EpCAM在前列腺癌发生发展中作用机制的初步研究

发布时间：2018-03-20 15:17

  本文选题：上皮细胞粘附分子　切入点：前列腺癌　出处：《苏州大学》2016年博士论文　论文类型：学位论文

【摘要】：目的:前列腺癌是目前西方社会导致死亡的第二大因素。尽管在过去数十年中,前列腺癌患者的总生存率有显著提高,但是最近的数据表明转移性前列腺癌患者生存的改善并没有对死亡率的下降产生显著影响。上皮细胞粘附分子(EpCAM,Epithelial cell adhesion molecule)是一种上皮细胞跨膜糖蛋白,最初是以一种肿瘤相关抗原而被发现的,由于EpCAM在多种肿瘤组织中的表达升高而备受关注。有较多研究成果显示EpCAM在肿瘤的发生、发展中发挥重要作用,有望作为肿瘤诊断的分子标志物和可能的治疗靶点。因此本文选择EpCAM作为研究对象,分析其在前列腺癌特别是雄激素依赖性前列腺癌发生与发展中的作用。方法:选取我中心近五年内接受前列腺癌根治术的50例患者的手术标本,标本包括癌和癌旁组织,均经冰冻连续切片首尾病理证实为癌或正常组织后留取标本,其中术前血清PSA≤10ng/ml 23例、PSA10ng/ml 27例,T2期18例、T3期24例、T4期8例,淋巴结转移18例,无远处转移病例。通过Western-blot方法和免疫组化检测50对癌和癌旁组织中EpCAM蛋白表达的差异,并且定期随访患者术后PSA等预后情况。在体外细胞水平,运用Western-blot方法检测3种人前列腺癌细胞株(LNCap、DU-145、PC-3)中EpCAM蛋白表达的水平,并分析其差异;设计shRNA,以慢病毒为载体构建EpCAM沉默的LNCap稳转细胞株,并运用流式细胞技术检测细胞周期,运用细胞功能学实验检测细胞增殖、侵袭和转移能力。通过Western-blot方法检测EpCAM及雄激素受体(Androgen receptor,AR)、前列腺特异性抗原(prostate specific antigen,PSA)水平,细胞周期相关蛋白,以及上皮-间质转化(EMT)蛋白标志物的表达水平,深入探讨EpCAM对前列腺癌细胞生物学特性的影响。结果:EpCAM在前列腺癌组织及癌旁组织中均有表达,但EpCAM在前列腺癌组织中的表达明显高于癌旁前列腺组织,差异具有统计学意义(P0.05)。EpCAM蛋白的高表达与前列腺癌的手术前血清中总的PSA水平、临床病理分级、淋巴结转移以及生化复发情况等具有明显相关性,且EpCAM高表达的前列腺癌患者,其生化复发时间显著短于EpCAM低表达的前列腺癌患者。3种前列腺癌细胞系DU-145、PC-3、LNCaP中雄激素依赖性前列腺癌细胞LNCaP的EpCAM表达明显高于其它两种雄激素抵抗性前列腺癌细胞(P0.05)。运用shRNA稳定转染LNCaP细胞后,相较空白对照组和阴性对照组,实验组EpCAM明显下调,而且细胞周期明显阻滞在G1期,且LNCaP细胞的增殖及侵袭能力明显受到抑制。LNCaP中沉默EpCAM的表达后,AR和PSA的表达水平显著降低,细胞周期相关蛋白Cycling D和E表达水平明显降低,上皮细胞特异性蛋白E-cadherin的水平上升而Vimentin、N-cadherin水平下降。结论:高表达水平的EpCAM与前列腺癌疾病的演变,特别是雄激素依赖性前列腺癌的发生发展有关。其过表达可促进前列腺癌细胞周期进展,增强前列腺癌细胞的增殖及侵袭能力,干扰EpCAM可能是激素依赖性前列腺癌治疗的一个靶点。此外,EpCAM的过表达可促进AR和PSA表达,可能与雄激素非依赖性前列腺癌的发生相关,因此EpCAM具有重要的研究价值,为将来前列腺癌细胞的发生和发展提供更多的理论依据,及为研制该阶段有效的药物提供了可靠的靶点。
[Abstract]:Objective: prostate cancer is the second leading cause of death in western society. Although in the past few decades, was significantly higher in patients with prostate cancer survival rates, but recent data suggest that prostate cancer metastasis survival patients do not have a significant impact on mortality decreased. The epithelial cell adhesion molecule (EpCAM, Epithelial cell adhesion molecule) is a kind of epithelial cell membrane glycoprotein, originally as a tumor associated antigen was found, due to the increased expression of EpCAM in tumor tissues and concern. There are more research results show that EpCAM in the incidence of cancer, plays an important role in the development, is expected to serve as molecular tumor diagnosis signs and possible therapeutic target. So this paper chooses EpCAM as the research object, analysis of the prostate cancer is androgen dependent prostate cancer occurrence and development Show in vitro. Methods: the center for radical prostatectomy in 50 cases of surgical specimens in the past five years, including specimens of carcinoma and adjacent tissues, were confirmed by pathology and frozen serial sections for cancer or normal tissue specimens, including preoperative serum PSA 10ng/ ml in 23 cases. PSA10ng/ml 27 cases, T2 18 cases, T3 24 cases, T4 8 cases, 18 cases with lymph node metastasis, distant metastasis cases. By using the methods of Western-blot and immunohistochemistry to detect EpCAM protein expression in 50 carcinoma and paracancerous tissues, and regular follow-up