药物重定位策略在筛选去势抵抗性前列腺癌药物中的应用与研究

发布时间：2017-12-28 20:43

本文关键词：药物重定位策略在筛选去势抵抗性前列腺癌药物中的应用与研究　出处：《第二军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景和目的我国前列腺癌的发病率快速上升,而且中晚期患者比例较高,预后偏差。对于晚期前列腺癌患者,目前治疗主要依赖于内分泌治疗,包括药物去势治疗、抗雄激素治疗、新一代雄激素生物合成抑制剂治疗,可有效控制患者病情进展。但绝大多数患者仍会出现去势抵抗,并伴有转移。因此,利用现有去势抵抗性前列腺癌的分子生物学研究基础,继续开发新型、有效的治疗药物,仍是该领域研究的的迫切任务。但是新药研发依然面临着研发周期长,人力、资金成本高、成功概率低等困难。在新药研发的多种不利因素的制约下,以及精准医疗对个性化用药的迫切需求,药物重定位(Drug Reposition)策略,也称作为老药新用,即整合挖掘现已有的药物未知的新用途,因其具有较高的投入产出效率,目前已经成为目前抗癌药物研发中的重要策略之一。公共数据库CMap(Connectivity map,CMap)是目前较为完善基于药物基因表达谱的药物研究平台,是药物重定位研究中重要的工具之一。本课题的研究目的主要就是应用药物重定位策略,基于基因表达谱,筛选可治疗去势抵抗性前列腺癌药物,并研究其作用机制。实验方法从NCBI网站的GEO免费共享数据库下载涵盖有激素敏感性前列腺癌原位病灶组织与去势抵抗性前列腺癌骨转移病灶组织基因表达谱数据的数据集GSE32269。利用R语言软件,通过线性回归和经验贝叶斯方法(limma)分析两类组织的差异基因。并行GO分析和KEGG通路分析。随后将差异基因转换为探针号,导入CMap数据库平台,进行基因表达谱比对、评分。我们挑选前列腺癌细胞系PC3的比对结果,以p≤0.05,enrichment≤-0.8,mean≤-0.6为药物筛选标准,共筛选到9个候选药物。结合CCK8细胞增殖实验,以及药物信息查询(Drugbank,Super Target),确定候选药物胍那苄。体外实验,主要通过CCK8细胞增殖实验,细胞克隆实验,流式细胞仪检测细胞凋亡,细胞划痕实验,transwell小室穿梭实验(有或无matrigel覆盖孔膜),western blot实验,RT-q PCR实验,免疫组化,免疫荧光等明确胍那苄对去势抵抗性前列腺癌有抑制细胞增殖、克隆形成,促进细胞凋亡,抑制细胞迁徙、侵袭等恶性生物学表型。在体外实验中,我们构建稳转荧光素酶和m Cherry荧光蛋白的PC3细胞,并主要通过构建基于PC3细胞的皮下移植瘤、胫骨平台注射小鼠模型,并用游标卡尺监测皮下移植瘤生长,应用活体成像仪检测肺、胫骨部位肿瘤负荷。实验结果(一)第一部分分析基因表达谱数据集GSE32269,发现两类组织的基因表达显著差异,在去势抵抗性前列腺癌骨转移病灶组织中,其中有93个基因显著上调表达,149个基因显著下调表达。GO分析显示,差异基因主要富集于细胞外区、结构分子活性、细胞粘附等。KEGG通路分析差异基因主要富集于细胞外基质互作,焦点粘附,谷胱甘肽代谢,氨基糖与核苷酸糖代谢。基因相应探针号导入CMap数据库,进行基因表达谱比对、评分。我们挑选前列腺癌细胞系PC3的比对结果,以p≤0.05,enrichment≤-0.8,mean≤-0.6为药物筛选标准,共筛选到9个候选药物。(二)第二部分CCK8和克隆形成实验表明胍那苄显著抑制去势抵抗性前列腺癌细胞增殖、克隆形成,且为呈浓度依赖性抑制。Annexin V-FITC/PI标记双标流式检测表明胍那苄显著促进去势抵抗性前列腺癌细胞凋亡和周期阻滞。细胞划痕实验和transwell小室穿梭实验表明,胍那苄可以抑制前列腺癌细胞迁移、侵袭能力。RT-q PCR,Western blot和免疫荧光实验表明胍那苄还可明显抑制前列腺癌细胞上皮间质化改变。基于PC3细胞的皮下移植瘤、胫骨平台注射骨转移裸鼠模型实验表明,胍那苄可明显抑制抑制瘤生长、转移灶肿瘤负荷增长。(三)第三部分RT-q PCR和Western blot实验表明去势抵抗性前列腺癌的GADD34的m RNA和蛋白水平表达均明显高于雄激素依赖前列腺癌细胞以及正常前列腺上皮细胞。GADD34在去势抵抗性前列腺癌细胞中的表达高于雄激素依赖前列腺癌及正常前列腺癌上皮细胞。且胍那苄可以抑制p-ei F2α的脱磷酸化。当敲减GADD34时,去势抵抗性前列腺癌细胞的增殖能力受到抑制,但与此同时,体外、体内实验都发现敲减GADD34后,肿瘤细胞对对胍那苄的敏感性也明显所降低。结论(一)去势抵抗性前列腺癌较激素敏感性前列腺癌基因表达有较大变化,基于该差异基因表达谱进行药物重定位研究是可行、有效的。(二)胍那苄在体内、体外显著抑制前列腺癌细胞增殖、转移,并促进其凋亡。(三)胍那苄主要通过结合GADD34(growth arrest and DNA damage inducible gene 34)来发挥抑癌作用。
[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).
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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