HepG2裸鼠皮下移植瘤生长。2.HS-1200干预使移植瘤血供明显减少,提示HS-1200可抑制移植瘤新生血管生成。3.对比移植瘤超微结构特点,证明HS-1200干预可诱导移植瘤细胞凋亡。4.根据免疫组化结果,推测HS-1200下调VEGF、bFGF表达,进而抑制移植瘤新生血管生成,表现抗肝癌及转移性肝癌的作用。
[Abstract]:Background and purpose: primary liver cancer is one of the most common malignant tumors and is the main cause of global cancer death. Its incidence is high and the prognosis is poor. Only in 2012, there are about 782500 new cases of primary liver cancer in the world, and about 745500 cases of hepatocellular carcinoma (hepatocellular carcinoma, HCC). The majority of patients with HCC. Due to the high metastasis rate and high recurrence rate of primary hepatocellular carcinoma (HCC), although considerable progress has been made in diagnosis and treatment in recent years, the mortality rate is still high. The number of drugs used to prevent liver cancer is limited and many drugs are still in clinical trial stage, so many drugs are still in the clinical trial stage, so The urgent task is to study and discover new liver cancer drugs and methods, and hope our research can provide new ideas for the prevention and treatment of liver cancer. The current anticancer effect of hydrophilic bile acids is getting more and more attention. Ursodeoxycholic acid (ursodeoxycholic acid, UDCA) belongs to hydrophilic bile acid, a lot of evidence suggests that it can inhibit in vitro Chenodeoxycholic acid (CDCA) is the most abundant primary bile acid known to reduce the risk of HCC in patients with hepatitis C and improve the prognosis of patients with primary biliary cirrhosis. It can reduce the dietary cholesterol absorption. HS-1200 is a new member of the hydrophilic bile acid. It has been reported that the HS-1200 {N-[(3, 5 beta, 7 alpha) -3,7- two hydroxy -24- oxycholestyl benzyl benzyl benzyl} of the oxydeoxycholic acid derivatives shows its anticancer activity in several human cancers, and HS-1200 can be used in human prostate cancer cells and bone meat. Tumor cells, breast cancer cells induce cancer cells to apoptosis and inhibit the proliferation of cancer cells, and can induce human hepatoma cell line BEL7402, HepG2 apoptosis. However, HS-1200 has no potential anti-cancer effect on liver cancer in vivo. Therefore, two animal models of liver cancer were established by oral and intraperitoneal injection of two kinds, respectively. Drug pathway to verify the effect of HS-1200 on liver cancer in vivo. Part 1 Experimental Study on the inhibition of two ethyl nitrosamine induced primary liver cancer by HS-1200 derivative of goose deoxycholic acid derivative: Although the molecular mechanism of the development of liver cancer is still unknown, DNA oxygenation injury is considered to be involved in human liver cancer and the occurrence of HCC. The important cause of progress. Oxidative stress, due to excessive production of active oxygen (reactive oxygen species, ROS) and other free radicals, is considered to cause genetic instability, resulting in the carcinogenic effect of.8- hydroxy deoxy guanosine (8-OHdG) as a widely accepted marker of R0S triggered oxidative damage DNA, and also identified as chronic C type C The.MTH1 (mutT homologl) gene, a risk factor for hepatitis virus infection, is the DNA repair gene of liver cells. The encoded MTH1 protein is beneficial to the repair of DNA damage induced by the incorporation of 8-OHdG into the DNA chain. The up regulation of MTH1 in the tumor tissue can eliminate the excess 8-OHdG in the tumor cells. It is now reported that the expression of MTH1 in some human cancers, such as kidney cancer, breast cancer, and colorectal cancer, has increased. In addition, it has also been reported that MTH1 is significantly increased in liver cancer tissue compared with adjacent non cancer tissue. However, MTH1 has been significantly increased. The exact role of HS-1200 in the development of liver cancer is not clear. We have tried to study the potential ability of HS-1200 to inhibit the occurrence of liver cancer and explore the role of MTH1 in the occurrence of liver cancer. In view of this, we established animal models of primary liver cancer induced by intraperitoneal injection of two ethyl nitrosamines (DEN). Different doses of HS-1200 were used to observe the effect of HS-1200 on liver function and liver cancer in model rats, and to study the level of MTH1mRNA in rat liver tissue, and to explore the anti tumor activity of HS-1200. Methods: 145 male Wistar rats were needed in the experiment, of which 50 rats were used to determine the potential hepatotoxicity and renal toxicity of HS-1200. 