Tipranavir抗肝癌机制初步研究

发布时间：2018-02-11 22:36

本文关键词： HCC 计算机辅助药物设计增殖抑制单克隆筛选 Western blot　出处：《重庆理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景肝癌是最常见且死亡率很高的恶性肿瘤之一,高居全球恶性肿瘤发病率第五位,中国癌症发病顺位第二位,严重威胁人类健康和生命。射频消融、化疗、手术切除、肝移植等是目前肝癌常用的治疗方法,然而,这些常用的治疗方法虽然可以治疗局部病灶,但患者术后易复发,并且对放疗、化疗易产生耐药性,预后很差。分子靶向抗肿瘤药物在提高疗效等方面具有一定的优越性,这种药物具有靶向分布特征,可通过减少给药剂量和次数来降低药物在全身各组织的分布,从而降低传统药物的全身性毒副作用并提高药物的治疗效率。所以,肝癌分子靶向药物的开发在抑制肝癌肿瘤增殖、延缓复发和转移等方面均有重要的意义。沉默信息调节因子1(silent information regulator1,SIRT1)是一类依赖于烟酰胺腺嘌呤二核苷酸(NAD+)的第Ⅲ类组蛋白去乙酰化酶。参与细胞能量代谢、应激耐受、分化、衰老和凋亡等多种生理活动。据文献报道,SIRT1在肝癌组织中的表达水平明显增高,本实验室通过虚拟筛选筛出了小分子化合物Tipranavir,通过通过体内外抗肝癌活性实验,发现Tipranavir对肝癌细胞有很强的增殖抑制作用。而据药典和文献报道,药物Tipranavir是CYP3A4肝药酶的抑制剂。是肝脏中最多的肝药酶。这些前期的研究结果为我们提供了思路,Tipranavir究竟是作用于SIRT1还是作用于CYP3A4,抑或是两者协同作用而导致对肝癌细胞增殖抑制,还是说可能还存在其他的作用靶点,是一个值得我们去深入探讨的问题。研究目的基于Sybyl2.1平台的Surflex-Dock模块和Discovery Studio3.0平台的Ligand Fit docking模块通过对接研究、比较Tipranavir分别和SIRT1晶体和CYP3A4的对接结果。构建肝癌细胞HepG2 SIRT1敲除细胞、肝癌细胞HepG2 CYP3A4敲除细胞,并进行单克隆筛选筛出单克隆敲除株。通过细胞实验验证Tipranavir对肝癌细胞敲除前后的抑制率变化,为研究Tipranavir抑制HCC的通路奠定基础。研究方法1.基于CADD平台将Tipranavir与SIRT1和CYP3A4蛋白晶体结构进行对接。2.构建HepG2细胞SIRT1和CYP3A4分别敲除细胞株及验证。3.筛选敲除细胞的单克隆细胞株及验证。4.CCK-8增殖抑制法检测Tipranavir对HepG2及其敲除细胞的增殖抑制率。5.以Tipranavir为配体基于id Target平台和Pharm Mapper平台通过反向分子对接查找肝癌相关的靶蛋白。研究成果本课题通过体外CCK-8增殖抑制法检测Tipranavir对HepG2及其敲除细胞的增殖抑制率,得出Tipranavir对SIRT1和CYP3A4敲除细胞均有一定的增殖抑制作用,但不明显。Tipranavir对正常肝细胞的增殖抑制作用远低于肝癌细胞。
[Abstract]:Background Hepatocellular carcinoma is one of the most common malignant tumors with high mortality rate, which ranks 5th in the world and ranks second in China, which is a serious threat to human health and life, radiofrequency ablation, chemotherapy and surgical resection. Liver transplantation is a common treatment for liver cancer at present. However, although these commonly used treatments can treat local lesions, patients are prone to relapse after operation, and are prone to develop drug resistance to radiotherapy and chemotherapy. The prognosis is very poor. Molecular targeted antitumor drugs have some advantages in improving the curative effect and so on. This kind of drug has the characteristics of targeted distribution, which can reduce the distribution of drugs in all tissues of the body by reducing the dosage and times of administration. Therefore, the development of molecular targeting drugs for liver cancer is to inhibit the proliferation of liver cancer tumors. Silent information regulator1SIRT1) is a kind of histone deacetylase dependent on nicotinamide adenine dinucleotide (NAD). It is involved in cell energy metabolism, stress tolerance and differentiation. It was reported that the expression level of SIRT1 in hepatocellular carcinoma was significantly increased. The small molecular compound Tipranavirus was screened by virtual screening in our laboratory, and the anti-hepatoma activity was tested in vivo and in vitro. It was found that Tipranavir had a strong inhibitory effect on the proliferation of hepatoma cells, and according to pharmacopoeia and literature reports, The drug Tipranavir is the inhibitor of liver drug enzyme of CYP3A4. It is the most common hepatic drug enzyme in the liver. These preliminary results provide us with some ideas as to whether Tipranavir acts on SIRT1, CYP3A4, or the synergistic effect of both on inhibiting the proliferation of hepatoma cells. Or that there may still exist other targets, which is a problem worthy of further discussion. The purpose of this study is to study the Surflex-Dock module based on Sybyl2.1 platform and the Ligand Fit docking module of Discovery Studio3.0 platform through docking. The docking results of Tipranavir with SIRT1 crystal and CYP3A4 were compared. HepG2 SIRT1 knockout cells and HepG2 CYP3A4 knockout cells were constructed. The inhibition rate of Tipranavir on hepatoma cells before and after knockout was verified by cell experiments. Methods 1. The crystal structure of Tipranavir with SIRT1 and CYP3A4 proteins was docked based on CADD platform. 2. HepG2 cell SIRT1 and CYP3A4 knockout cell lines were constructed and verified. 3. Single gram of knockout cells was screened. 2. The proliferation inhibition rate of Tipranavir on HepG2 and its knockout cells was determined by CCK-8 proliferation inhibition assay. 5. Tipranavir as ligand was used as ligand to find the target proteins related to HCC by reverse molecular docking based on id Target platform and Pharm Mapper platform. In this study, the inhibitory rate of Tipranavir on HepG2 and its knockout cells was detected by CCK-8 proliferation inhibition assay in vitro. The results showed that Tipranavir could inhibit the proliferation of both SIRT1 and CYP3A4 knockout cells, but the inhibitory effect of Tipranavir on normal hepatocytes was much lower than that on HCC cells.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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