PRDX2对结肠癌干细胞特性的调控作用及机制研究

发布时间：2018-01-01 18:05

本文关键词：PRDX2对结肠癌干细胞特性的调控作用及机制研究　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：结肠癌是全球范围内的高发肿瘤之一。在我国结肠癌的发病率逐年增高,威胁到更多人的生命健康。根据中国肿瘤登记中心的统计数据,在2015年结直肠癌新发病例估算为37.63万人,占同年总新发肿瘤病例的8.7%,排第五位,结直肠癌死亡病例估算为19.10万人,占同年总死亡肿瘤病例的6.8%,排第五位。目前结直肠癌的治疗仍以传统手术、辅助化疗为主,但是中晚期结直肠癌疗效并不理想,为了提升结直肠癌的治疗效果,有必要深入理解结直肠癌发生发展和转移复发的机制。对于结直肠癌的标准化治疗所获得的疗效有限,目前研究认为可能归因于结肠癌中存在少量高耐药低增殖的肿瘤干细胞。肿瘤干细胞具有一系列独有的生物学特性,包括自我更新、多向分化潜能、无限增殖、转移和耐药,被认为是肿瘤发生发展、侵袭转移以及复发的“种子细胞”。肿瘤微环境是肿瘤细胞赖以生存的环境,同样地,肿瘤干细胞也有相应的微环境,称为肿瘤干细胞龛,在维持肿瘤干细胞特性方面发挥重要作用。肿瘤干细胞龛中氧化还原平衡的改变有可能对干细胞特性产生影响。过氧化物还原酶(PRDXs)是细胞中的一个抗氧化酶超家族。在这个家族中PRDX2是一个典型的2-Cys过氧化物还原酶,研究发现PRDX2在结肠癌组织中表达量高于癌旁组织,而且prdx2敲减能够抑制结肠癌细胞生长,促进凋亡。然而prdx2作为氧化还原平衡的重要调节因子,与结肠癌干细胞特性之间的关系还没有研究清楚。目的:探讨干扰prdx2基因表达对结肠癌干细胞特性的影响及其分子机制。方法:1)免疫组化检测10例人结肠癌组织样本中的prdx2和cd133的蛋白表达,用imageproplus软件进行光密度分析获得蛋白半定量数据,对两组数据进行pearson相关性分析;用免疫磁珠法分选结肠癌细胞中的cd133-和cd133+细胞,用流式细胞仪检测cd133+细胞比例以鉴定分选效率;分别从三株结肠癌细胞中分选出cd133-和cd133+细胞,用westernblot检测cd133-和cd133+细胞中prdx2、cd44和cd133的蛋白表达量;在三种结肠癌细胞系中,用免疫荧光法检测结肠癌细胞和结肠癌干细胞微球体中prdx2和cd133的蛋白表达情况。2)建立prdx2干扰质粒载体并用慢病毒包装,通过westernblot外源筛选有效载体,qrt-pcr内源验证敲减效率;通过慢病毒转染在三株结肠癌细胞系中分别建立空载慢病毒组(cont组)和prdx2敲减组(shprdx2组),用荧光显微镜观察慢病毒转染率,用westernblot和qrt-pcr检测两组细胞中prdx2在mrna和蛋白水平的差异;用流式细胞仪检测两组细胞中cd133+细胞的比例,用westernblot和qrt-pcr检测两组细胞中cd44、cd133、lgr5、cxcr4、epcam和nanog的蛋白和mrna的表达量,用成球试验观察两组细胞中干细胞微球体形成数量的差异,用药物毒性实验检测两组细胞对5-fu敏感性的差异,用成瘤实验检测两组中的cd133+细胞在裸鼠皮下形成移植瘤的体积差异。3)建立prdx2过表达质粒载体并用慢病毒包装,用westernblot鉴定其有效性;通过慢病毒转染在三株结肠癌细胞系中分别建立空载慢病毒组(cont组)和prdx2过表达组(prdx2组),用荧光显微镜观察慢病毒转染率,用westernblot检测两组细胞中prdx2蛋白表达量的差异;用流式细胞仪检测两组细胞中cd133+细胞的比例,用westernblot检测两组细胞中cd44、cd133和nanog的蛋白表达量差异,用成球试验观察两组细胞中干细胞微球体形成数量的差异。4)选择ht29细胞,在空载慢病毒组(cont组)、prdx2敲减组(shprdx2组)和prdx2过表达组(prdx2组)中分别用免疫磁珠法分选出cd133+细胞,对于三个组别的cd133+细胞,用westernblot检测hedgehog/gli1通路关键蛋白smo和gli1的蛋白表达量;用dmso或者smo抑制剂cyclopamine对ht29细胞进行干预并分为dmso组和cyclopamine组,用westernblot检测干性标志物cd44和cd133的蛋白表达量。结果:1)在10例人结肠癌组织样本中,prdx2和cd133的蛋白表达量之间存在线性正相关性,pearson相关系数为0.7863,p=0.007。免疫磁珠法分选出的cd133+细胞中cd133+细胞的比例为93.10%,cd133-细胞中cd133+细胞的比例为1.06%。在sw620、ht29、hct116三种结肠癌细胞系中,cd133+细胞中prdx2的蛋白表达量均显著高于cd133-细胞,p0.05。prdx2蛋白表达主要定位在细胞质中,cd133蛋白表达主要定位在细胞膜。2)在空载慢病毒组(cont组)和prdx2敲减组(shprdx2组)中荧光转染率均大于95%,shprdx2组中prdx2的mrna和蛋白表达量均显著低于cont组;与cont组相比,shprdx2组中cd133+细胞群体比例显著降低,干细胞表面标志物cd44、cd133、lgr5、epcam和干细胞相关转录因子nanog的mrna和蛋白表达量显著减少,微球体形成量显著减少,cd133+细胞对5-fu的敏感性显著增加,cd133+细胞形成裸鼠皮下移植瘤的体积显著变小,p0.05。3)在空载慢病毒组(cont组)和prdx2过表达组(prdx2组)中荧光转染率均大于95%,prdx2组中prdx2的mrna和蛋白表达量均显著高于cont组;与cont组相比,prdx2组中cd133+细胞群体比例显著增高,干细胞表面标志物cd44、cd133和干细胞相关转录因子nanog的蛋白表达量显著增加,微球体形成量显著增加,p0.05。4)在ht29细胞分选出的cd133+细胞中,与cont组相比,shprdx2组的smo和gli1蛋白表达量均显著降低,prdx2组的smo和gli1蛋白表达量均显著增加,p0.05;在ht29细胞系中,与dmso组相比,cyclopamine组的smo和gli1蛋白表达量均显著降低,同时干细胞表面标志物cd44和cd133蛋白表达量均显著降低,p0.05。结论:prdx2可能通过hedgehog/gli1信号通路维持结肠癌干细胞特性。
[Abstract]:Colon cancer is one of the worldwide high incidence of tumors. In the pathogenesis of colorectal cancer in China increased year by year, more threat to people's lives and health. According to the statistical data of Chinese cancer registry in 2015, new cases of colorectal cancer is estimated to be 376 thousand and 300 people, accounting for the same year the total new tumors were 8.7%, fifth a colorectal cancer deaths estimated 191 thousand people, accounting for the same year the total death of the tumor was 6.8%, ranked fifth. The current treatment of colorectal cancer with traditional surgery, adjuvant chemotherapy in advanced colorectal cancer, but the effect is not ideal, to enhance the treatment effect of colorectal cancer, it is necessary to understand the mechanism of the occurrence. The development