20 experimental models of primary liver cancer induced by intraperitoneal injection of DEN for DEN induced primary liver cancer. After the safety non-toxic evaluation of HS-1200 and the successful modeling of the rat model of liver cancer, the following 75 rats were divided into 5 groups randomly: the control group (N = 15), the HS-1200 group (N = 15), the HCC (N = 15), HC, HC C+ low dose HS-1200 group (N = 15) and HCC+ high dose HS-1200 (N = 15) group. The latter three groups were injected intraperitoneally with DEN to establish rat liver cancer model. At the same time, the four groups of rats were treated with different doses of HS-1200 by oral administration of the stomach daily for.20 weeks, and the rats were killed and weighed, and the weight of the rats was weighed, and the weight of the liver was calculated. The weight of the rats was calculated and the weight of the liver was calculated. Calculation of the weight of the liver was taken and calculated. The weight of the rat was calculated. The weight of the rat was weighed and the liver was weighed and calculated. Liver weight / weight ratio (liver coefficient) and nodule count and pathological examination. The level of AFP, ALT and AST in serum was detected by ELISA method in rat heart. RNA was extracted from liver tissues of rats in each group, and the expression of MTH1mRNA in liver tissues of each group was detected by real-time quantitative fluorescence quantitative RT-PCR. Results: HCC group at 1.20 weekend, HCC+ low The rat primary liver cancer model was successfully established in the dose HS-1200 group and the high dose HCC+ HS-1200 group. The tumor formation rate of each group was 93% (14/15), 73% (11/15), and 53% (8/15). There was no significant difference between the weight of the control group and the HS-1200 group, the liver weight and the liver coefficient, and the weight of the HCC group, the HCC+ low dose HS-1200 group and the HCC+ high dose HS-1200 group were all obvious. Lower than the control group (P0.05), but compared with the HCC group, the body weight and liver coefficient decreased (P0.05) after the animals received low or high dose HS-1200, and the weight of the HCC+ high dose HS-1200 group was significantly higher than that of the low HCC+ dose HS-1200 group, and the liver weight and liver coefficient decreased significantly (P0.05).2. control group and HS-1200 group. The liver pathomorphology of normal liver.HE showed normal liver lobular structure in control group and HS-1200 group, without degeneration and necrosis. In group HCC, normal hepatic lobule structure disappeared, false lobule formed, liver cell cord arranged disorder, cancer nests were visible, tumor cells had obvious heteromorphosis and visible necrosis; HS-12 00 after intervention, the formation of cancer nests and hemorrhagic necrosis were reduced, the pathological changes of liver were obviously improved. The structure of normal lobule in HCC+ low dose HS-1200 group disappeared, the structure of false lobule, tumor nest and hemorrhagic necrosis were seen, and the pathological improvement of liver in HCC+ high dose HS-1200 group was more significant, only false lobules, no obvious cancer nests, and hemorrhagic necrosis were rare.3. and pairs. The levels of ALT, AST and AFP in the serum of HCC rats increased significantly (compared with those of the control group), which meant the deterioration of liver function. After HS-1200 intervention, the increase of various indexes was reversed in varying degrees, compared with those in the HCC group (compared with the P0.05 and HCC group), and the decrease of each index in the HS-1200 high dose group was more obvious. The expression level of MTH1mRNA in the liver of normal control group was 1 as reference, and the relative expression of HCC group was 16.23 + 0.74 (P0.05). HS-1200 treatment significantly reversed the up expression of MTH1 mRNA. High dose HS-1200 seemed to have higher efficacy in this area (HCC+ low dose HS-1200 group, 9.48 + 0.46; HCC+ high dose HS-1200 group, 6.13 + 0.33; 6.13 + 0.33; Compared with group HCC) conclusion: 1.HS-1200 has no obvious liver, renal toxicity, and ensures the safety of this drug,.DEN intraperitoneal injection can successfully establish rat primary liver cancer model.2.HS-1200 can reduce the rate of liver cancer induced by