前列腺癌中封闭STAT3信号通路靶向肿瘤起源细胞和已分化细胞的研究

发布时间：2018-03-09 18:52

本文选题：STAT3　切入点：TICs　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：研究目的：尽管目前有许多前列腺癌相关的研究,但和前列腺癌进展相关的基因改变和生物学通路改变仍不明了。我们研究的主要目的是探索STAT3信号通路对前列腺癌肿瘤细胞以及肿瘤起源细胞的影响。研究方法：收集2012年5月至2014年1月间进行手术治疗的14例前列腺癌肿瘤组织及配对癌旁组织。应用Western blotting和酶联免疫吸附测定(ELISA)检测基因的蛋白表达水平；用STAT3小分子抑制剂Stattic作用于PC3M-1E8细胞,应用流式细胞仪检测细胞凋亡及细胞周期；应用细胞免疫荧光技术和免疫荧光激光扫描共聚焦显微镜检测pSTAT3在细胞核内的分布;软琼脂糖凝胶克隆形成实验检测肿瘤细胞体外增殖;ALDEFLUOR assay检测列腺癌细胞系PC3M-1E8及临床样本中ALDH酶活性；应用流式细胞仪分选ALDH高表达(ALDHhigh)细胞亚群；流式细胞仪检测细胞凋亡。构建沉默STAT3表达的shRNA慢病毒载体并转染PC3M-1E8细胞。裸鼠皮下成瘤检测细胞成瘤能力：用细胞异种移植模型(cell-line xenograft)和人源性肿瘤异种移植模型(patient-derived tumor xenograft,PDTX)检测肿瘤增殖能力。研究结果：低剂量Stattic能诱导PC3M-1E8细胞出现细胞周期的S期阻滞;相对高剂量Stattic能造成细胞大量凋亡。前列腺癌肿瘤组织及细胞系PC3M-1E8中均存在ALDHhigh细胞亚群,ALDHhigh细胞群中pSTAT3表达量高于non-ALDHhigh细胞群。Stattic能封闭ALDHhigh细胞亚群中STAT3活化并杀伤ALDHhigh细胞亚群。IL-6能介导前列腺癌细胞系和前列腺癌原代细胞中的non-TICs向TICs转化。封闭STAT3活化能抑制IL-6介导的TICs转化现象。敲减STAT3表达或者封闭STAT3活化抑制前列腺癌的肿瘤生长。在前列腺癌细胞异种移植模型和患者来源的肿瘤异种移植模型中,封闭STAT3信号通路活化能同时显著杀伤前列腺癌肿瘤起源细胞和体肿瘤细胞。研究结论：封闭STAT3信号活化能同时靶向并杀伤前列腺癌中已分化的肿瘤细胞和肿瘤起源细胞。因此,封闭STAT3信号活化是潜在的前列腺癌治疗靶点。
[Abstract]:Objective: although there are a lot of prostate cancer related studies, However, the genetic changes and biological pathway changes associated with the progression of prostate cancer are still unclear. The main purpose of our study is to explore the effects of STAT3 signaling pathway on prostate cancer tumor cells and tumor-derived cells. From May 2012 to January 2014, 14 cases of prostate cancer tumor tissues and matched adjacent tissues were collected. Western blotting and enzyme linked immunosorbent assay (Elisa) were used to detect the protein expression level of prostatic carcinoma. The apoptosis and cell cycle of PC3M-1E8 cells were detected by flow cytometry with Stattic, a small molecule inhibitor of STAT3, and the distribution of pSTAT3 in the nucleus was detected by cell immunofluorescence and immunofluorescence laser scanning confocal microscopy. Soft agarose gel clone forming assay was used to detect the proliferation of tumor cells in vitro and the activity of ALDH enzyme in PC3M-1E8 and clinical samples, and the subsets of high expression ALDH cells were separated by flow cytometry. Flow cytometry was used to detect apoptosis. ShRNA lentivirus vector which silenced STAT3 expression was constructed and transfected into PC3M-1E8 cells. In nude mice, the ability of subcutaneous tumorigenesis was detected by cell line xenograftg) and human tumor xenograftmodel. Results: low dose Stattic could induce S-phase arrest of PC3M-1E8 cell cycle. The relative high dose of Stattic can induce a large number of apoptosis. The expression of pSTAT3 in ALDHhigh cell subgroup is higher than that in non-ALDHhigh cell group. Stattic can block STAT3 activation in ALDHhigh cell subgroup and kill ALDHhigh in prostate cancer tumor tissue and cell line PC3M-1E8. Cell subsets. IL-6 can mediate the transformation of non-TICs from prostate cancer cell line and primary prostate cancer cell line to TICs. Blocking STAT3 activation can inhibit TICs transformation induced by IL-6. Knock down STAT3 expression or block STAT3 activation to inhibit prostate cancer swelling. Tumor growth. In xenotransplantation models of prostate cancer cells and tumor-derived xenografts from patients, Blocking the activation energy of STAT3 signaling pathway can significantly kill both tumor and body tumor cells in prostate cancer. Conclusion: blocking STAT3 signal activation energy simultaneously targets and kills differentiated tumor cells and tumor derived cells in prostate cancer. Blocking STAT3 signal activation is a potential target for prostate cancer therapy.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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