and metastasis of recurrent rectal cancer. The curative effect obtained for colorectal cancer standardized treatment is limited, current research that may be attributed to colon cancer in the presence of a small amount of high resistance low proliferation of tumor stem cells . tumor stem has a series of unique biological characteristics of cells, including self-renewal, differentiation, proliferation, metastasis and drug resistance, is considered to be the tumor development, invasion and metastasis and recurrence of the "seed cells". The tumor microenvironment is a tumor cell survival environment, similarly, tumor stem cells are also micro the environment, called cancer stem cell niche, play an important role in maintaining the characteristics of tumor stem cells. Tumor stem cell niche in the oxidation reduction equilibrium changes may influence the properties of stem cells. Reduction of peroxide enzyme (PRDXs) is a superfamily of antioxidant enzymes in the cell. In this family is PRDX2 a typical 2-Cys peroxidoxin, the study found that the expression of PRDX2 in colon cancer was higher than that in paracancerous tissue, and knockdown of prdx2 can inhibit the growth of colon cancer cells, promote apoptosis. However, PRDX 2 as an important regulator of the redox balance, and colon cancer stem cell characteristics between has not been studied clearly. Objective: To investigate the effect of prdx2 gene expression characteristics of colon cancer stem cells and its molecular mechanism. Methods: 1) was detected by immunohistochemistry in 10 cases of human colon cancer tissue samples of prdx2 and CD133 the expression of the optical density analysis of protein semi quantitative data by imageproplus software, Pearson correlation analysis was performed between the two groups of data; using immunomagnetic cell sorting in colorectal cancer cells cd133- and cd133+ cells, with the proportion of cd133+ cells by flow cytometry to identify the separation efficiency; respectively from the colon cancer cell lines by three cd133- and cd133+ cells detected by Westernblot cd133- and cd133+ prdx2 cells, expression of CD44 and CD133 protein; in three colon cancer cell lines, by immunofluorescence detection of colon cancer Cells and colon cancer stem cells prdx2 and CD133 protein microspheres in the expression of.2) to establish prdx2 interference plasmid vector and lentiviral packaging by exogenous Westernblot screening of the effective carrier, qRT-PCR knockdown of endogenous verification efficiency; establish no-load lentivirus group in three strains of colon cancer cells by lentiviral transfection (cont group) and prdx2 knockdown group (shprdx2 group), observed by fluorescence microscopy and lentiviral transfection efficiency, with the difference between Westernblot and qRT-PCR for detection of prdx2 in the two groups at the level of mRNA and protein ratio; flow cytometry cd133+ cells in two groups of cells, Westernblot and qRT-PCR in two groups were detected in CD44 cells. CD133, Lgr5, CXCR4, EpCAM expression and Nanog protein and mRNA, observe the cell number of microspheres formed in two groups of cells with different ball test, the sensitivity of 5-FU of two groups of cell drug toxicity test The difference in formation volume differences in transplantation tumor of.3 in nude mice with cd133+ cell tumor assay in two groups) to establish prdx2 over expression vector and lentiviral packaging, identification of the effectiveness of Westernblot were established; no-load lentivirus group in three strains of colon cancer cells by lentiviral transfection (cont