DEN, and reduce the content of AFP in the rat serum, indicating that HS-1200 has an inhibitory effect on the occurrence of primary liver cancer in rats. .3.HS-1200 can reduce the level of AST and ALT in rat serum and reduce the expression of MTH1mRNA in the liver of rats, indicating that the inhibitory effect of HS-1200 on the occurrence of primary liver cancer in rats is related to the improvement of rat liver function inhibition inflammation and inhibition of oxidative stress. The second part of the goose deoxycholic acid derivative HS-1200 inhibits the transplanted tumor of the liver cancer of HepG2 nude mice. Objective: in order to further study the effect of HS-1200 on the angiogenesis and metastatic liver cancer of liver cancer, we inoculated BALB/C nude mice through the tumor and replicated the subcutaneous transplantation tumor model of human hepatoma cell line HepG2 in nude mice. The effects of HS-1200 intervention on the tissue structure, ultrastructure and angiogenesis of the transplanted tumor were observed. The effect of HS-1200 on the angiogenic factor VEGF, bFGF expression and angiogenesis of hepatocellular carcinoma, thus further accumulating the experimental basis for the treatment of HS-1200 in the treatment of liver cancer and metastasis. The study method: the cell density of human hepatocellular carcinoma cell line HepG2 is 1.5 * 107/ml, and the cell suspension is inoculated subcutaneously in the armpit of nude mice. When the diameter of the tumor was about 10mm, the tumor tissue was stripped and the tumor tissue was inoculated subcutaneously in the left armpit of more than 60 nude mice with each size of 2 x 2 x 2mm3. After the tumor was inoculated to a long diameter of about 10mm, the tumor bearing nude mice were randomly divided into the control group (N=20), the 20mg/kg low dose HS-1200 group (N=20) and the 60 mg/kg high dose HS-1200 group (N=20). The latter two groups were injected with the corresponding dose of the corresponding dose. HS-1200, once a day, the control group of nude mice was treated with the same amount of Sterile Water for Injection for 2 weeks. Then the nude mice were killed and the transplanted tumor was stripped and measured and weighed. The tumor volume was calculated. The tumor weight inhibition rate of the low, high dose HS-1200 treatment group was calculated. The tissue structure and ultrastructure of the transplanted tumor were observed by light and electron microscopy, and the ultrastructure and ultrastructure were observed by light microscope and electron microscope. VEGF and bFGF expression were detected by the immunohistochemical method. Results: 1. low, high dose HS-1200 group was significantly smaller than the control group, the tumor suppressor rate was 38.23%, the 47.05%.2. light microscope was observed respectively. The control group found the vascular proliferation active, the tumor cell nests distributed in the tumor cells, and the cells showed the number of blood vessels in the obvious heteromorphic.HS-1200 intervention group. HS-1200 high dose group was more obvious.3. transmission electron microscopy, and the control group showed peculiar ultrastructural characteristics of malignant tumor such as cell morphology and nuclear large malformation. Low, high dose HS-1200 group saw apoptotic cells in different period, apoptotic bodies, necrotic tumor cells and cell disintegration fragments. The results showed that the.4. immunohistochemical results of high dose HS-1200 group showed that the positive expression of VEGF in the transplanted tumor of HS-1200 group was lower than that of the control group (P0.05), and the HS-1200 high dose group was more obvious. Conclusion: 1.HS-1200 could inhibit the growth of the transplanted tumor of human liver cancer cell line, the growth.2.HS-1200 intervention to make the transplanted tumor blood supply. The results suggest that HS-1200 can inhibit the ultrastructural characteristics of the neovascularization of the transplanted tumor, and that the HS-1200 intervention can induce the apoptosis of the transplanted tumor cell.4.. According to the immunohistochemical results, it is suggested that HS-1200 can down regulate the VEGF, bFGF expression, and then inhibit the neovascularization of the transplanted tumor, and show the role of anti hepatoma and metastatic liver cancer.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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