HP1γ在膀胱尿路上皮癌中的表达情况及其对膀胱癌细胞生物学行为的影响

发布时间：2018-02-23 18:40

  本文关键词： 膀胱尿路上皮癌 HP1γ RNA干扰 糖酵解　出处：《南京大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的1、探讨HP1γ在膀胱尿路上皮癌中的表达情况,并研究HPly与其他临床、病理参数,以及预后的关系；2、通过体外实验研究HPly对膀胱癌细胞系T24及5637增殖能力、克隆形成能力及迁移能力的影响,并对可能的机制进行初步探讨。为膀胱癌防治提供新的靶点。方法1、应用蛋白质印迹法(Westernblot)检测30对膀胱尿路上皮癌组织及其癌旁正常的尿路上皮组织中HPly的表达情况；应用免疫组织化学染色技术(IHC)检测62例膀胱尿路上皮癌组织中HP1γ以及Ki67的表达情况,应用统计学方法分析其表达量与临床、病理参数之间的关系,以及对患者预后的影响。2、体外实验中,应用慢病毒介导的HPly特异性shRNA干扰膀胱癌细胞系T24、5637中HP1γ的表达,通过MTT实验研究HPly对膀胱癌细胞增值能力的影响；通过流式细胞术研究HPly对膀胱癌细胞的细胞周期的影响；通过克隆形成实验研究HPly对膀胱癌细胞癌细胞克隆形成能力的影响；通过划痕实验研究HPly对膀胱癌细胞迁移能力的影响。同时通过应用Real-Time PCR检测敲降HPly对糖酵解相关基因的表达情况的影响。结果1、Weaternblot组30对膀胱癌组织及其癌旁正常尿路上皮中,19对(63.3%)中膀胱癌组织中HPly的表达量高于癌旁正常尿路上皮组织中的表达量。IHC组62例膀胱癌组织中HPly的表达与肿瘤是否浸润肌层、是否区域淋巴结转移有关,与Ki67的表达量呈正相关。多因素生存分析结果显示,HPly的表达情况、是否淋巴结转移以及性别是膀胱癌独立的预后因子。2、体外实验证实,敲降HPly后,与对照组相比,MTT实验显示膀胱癌细胞增殖速度下降；克隆形成实验中,实验组形成的克隆数目少而且体积小；划痕实验中,实验组膀胱癌细胞迁移速度变慢,划痕融合率较对照组下降；流式细胞术检测结果显示G2/M期细胞分布比例升高,而S期细胞分布比例下降,G0/G1期细胞分布比例无明显变化,即细胞周期阻滞在G2/M期。进一步研究发现,实验组中膀胱癌细胞有氧糖酵解途径的关键基因Glut1、HK2、PGK、LDHA表达量较对照组下降。结论1、膀胱尿路上皮癌中HP1γ异常高表达,与肿瘤分期及Ki67的表达量相关,是独立的预后因子,提示HP1γ可能可能参与了膀胱癌的发生和发展,可能是一个新的肿瘤标记物,将有助于膀胱癌的预后评估。2、体外实验证实敲降HPly可以抑制膀胱癌细胞的增殖、迁移及克隆形成能力,有可能成为治疗膀胱癌的靶点。
[Abstract]:Objective 1 to investigate the expression of HP1 纬 in bladder urothelial carcinoma, and to study the relationship between HPly and other clinical, pathological parameters and prognosis. The proliferative ability of HPly on bladder cancer cell lines T24 and 5637 was studied in vitro. The effects of cloning ability and migration ability, To provide a new target for the prevention and treatment of bladder cancer. Methods 1. Western blot was used to detect the expression of HPly in 30 cases of bladder urothelial carcinoma and its adjacent normal urothelial tissue. The expression of HP1 纬 and Ki67 in 62 cases of bladder urothelial carcinoma was detected by immunohistochemical staining. The relationship between the expression of HP1 纬 and clinical and pathological parameters was analyzed by statistical method. In vitro, HPly specific shRNA mediated by lentivirus was used to interfere the expression of HP1 纬 in bladder cancer cell line T24n5637. The effect of HPly on the proliferation of bladder cancer cells was studied by MTT assay. The effect of HPly on the cell cycle of bladder cancer cells was studied by flow cytometry, and the effect of HPly on the clone forming ability of bladder cancer cells was studied by clone formation assay. The effect of HPly on the migration ability of bladder cancer cells was studied by scratch test, and the expression of glycolytic related genes was detected by Real-Time PCR. Results 1. The expression of HPly in bladder cancer tissues was higher than that in adjacent normal urothelial epithelium tissues. The expression of HPly in 62 cases of bladder cancer tissues and whether the tumor infiltrated the myometrium or not was higher than that in the adjacent normal urothelial epithelium tissues. Multivariate survival analysis showed that the expression of HPly, lymph node metastasis and sex were the independent prognostic factors of bladder cancer. Compared with the control group, MTT assay showed that the proliferation rate of bladder cancer cells decreased; in the clone formation test, the number of clones in the experimental group was small and the number of clones was small; in the scratch test, the migration rate of bladder cancer cells in the experimental group became slower. The rate of scratch fusion was lower than that of the control group, flow cytometry showed that the proportion of cell distribution in G _ 2 / M phase was increased, but the cell distribution ratio in S phase was not significantly changed in G _ 0 / G _ 1 phase. That is, cell cycle arrest occurred in G _ 2 / M phase. Further study showed that the expression of HP1 纬 in bladder cancer cells was significantly higher than that in control group, which was the key gene of aerobic glycolytic pathway in bladder cancer cells. Conclusion 1, the expression of HP1 纬 in bladder urothelial carcinoma was significantly higher than that in control group. HP1 纬 may be involved in the occurrence and development of bladder cancer and may be a new tumor marker. In vitro, knock down HPly can inhibit the proliferation, migration and clone formation of bladder cancer cells, and may become a target for the treatment of bladder cancer.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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