本文關鍵詞：PRDX2對結(jié)腸癌干細胞特性的調(diào)控作用及機制研究　出處：《重慶醫(yī)科大學》2017年博士論文　論文類型：學位論文

  更多相關文章： PRDX2 干細胞特性 腫瘤干細胞 Hedgehog 結(jié)腸癌

【摘要】：結(jié)腸癌是全球范圍內(nèi)的高發(fā)腫瘤之一。在我國結(jié)腸癌的發(fā)病率逐年增高,威脅到更多人的生命健康。根據(jù)中國腫瘤登記中心的統(tǒng)計數(shù)據(jù),在2015年結(jié)直腸癌新發(fā)病例估算為37.63萬人,占同年總新發(fā)腫瘤病例的8.7%,排第五位,結(jié)直腸癌死亡病例估算為19.10萬人,占同年總死亡腫瘤病例的6.8%,排第五位。目前結(jié)直腸癌的治療仍以傳統(tǒng)手術、輔助化療為主,但是中晚期結(jié)直腸癌療效并不理想,為了提升結(jié)直腸癌的治療效果,有必要深入理解結(jié)直腸癌發(fā)生發(fā)展和轉(zhuǎn)移復發(fā)的機制。對于結(jié)直腸癌的標準化治療所獲得的療效有限,目前研究認為可能歸因于結(jié)腸癌中存在少量高耐藥低增殖的腫瘤干細胞。腫瘤干細胞具有一系列獨有的生物學特性,包括自我更新、多向分化潛能、無限增殖、轉(zhuǎn)移和耐藥,被認為是腫瘤發(fā)生發(fā)展、侵襲轉(zhuǎn)移以及復發(fā)的“種子細胞”。腫瘤微環(huán)境是腫瘤細胞賴以生存的環(huán)境,同樣地,腫瘤干細胞也有相應的微環(huán)境,稱為腫瘤干細胞龕,在維持腫瘤干細胞特性方面發(fā)揮重要作用。腫瘤干細胞龕中氧化還原平衡的改變有可能對干細胞特性產(chǎn)生影響。過氧化物還原酶(PRDXs)是細胞中的一個抗氧化酶超家族。在這個家族中PRDX2是一個典型的2-Cys過氧化物還原酶,研究發(fā)現(xiàn)PRDX2在結(jié)腸癌組織中表達量高于癌旁組織,而且prdx2敲減能夠抑制結(jié)腸癌細胞生長,促進凋亡。然而prdx2作為氧化還原平衡的重要調(diào)節(jié)因子,與結(jié)腸癌干細胞特性之間的關系還沒有研究清楚。目的:探討干擾prdx2基因表達對結(jié)腸癌干細胞特性的影響及其分子機制。方法:1)免疫組化檢測10例人結(jié)腸癌組織樣本中的prdx2和cd133的蛋白表達,用imageproplus軟件進行光密度分析獲得蛋白半定量數(shù)據(jù),對兩組數(shù)據(jù)進行pearson相關性分析;用免疫磁珠法分選結(jié)腸癌細胞中的cd133-和cd133+細胞,用流式細胞儀檢測cd133+細胞比例以鑒定分選效率;分別從三株結(jié)腸癌細胞中分選出cd133-和cd133+細胞,用westernblot檢測cd133-和cd133+細胞中prdx2、cd44和cd133的蛋白表達量;在三種結(jié)腸癌細胞系中,用免疫熒光法檢測結(jié)腸癌細胞和結(jié)腸癌干細胞微球體中prdx2和cd133的蛋白表達情況。2)建立prdx2干擾質(zhì)粒載體并用慢病毒包裝,通過westernblot外源篩選有效載體,qrt-pcr內(nèi)源驗證敲減效率;通過慢病毒轉(zhuǎn)染在三株結(jié)腸癌細胞系中分別建立空載慢病毒組(cont組)和prdx2敲減組(shprdx2組),用熒光顯微鏡觀察慢病毒轉(zhuǎn)染率,用westernblot和qrt-pcr檢測兩組細胞中prdx2在mrna和蛋白水平的差異;用流式細胞儀檢測兩組細胞中cd133+細胞的比例,用westernblot和qrt-pcr檢測兩組細胞中cd44、cd133、lgr5、cxcr4、epcam和nanog的蛋白和mrna的表達量,用成球試驗觀察兩組細胞中干細胞微球體形成數(shù)量的差異,用藥物毒性實驗檢測兩組細胞對5-fu敏感性的差異,用成瘤實驗檢測兩組中的cd133+細胞在裸鼠皮下形成移植瘤的體積差異。