發(fā)布時間：2017-12-28 20:43

  本文關鍵詞：藥物重定位策略在篩選去勢抵抗性前列腺癌藥物中的應用與研究　出處：《第二軍醫(yī)大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 去勢抵抗性前列腺癌 骨轉移 藥物重定位 聯(lián)系圖 胍那芐

【摘要】：研究背景和目的我國前列腺癌的發(fā)病率快速上升,而且中晚期患者比例較高,預后偏差。對于晚期前列腺癌患者,目前治療主要依賴于內(nèi)分泌治療,包括藥物去勢治療、抗雄激素治療、新一代雄激素生物合成抑制劑治療,可有效控制患者病情進展。但絕大多數(shù)患者仍會出現(xiàn)去勢抵抗,并伴有轉移。因此,利用現(xiàn)有去勢抵抗性前列腺癌的分子生物學研究基礎,繼續(xù)開發(fā)新型、有效的治療藥物,仍是該領域研究的的迫切任務。但是新藥研發(fā)依然面臨著研發(fā)周期長,人力、資金成本高、成功概率低等困難。在新藥研發(fā)的多種不利因素的制約下,以及精準醫(yī)療對個性化用藥的迫切需求,藥物重定位(Drug Reposition)策略,也稱作為老藥新用,即整合挖掘現(xiàn)已有的藥物未知的新用途,因其具有較高的投入產(chǎn)出效率,目前已經(jīng)成為目前抗癌藥物研發(fā)中的重要策略之一。公共數(shù)據(jù)庫CMap(Connectivity map,CMap)是目前較為完善基于藥物基因表達譜的藥物研究平臺,是藥物重定位研究中重要的工具之一。本課題的研究目的主要就是應用藥物重定位策略,基于基因表達譜,篩選可治療去勢抵抗性前列腺癌藥物,并研究其作用機制。實驗方法從NCBI網(wǎng)站的GEO免費共享數(shù)據(jù)庫下載涵蓋有激素敏感性前列腺癌原位病灶組織與去勢抵抗性前列腺癌骨轉移病灶組織基因表達譜數(shù)據(jù)的數(shù)據(jù)集GSE32269。利用R語言軟件,通過線性回歸和經(jīng)驗貝葉斯方法(limma)分析兩類組織的差異基因。并行GO分析和KEGG通路分析。隨后將差異基因轉換為探針號,導入CMap數(shù)據(jù)庫平臺,進行基因表達譜比對、評分。我們挑選前列腺癌細胞系PC3的比對結果,以p≤0.05,enrichment≤-0.8,mean≤-0.6為藥物篩選標準,共篩選到9個候選藥物。結合CCK8細胞增殖實驗,以及藥物信息查詢(Drugbank,Super Target),確定候選藥物胍那芐。體外實驗,主要通過CCK8細胞增殖實驗,細胞克隆實驗,流式細胞儀檢測細胞凋亡,細胞劃痕實驗,transwell小室穿梭實驗(有或無matrigel覆蓋孔膜),western blot實驗,RT-q PCR實驗,免疫組化,免疫熒光等明確胍那芐對去勢抵抗性前列腺癌有抑制細胞增殖、克隆形成,促進細胞凋亡,抑制細胞遷徙、侵襲等惡性生物學表型。在體外實驗中,我們構建穩(wěn)轉熒光素酶和m Cherry熒光蛋白的PC3細胞,并主要通過構建基于PC3細胞的皮下移植瘤、脛骨平臺注射小鼠模型,并用游標卡尺監(jiān)測皮下移植瘤生長,應用活體成像儀檢測肺、脛骨部位腫瘤負荷。實驗結果(一)第一部分分析基因表達譜數(shù)據(jù)集GSE32269,發(fā)現(xiàn)兩類組織的基因表達顯著差異,在去勢抵抗性前列腺癌骨轉移病灶組織中,其中有93個基因顯著上調(diào)表達,149個基因顯著下調(diào)表達。GO分析顯示,差異基因主要富集于細胞外區(qū)、結構分子活性、細胞粘附等。KEGG通路分析差異基因主要富集于細胞外基質(zhì)互作,焦點粘附,谷胱甘肽代謝,氨基糖與核苷酸糖代謝。基因相應探針號導入CMap數(shù)據(jù)庫,進行基因表達譜比對、評分。我們挑選前列腺癌細胞系PC3的比對結果,以p≤0.05,enrichment≤-0.8,mean≤-0.6為藥物篩選標準,共篩選到9個候選藥物。(二)第二部分CCK8和克隆形成實驗表明胍那芐顯著抑制去勢抵抗性前列腺癌細胞增殖、克隆形成,且為呈濃度依賴性抑制。Annexin V-FITC/PI標記雙標流式檢測表明胍那芐顯著促進去勢抵抗性前列腺癌細胞凋亡和周期阻滯。細胞劃痕實驗和transwell小室穿梭實驗表明,胍那芐可以抑制前列腺癌細胞遷移、侵襲能力。RT-q PCR,Western blot和免疫熒光實驗表明胍那芐還可明顯抑制前列腺癌細胞上皮間質(zhì)化改變。基于PC3細胞的皮下移植瘤、脛骨平臺注射骨轉移裸鼠模型實驗表明,胍那芐可明顯抑制抑制瘤生長、轉移灶腫瘤負荷增長。(三)第三部分RT-q PCR和Western blot實驗表明去勢抵抗性前列腺癌的GADD34的m RNA和蛋白水平表達均明顯高于雄激素依賴前列腺癌細胞以及正常前列腺上皮細胞。GADD34在去勢抵抗性前列腺癌細胞中的表達高于雄激素依賴前列腺癌及正常前列腺癌上皮細胞。且胍那芐可以抑制p-ei F2α的脫磷酸化。當敲減GADD34時,去勢抵抗性前列腺癌細胞的增殖能力受到抑制,但與此同時,體外、體內(nèi)實驗都發(fā)現(xiàn)敲減GADD34后,腫瘤細胞對對胍那芐的敏感性也明顯所降低。結論(一)去勢抵抗性前列腺癌較激素敏感性前列腺癌基因表達有較大變化,基于該差異基因表達譜進行藥物重定位研究是可行、有效的。(二)胍那芐在體內(nèi)、體外顯著抑制前列腺癌細胞增殖、轉移,并促進其凋亡。(三)胍那芐主要通過結合GADD34(growth arrest and DNA damage inducible gene 34)來發(fā)揮抑癌作用。
[Abstract]:Background and objective the incidence of prostate cancer in China is rising rapidly, and the proportion of patients in the middle and late stages is high and the prognosis is deviant. For advanced prostate cancer, the current treatment is mainly dependent on endocrine therapy, including drug castration, androgen deprivation therapy, and new generation of androgen biosynthesis inhibitor therapy, which can effectively control the progress of patients. But most patients still have castrated resistance accompanied by metastasis. Therefore, using the existing molecular biology research basis of castration resistant prostate cancer, and developing