group) and prdx2 overexpression group (prdx2 group), observed by fluorescence microscopy and lentiviral transfection rate and the expression level of prdx2 protein between two groups of cells detected by Westernblot; the proportion of using flow cytometry cd133+ cells in the two groups, two groups of CD44 cells detected by Westernblot, the expression of CD133 and Nanog the amount of protein differences were observed, mammosphere formation number difference of.4 in two groups of cells with stem ball test) HT29 cells in empty lentivirus group (cont group), prdx2 group (group shprdx2) knockdown and overexpression of prdx2 group (prdx2 group) respectively. Select the cd133+ cells by immunomagnetic beads method, for the three groups of the cd133+ cells, the expression of hedgehog/gli1 protein detected by Westernblot pathway and Gli1 protein SMO; DMSO or SMO inhibitor cyclopamine on HT29 cells were intervened and divided into DMSO group and cyclopamine group, marker CD44 expression and CD133 protein were detected by Westernblot dry. Results: 1) in 10 cases of human colon cancer tissue samples, the expression of prdx2 and CD133 protein had positive linear relationship between Pearson, the correlation coefficient was 0.7863, cd133+ cells p=0.007. immunomagnetic cell sorting of CD133 + cells in the ratio of 93.10%, cd133+ cells and cd133- cells in the ratio of 1.06%. in SW620 HT29, HCT116, three colon cancer cell lines, the expression of prdx2 protein in cd133+ cells was significantly higher than that of cd133- cells, the expression of p0.05.prdx2 protein was mainly localized in the cytoplasm, CD133 Protein expression was mainly localized in the cell membrane of.2) in empty lentivirus group (cont group) and prdx2 knockdown group (group shprdx2) fluorescence transfection rate was greater than 95%, the mRNA and protein expression of prdx2 in shprdx2 group were significantly lower than that of cont group; compared with cont group, shprdx2 group in cd133+ cell group was significantly lower stem cell surface markers CD44, CD133, Lgr5, EpCAM and mRNA protein and transcription factor Nanog related stem cell expression was significantly reduced, mammosphere Chengliang significantly reduce the sensitivity of cd133+ cells to 5-FU significantly increased the formation of cd133+ cells of nude mice subcutaneous transplantation tumor volume was significantly smaller, p0.05.3) in slow load virus group (cont group) and prdx2 group (group prdx2) expression in fluorescence transfection rate was greater than 95%, the mRNA and protein expression of prdx2 in prdx2 group was significantly higher than that of cont group; compared with cont group, prdx2 group in cd133+ group was significantly higher than in stem cells. The cell surface markers CD44, CD133 and transcription factor Nanog related stem cell protein expression was significantly increased, mammosphere Chengliang increased significantly, p0.05.4) in HT29 cells sorted cd133+ cells, compared with the cont group, the expression of SMO and Gli1 protein in shprdx2 group decreased significantly, and the expression of prdx2 group SMO and Gli1 the amount of protein was increased significantly in P0.05; HT29 cell line, compared with the DMSO group, the expression of SMO and Gli1 protein in cyclopamine group decreased significantly, while the stem cell surface marker CD44 expression and CD133 protein decreased significantly. Conclusion: prdx2 may p0.05. through hedgehog/gli1 signaling pathway to maintain the characteristics of cancer stem cell junction.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.35

【相似文献】