3)建立prdx2過表達質(zhì)粒載體并用慢病毒包裝,用westernblot鑒定其有效性;通過慢病毒轉(zhuǎn)染在三株結(jié)腸癌細胞系中分別建立空載慢病毒組(cont組)和prdx2過表達組(prdx2組),用熒光顯微鏡觀察慢病毒轉(zhuǎn)染率,用westernblot檢測兩組細胞中prdx2蛋白表達量的差異;用流式細胞儀檢測兩組細胞中cd133+細胞的比例,用westernblot檢測兩組細胞中cd44、cd133和nanog的蛋白表達量差異,用成球試驗觀察兩組細胞中干細胞微球體形成數(shù)量的差異。4)選擇ht29細胞,在空載慢病毒組(cont組)、prdx2敲減組(shprdx2組)和prdx2過表達組(prdx2組)中分別用免疫磁珠法分選出cd133+細胞,對于三個組別的cd133+細胞,用westernblot檢測hedgehog/gli1通路關鍵蛋白smo和gli1的蛋白表達量;用dmso或者smo抑制劑cyclopamine對ht29細胞進行干預并分為dmso組和cyclopamine組,用westernblot檢測干性標志物cd44和cd133的蛋白表達量。結(jié)果:1)在10例人結(jié)腸癌組織樣本中,prdx2和cd133的蛋白表達量之間存在線性正相關性,pearson相關系數(shù)為0.7863,p=0.007。免疫磁珠法分選出的cd133+細胞中cd133+細胞的比例為93.10%,cd133-細胞中cd133+細胞的比例為1.06%。在sw620、ht29、hct116三種結(jié)腸癌細胞系中,cd133+細胞中prdx2的蛋白表達量均顯著高于cd133-細胞,p0.05。prdx2蛋白表達主要定位在細胞質(zhì)中,cd133蛋白表達主要定位在細胞膜。2)在空載慢病毒組(cont組)和prdx2敲減組(shprdx2組)中熒光轉(zhuǎn)染率均大于95%,shprdx2組中prdx2的mrna和蛋白表達量均顯著低于cont組;與cont組相比,shprdx2組中cd133+細胞群體比例顯著降低,干細胞表面標志物cd44、cd133、lgr5、epcam和干細胞相關轉(zhuǎn)錄因子nanog的mrna和蛋白表達量顯著減少,微球體形成量顯著減少,cd133+細胞對5-fu的敏感性顯著增加,cd133+細胞形成裸鼠皮下移植瘤的體積顯著變小,p0.05。3)在空載慢病毒組(cont組)和prdx2過表達組(prdx2組)中熒光轉(zhuǎn)染率均大于95%,prdx2組中prdx2的mrna和蛋白表達量均顯著高于cont組;與cont組相比,prdx2組中cd133+細胞群體比例顯著增高,干細胞表面標志物cd44、cd133和干細胞相關轉(zhuǎn)錄因子nanog的蛋白表達量顯著增加,微球體形成量顯著增加,p0.05。4)在ht29細胞分選出的cd133+細胞中,與cont組相比,shprdx2組的smo和gli1蛋白表達量均顯著降低,prdx2組的smo和gli1蛋白表達量均顯著增加,p0.05;在ht29細胞系中,與dmso組相比,cyclopamine組的smo和gli1蛋白表達量均顯著降低,同時干細胞表面標志物cd44和cd133蛋白表達量均顯著降低,p0.05。結(jié)論:prdx2可能通過hedgehog/gli1信號通路維持結(jié)腸癌干細胞特性。
[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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.35

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