【作者】 黄仁伦；

【导师】 向松涛；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2020， 硕士

【摘要】 目的:前列腺癌干细胞(Prostate cancer stem cells,PCSCs)在前列腺癌的进展和转移中起着关键作用,肿瘤相关巨噬细胞(Tumor-associated macrophages,TAMs)是肿瘤微环境中最丰富的免疫细胞群。系统研究PCSCs与TAMs之间的相互作用网络可能有助于寻找抑制PCSCs和前列腺癌转移的关键靶点,进一步探讨扶正抑瘤方通过干预TAMs功能抑制PCSCs自我更新和PCa转移的作用。方法:1.采用细胞活力测定实验、平板克隆实验测定CCL5或扶正抑瘤方对前列腺癌细胞增殖活性的影响。2.采用划痕愈合试验和transwell试验测量外源性措施干预前列腺癌细胞后的迁移和侵袭能力变化。3.采用蛋白免疫印迹实验检测蛋白的表达。4.采用成球实验、流式细胞术测定前列腺癌干细胞的自我更新活性。5.采用QPCR筛选CCL5的下游应答基因。6.通过质粒转染和siRNA干扰验证CCL5与下游通路的关系。7.临床组织样本和生物信息学分析CCL5和前列腺癌病理特征的相关性。8.异种移植瘤动物模型检测TAMs在体内对前列腺癌的干预作用;致瘤性实验检测TAMs在体内对前列腺癌干细胞成瘤性的干预作用。结果:1.在体外研究中,TAMs分泌的CCL5可以显著促进前列腺癌细胞的转移以及PCSCs亚群的形成。2.STAT3是CCL5干预前列腺癌细胞后最重要的应答基因。3.CCL5通过激活CCR5/β-catenin/STAT3通路促进前列腺癌转移和PCSCs亚群的形成。4.敲低CCL5表达能够减弱TAMs对前列腺癌生长、骨转移以及PCSCs活性的促进作用。5.CCL5表达与前列腺癌晚期临床病理特征及不良预后呈正相关。6.扶正抑瘤方抑制体外TAMs和前列腺癌细胞共培养体系中PCSCs亚群的形成。7.扶正抑瘤方通过阻滞TAMs/CCL5通路抑制前列腺癌在体内的生长和骨转移作用。结论:TAMs/CCL5可以通过激活β-catenin/STAT3信号通路促进PCSCs自我更新和前列腺癌转移。此外,扶正抑瘤方通过阻滞TAMs/CCL5通路抑制PCSCs自我更新和前列腺癌转移。本研究为开发TAMs/CCL5作为清除PCSCs和预防前列腺癌发生转移的潜在分子靶点提供了新的理论基础。更多还原

【Abstract】 ObjectiveProstate cancer stem cells(PCSCs)play a critical role in prostate cancer progression and metastasis,which remains an obstacle for successful prostate cancer treatment.Tumor-associated macrophages(TAMs)are the most abundant immune cell population within the tumor microenvironment.Systematic investigation of the interaction and network signaling between PCSCs and TAMs may help in searching for the critical target to suppress PCSCs and metastasis.And the anti-tumor effect of FuZheng YiLiu formula(FZYL)in PCa stem cells self-renewal and metastasis was researched.Methods1.The effects of CCL5 on the proliferation of prostate cancer cells were investigated using CCK-8 method or colony formation assay.2.Wound healing assay and transwell assay were conducted to measure the migration and invasion ability changes of prostate cancer cells after exogenous interventions as indicated,respectively.3.Western blotting was used to measure the expression of protein.4.The self-renewal activity of prostate cancer stem cells were investigated using mammosphere formation assay and flow cytometry.5.The downstream response genes of CCL5 were investigated using QPCR.6.The relationship between CCL5 and downstream pathways was verified using plasmid transfection and siRNA interference.7.The correlation between CCL5 and pathological features of prostate cancer were investigated using tissue microarray analysis and bioinformatic analysis.8.The effect of TAMs on prostate cancer in vivo were investigated using Xenograft tumor animal model.Results1.TAMs-secreted CCL5 promotes the invasion and the PCSCs subpopulation of prostate cancer cells in vitro.2.STAT3 is the primary gene responsive for CCL5 stimulation in prostate cancer.3.CCL5 promotes prostate cancer invasion and PCSCs selfrenewal via activating the.CCR5/β-catenin/STAT3 pathway.4.CCL5 knockdown in TAMs suppresses prostate cancer growth,bone metastasis and PCSCs activity in vivo.5.CCL5 expression is positively correlated with advanced clinicopathological characteristics and poor prognosis of prostate cancer.6.The FZYL formula can inhibit the PCSCs subpopulation of prostate cancer cells in the co-culture system of TAMs and prostate cancer cells in vitro.7.The FZYL formula can inhibit the growth and bone metastasis of prostate cancer cells by blocking the effect of TAMs/CCL5 pathway in vivo.ConclusionTAMs/CCL5 could promote PCSCs self-renewal and prostate cancer metastasis via activating β-catenin/STAT3 signaling.Interestingly,it could be blocked by using FZYL.This study provides a novel rationale for developing TAMs/CCL5 as a potential molecular target for PCSCs elimination and metastatic prostate cancer prevention.更多还原