本文關(guān)鍵詞： HCC 計(jì)算機(jī)輔助藥物設(shè)計(jì) 增殖抑制 單克隆篩選 Western blot　出處：《重慶理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景肝癌是最常見(jiàn)且死亡率很高的惡性腫瘤之一,高居全球惡性腫瘤發(fā)病率第五位,中國(guó)癌癥發(fā)病順位第二位,嚴(yán)重威脅人類健康和生命。射頻消融、化療、手術(shù)切除、肝移植等是目前肝癌常用的治療方法,然而,這些常用的治療方法雖然可以治療局部病灶,但患者術(shù)后易復(fù)發(fā),并且對(duì)放療、化療易產(chǎn)生耐藥性,預(yù)后很差。分子靶向抗腫瘤藥物在提高療效等方面具有一定的優(yōu)越性,這種藥物具有靶向分布特征,可通過(guò)減少給藥劑量和次數(shù)來(lái)降低藥物在全身各組織的分布,從而降低傳統(tǒng)藥物的全身性毒副作用并提高藥物的治療效率。所以,肝癌分子靶向藥物的開(kāi)發(fā)在抑制肝癌腫瘤增殖、延緩復(fù)發(fā)和轉(zhuǎn)移等方面均有重要的意義。沉默信息調(diào)節(jié)因子1(silent information regulator1,SIRT1)是一類依賴于煙酰胺腺嘌呤二核苷酸(NAD+)的第Ⅲ類組蛋白去乙酰化酶。參與細(xì)胞能量代謝、應(yīng)激耐受、分化、衰老和凋亡等多種生理活動(dòng)。據(jù)文獻(xiàn)報(bào)道,SIRT1在肝癌組織中的表達(dá)水平明顯增高,本實(shí)驗(yàn)室通過(guò)虛擬篩選篩出了小分子化合物Tipranavir,通過(guò)通過(guò)體內(nèi)外抗肝癌活性實(shí)驗(yàn),發(fā)現(xiàn)Tipranavir對(duì)肝癌細(xì)胞有很強(qiáng)的增殖抑制作用。而據(jù)藥典和文獻(xiàn)報(bào)道,藥物Tipranavir是CYP3A4肝藥酶的抑制劑。是肝臟中最多的肝藥酶。這些前期的研究結(jié)果為我們提供了思路,Tipranavir究竟是作用于SIRT1還是作用于CYP3A4,抑或是兩者協(xié)同作用而導(dǎo)致對(duì)肝癌細(xì)胞增殖抑制,還是說(shuō)可能還存在其他的作用靶點(diǎn),是一個(gè)值得我們?nèi)ド钊胩接懙膯?wèn)題。研究目的基于Sybyl2.1平臺(tái)的Surflex-Dock模塊和Discovery Studio3.0平臺(tái)的Ligand Fit docking模塊通過(guò)對(duì)接研究、比較Tipranavir分別和SIRT1晶體和CYP3A4的對(duì)接結(jié)果。構(gòu)建肝癌細(xì)胞HepG2 SIRT1敲除細(xì)胞、肝癌細(xì)胞HepG2 CYP3A4敲除細(xì)胞,并進(jìn)行單克隆篩選篩出單克隆敲除株。通過(guò)細(xì)胞實(shí)驗(yàn)驗(yàn)證Tipranavir對(duì)肝癌細(xì)胞敲除前后的抑制率變化,為研究Tipranavir抑制HCC的通路奠定基礎(chǔ)。研究方法1.基于CADD平臺(tái)將Tipranavir與SIRT1和CYP3A4蛋白晶體結(jié)構(gòu)進(jìn)行對(duì)接。2.構(gòu)建HepG2細(xì)胞SIRT1和CYP3A4分別敲除細(xì)胞株及驗(yàn)證。3.篩選敲除細(xì)胞的單克隆細(xì)胞株及驗(yàn)證。4.CCK-8增殖抑制法檢測(cè)Tipranavir對(duì)HepG2及其敲除細(xì)胞的增殖抑制率。5.以Tipranavir為配體基于id Target平臺(tái)和Pharm Mapper平臺(tái)通過(guò)反向分子對(duì)接查找肝癌相關(guān)的靶蛋白。研究成果本課題通過(guò)體外CCK-8增殖抑制法檢測(cè)Tipranavir對(duì)HepG2及其敲除細(xì)胞的增殖抑制率,得出Tipranavir對(duì)SIRT1和CYP3A4敲除細(xì)胞均有一定的增殖抑制作用,但不明顯。Tipranavir對(duì)正常肝細(xì)胞的增殖抑制作用遠(yuǎn)低于肝癌細(xì)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.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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