new and effective therapeutic drugs is still an urgent task in this field. However, the new drug research and development still faces the difficulties of long research and development cycle, high cost of manpower, capital and low probability of success. In the control of various unfavorable factors of drug development, and the urgent need for accurate medical personalized medicine, drug repositioning (Drug Reposition) strategy, also known as the new use of old drugs, which is the integration of new uses of existing mining unknown drugs, because of its high input-output efficiency, has become one of the important in the current strategy of anticancer drug development. Public database CMap (Connectivity map, CMap) is a relatively perfect drug research platform based on drug gene expression profile. It is one of the important tools in drug reorientation research. The purpose of this research is mainly to apply drug repositioning strategy, based on gene expression profile, to screen castrated resistant prostate cancer drugs, and to study its mechanism. The experimental method was downloaded from the GEO free sharing database of NCBI website, and the data set of gene expression data covering hormone sensitive prostate cancer in situ lesion tissue and castration resistant prostate cancer metastasis tissue was GSE32269. The differential genes of two types of tissues were analyzed by linear regression and empirical Bayesian method (limma) by using R language software. Parallel GO analysis and KEGG path analysis. Then the differential gene was converted into a probe number, and the CMap database platform was introduced to compare and score the gene expression profiles. Results we selected prostate cancer cell line PC3, with P = 0.05, enrichment = -0.8, mean = -0.6 for drug screening standard, 9 drug candidates were screened. In combination with the CCK8 cell proliferation test and the drug information query (Drugbank, Super Target), the candidate drug guananbenzyl was determined. In vitro experiments, mainly through the experiment of CCK8 cell proliferation, clonogenic assays, cell apoptosis was detected by flow cytometry, cell scratch test and Transwell chamber shuttle test (with or without Matrigel coverage, Western Kong Mo) blot experiment, RT-q PCR experiment, immunohistochemistry, immunofluorescence clearly guanabenz inhibition of cell proliferation and clone the formation of castration resistant prostate cancer, promote cell apoptosis, inhibit cell migration and invasion of malignant phenotype. In vitro, we construct stable fluorescent protein Cherry luciferase and m PC3 cells, and mainly through the construction of subcutaneous transplantation tumor cells, PC3 tibial plateau injection mouse model based on monitoring and subcutaneous tumor growth using vernier caliper, imaging instrument to detect lung tumor load, tibia. Results: (1) the first part analyzed the gene expression data set GSE32269. It was found that there were significant differences in gene expression between two kinds of