of patients with postoperative prognosis in PSA. In vitro, using Western-blot method to detect 3 kinds of human prostate cancer cell lines (LNCap, DU-145, PC-3) level of expression of EpCAM protein, and to analyze the difference; design of shRNA, a lentivirus vector stably transfected cell line EpCAM silencing of LNCap, and using flow cytometry. Cell cycle, cell function by cell proliferation assay, invasion and metastasis. Detected by Western-blot EpCAM and androgen receptor (Androgen receptor, AR), prostate specific antigen (prostate specific, antigen, PSA) level, cell cycle related protein, and epithelial mesenchymal transition (EMT) protein expression level mark the in-depth study of effect of EpCAM on cell biological characteristics of prostate cancer. The results showed the expression of EpCAM in prostate cancer tissues and paracancerous tissues, but the expression of EpCAM in prostate cancer tissues was significantly higher than adjacent noncancerous prostate tissue, the difference was statistically significant (P0.05) and the high expression of.EpCAM protein in prostate cancer surgery the serum total PSA level, clinical pathological grading, lymph node metastasis and biochemical recurrence and had a significant correlation, and the high expression of EpCAM in prostate cancer patients, the biochemical The recurrence time was significantly shorter than that of.3 in patients with prostate cancer DU-145 and prostate cancer cell lines with low expression of EpCAM PC-3 and LNCaP in androgen independent prostate cancer cell LNCaP EpCAM expression was significantly higher than the other two androgen resistant prostate cancer cells (P0.05). Using shRNA stable transfected LNCaP cells compared with the blank control group and negative control group the experimental group, EpCAM was significantly decreased, and the cell cycle was arrested in G1 phase, and the proliferation and invasion of LNCaP cells was significantly inhibited to silence the expression of EpCAM in.LNCaP, the expression level of AR and PSA were significantly decreased and the expression of cell cycle related protein Cycling, D and E were significantly decreased, increased levels of epithelial cell specific protein E-cadherin and Vimentin, N-cadherin levels decreased. Conclusion: the evolution of a high level of EpCAM expression in prostate cancer and disease, especially in androgen dependent prostate cancer Related to the development. Its overexpression may promote the progression of prostate cancer cell cycle, enhance the ability of proliferation and invasion of prostate cancer cells, EpCAM interference may be a target for hormone dependent prostate cancer treatment. In addition, the overexpression of EpCAM may promote the expression of AR and PSA may be related to androgen independent prostate cancer therefore, EpCAM has important research value, provides theoretical basis for the occurrence and development of prostate cancer cells in the future, and for the development of the effective drug target provides reliable.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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