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

  更多相關(guān)文章： 去勢(shì)抵抗性前列腺癌 骨轉(zhuǎn)移 藥物重定位 聯(lián)系圖 胍那芐

【摘要】：研究背景和目的我國(guó)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(yù)后偏差。對(duì)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(shì)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(jìn)展。但絕大多數(shù)患者仍會(huì)出現(xiàn)去勢(shì)抵抗,并伴有轉(zhuǎn)移。因此,利用現(xiàn)有去勢(shì)抵抗性前列腺癌的分子生物學(xué)研究基礎(chǔ),繼續(xù)開發(fā)新型、有效的治療藥物,仍是該領(lǐng)域研究的的迫切任務(wù)。但是新藥研發(fā)依然面臨著研發(fā)周期長(zhǎng),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(zhǔn)醫(yī)療對(duì)個(gè)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(kù)CMap(Connectivity map,CMap)是目前較為完善基于藥物基因表達(dá)譜的藥物研究平臺(tái),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(yīng)用藥物重定位策略,基于基因表達(dá)譜,篩選可治療去勢(shì)抵抗性前列腺癌藥物,并研究其作用機(jī)制。實(shí)驗(yàn)方法從NCBI網(wǎng)站的GEO免費(fèi)共享數(shù)據(jù)庫(kù)下載涵蓋有激素敏感性前列腺癌原位病灶組織與去勢(shì)抵抗性前列腺癌骨轉(zhuǎn)移病灶組織基因表達(dá)譜數(shù)據(jù)的數(shù)據(jù)集GSE32269。利用R語(yǔ)言軟件,通過線性回歸和經(jīng)驗(yàn)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(zhuǎn)換為探針號(hào),導(dǎo)入CMap數(shù)據(jù)庫(kù)平臺(tái),進(jìn)行基因表達(dá)譜比對(duì)、評(píng)分。我們挑選前列腺癌細(xì)胞系PC3的比對(duì)結(jié)果,以p≤0.05,enrichment≤-0.8,mean≤-0.6為藥物篩選標(biāo)準(zhǔn),共篩選到9個(gè)候選藥物。結(jié)合CCK8細(xì)胞增殖實(shí)驗(yàn),以及藥物信息查詢(Drugbank,Super Target),確定候選藥物胍那芐。體外實(shí)驗(yàn),主要通過CCK8細(xì)胞增殖實(shí)驗(yàn),細(xì)胞克隆實(shí)驗(yàn),流式細(xì)胞儀檢測(cè)細(xì)胞凋亡,細(xì)胞劃痕實(shí)驗(yàn),transwell小室穿梭實(shí)驗(yàn)(有或無matrigel覆蓋孔膜),western blot實(shí)驗(yàn),RT-q PCR實(shí)驗(yàn),免疫組化,免疫熒光等明確胍那芐對(duì)去勢(shì)抵抗性前列腺癌有抑制細(xì)胞增殖、克隆形成,促進(jìn)細(xì)胞凋亡,抑制細(xì)胞遷徙、侵襲等惡性生物學(xué)表型。在體外實(shí)驗(yàn)中,我們構(gòu)建穩(wěn)轉(zhuǎn)熒光素酶和m Cherry熒光蛋白的PC3細(xì)胞,并主要通過構(gòu)建基于PC3細(xì)胞的皮下移植瘤、脛骨平臺(tái)注射小鼠模型,并用游標(biāo)卡尺監(jiān)測(cè)皮下移植瘤生長(zhǎng),應(yīng)用活體成像儀檢測(cè)肺、脛骨部位腫瘤負(fù)荷。實(shí)驗(yàn)結(jié)果(一)第一部分分析基因表達(dá)譜數(shù)據(jù)集GSE32269,發(fā)現(xiàn)兩類組織的基因表達(dá)顯著差異,在去勢(shì)抵抗性前列腺癌骨轉(zhuǎn)移病灶組織中,其中有93個(gè)基因顯著上調(diào)表達(dá),149個(gè)基因顯著下調(diào)表達(dá)。GO分析顯示,差異基因主要富集于細(xì)胞外區(qū)、結(jié)構(gòu)分子活性、細(xì)胞粘附等。KEGG通路分析差異基因主要富集于細(xì)胞外基質(zhì)互作,焦點(diǎn)粘附,谷胱甘肽代謝,氨基糖與核苷酸糖代謝。基因相應(yīng)探針號(hào)導(dǎo)入CMap數(shù)據(jù)庫(kù),進(jìn)行基因表達(dá)譜比對(duì)、評(píng)分。我們挑選前列腺癌細(xì)胞系PC3的比對(duì)結(jié)果,以p≤0.05,enrichment≤-0.8,mean≤-0.6為藥物篩選標(biāo)準(zhǔn),共篩選到9個(gè)候選藥物。(二)第二部分CCK8和克隆形成實(shí)驗(yàn)表明胍那芐顯著抑制去勢(shì)抵抗性前列腺癌細(xì)胞增殖、克隆形成,且為呈濃度依賴性抑制。Annexin V-FITC/PI標(biāo)記雙標(biāo)流式檢測(cè)表明胍那芐顯著促進(jìn)去勢(shì)抵抗性前列腺癌細(xì)胞凋亡和周期阻滯。細(xì)胞劃痕實(shí)驗(yàn)和transwell小室穿梭實(shí)驗(yàn)表明,胍那芐可以抑制前列腺癌細(xì)胞遷移、侵襲能力。RT-q PCR,Western blot和免疫熒光實(shí)驗(yàn)表明胍那芐還可明顯抑制前列腺癌細(xì)胞上皮間質(zhì)化改變�；赑C3細(xì)胞的皮下移植瘤、脛骨平臺(tái)注射骨轉(zhuǎn)移裸鼠模型實(shí)驗(yàn)表明,胍那芐可明顯抑制抑制瘤生長(zhǎng)、轉(zhuǎn)移灶腫瘤負(fù)荷增長(zhǎng)。(三)第三部分RT-q PCR和Western blot實(shí)驗(yàn)表明去勢(shì)抵抗性前列腺癌的GADD34的m RNA和蛋白水平表達(dá)均明顯高于雄激素依賴前列腺癌細(xì)胞以及正常前列腺上皮細(xì)胞。GADD34在去勢(shì)抵抗性前列腺癌細(xì)胞中的表達(dá)高于雄激素依賴前列腺癌及正常前列腺癌上皮細(xì)胞。且胍那芐可以抑制p-ei F2α的脫磷酸化。當(dāng)敲減GADD34時(shí),去勢(shì)抵抗性前列腺癌細(xì)胞的增殖能力受到抑制,但與此同時(shí),體外、體內(nèi)實(shí)驗(yàn)都發(fā)現(xiàn)敲減GADD34后,腫瘤細(xì)胞對(duì)對(duì)胍那芐的敏感性也明顯所降低。結(jié)論(一)去勢(shì)抵抗性前列腺癌較激素敏感性前列腺癌基因表達(dá)有較大變化,基于該差異基因表達(dá)譜進(jìn)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(xì)胞增殖、轉(zhuǎn)移,并促進(jìn)其凋亡。(三)胍那芐主要通過結(jié)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(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25

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