tissues. In 93 cases of castration resistant prostate cancer, 93 genes were up-regulated and 149 genes were down regulated. GO analysis showed that the differentially expressed genes were mainly enriched in the extracellular domain, the activity of structural molecules, and cell adhesion. The differential gene of KEGG pathway is mainly enriched in the interaction of extracellular matrix, focal adhesion, glutathione metabolism, amino sugar and nucleotides glucose metabolism. The corresponding probe number of the gene was introduced into the CMap database to compare and score the gene expression profiles. Results we selected prostate cancer cell line PC3, with P = 0.05, enrichment = -0.8, mean = -0.6 for drug screening standard, 9 drug candidates were screened. (two) in the second part, CCK8 and clonogenic assay showed that guanagin inhibited the proliferation and clone formation of castrated resistant prostate cancer cells in a concentration dependent manner. Annexin V-FITC/PI labeled double standard flow test showed that guananbenzyl significantly promoted the apoptosis and cycle arrest of castrated resistant prostate cancer cells. The cell scratch test and the Transwell cell shuttle test showed that guananbenzyl could inhibit the migration and invasion of prostate cancer cells. RT-q PCR, Western blot and immunofluorescence experiments showed that guananbenzyl could also significantly inhibit the epithelial changes in the epithelial cells of prostate cancer cells. Based on PC3 cell subcutaneous tumor and bone metastasis from tibial plateau, nagb inhibited the growth of tumor and the growth of metastatic tumor. (three) the third part of RT-q PCR and Western blot experiments showed that the expression of M RNA and protein of GADD34 in castrated resistant prostate cancer was significantly higher than that in androgen dependent prostate cancer cells and normal prostate epithelial cells. The expression of GADD34 in the castrated resistant prostate cancer cells is higher than that of androgen dependence on prostate cancer and normal prostate cancer epithelial cells. And guananbenzyl can inhibit dephosphorylation of p-ei F2 alpha. When knocking down GADD34, the proliferation ability of castrated resistant prostate cancer cells was inhibited, but at the same time, in vitro and in vivo experiments found that after knocking down GADD34, the sensitivity of tumor cells to guanidine was also significantly reduced. Conclusion (1) the gene expression of castrated resistant prostate cancer is larger than that of hormone sensitive prostate cancer. Based on the differential gene expression profile, drug relocation is feasible and effective. (two) guananbenzyl significantly inhibits the proliferation and metastasis of prostate cancer cells in vivo and in vitro, and promotes its apoptosis. (three) guananbenzyl is inhibited mainly by combining with GADD34 (growth arrest and DNA damage inducible gene 34).
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R737.25

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