【作者】 赵媛媛；

【导师】 王毅；

【作者基本信息】 吉林大学 ， 再生医学， 2017， 硕士

【摘要】 结肠癌(Colorectal cancer)是世界上最常见的消化道恶性肿瘤之一,其死亡率居肿瘤相关死亡率的第三位。在我国,结肠癌的发病率及死亡率也呈逐年上升的趋势。目前针对结肠癌的治疗手段仍以外科手术切除联合放化疗为主,但是患者仍会出现术后肿瘤复发和转移,严重危害患者的健康与生命。因此,急需寻找更合适及更有效的治疗结肠癌的方法。近年来,随着对恶性肿瘤的深入研究及肿瘤干细胞理论的提出,人们发现,肿瘤中存在极少数具有“干性”,能够自我更新并具有多向分化潜能的肿瘤干细胞(Cancer stem cell,CSC),与肿瘤发生发展、转移及复发密切相关。肿瘤干细胞理论认为,肿瘤干细胞是肿瘤的启动细胞,可能是恶性肿瘤形成的根源,只有靶向治疗肿瘤干细胞,才能最终治愈肿瘤。干性基因在干细胞的自我更新和分化中发挥关键作用,其中干细胞转录因子Nanog不仅在维持胚胎干细胞多能性和自我更新方面发挥重要作用,而且在多种肿瘤中异常表达。研究发现,Nanog的高表达与肿瘤不良预后密切相关,提示Nanog可能通过维持肿瘤干细胞的干性促进肿瘤的发生发展。因此,为了探究Nanog对结肠癌干细胞的生物学影响,本实验以Ep CAM和CD44为肿瘤干细胞筛选标志,通过免疫磁珠法从人结肠癌细胞系HCT-116中筛选出Ep CAM+CD44+HCT-116的人结肠癌干细胞(Colorectal cancer stem cell,CCSC),利用RNA干扰(RNA interference,RNAi)沉默结肠癌干细胞中Nanog的表达,探究其对结肠癌干细胞生物学行为的影响。结果显示,筛选获得的Ep CAM+CD44+HCT-116细胞在无血清DMEM/F12培养基中悬浮生长,由单个细胞逐渐长成肿瘤微球;经含血清培养基诱导后,细胞微球呈现贴壁分化状态。Real-time PCR检测结果显示:Ep CAM+CD44+HCT-116细胞中Nanog的m RNA表达显著高于未分选的HCT-116细胞(P<0.01);Ep CAM+CD44+HCT-116经Nanog si RNA作用48h之后,细胞中Nanog基因的m RNA和蛋白表达水平均显著下降(P<0.01,P<0.05)。MTS增殖实验显示Nanog基因沉默可显著抑制Ep CAM+CD44+HCT-116增殖(P<0.01)。Annexin V-FITC/PI双染法从细胞膜的完整程度上区分早、晚期凋亡细胞,结果显示:Ep CAM+CD44+HCT-116中Nanog基因沉默后,早、晚期凋亡细胞比例增加;JC-1法通过细胞膜电位的变化反映细胞凋亡情况,结果显示:Nanog基因沉默后细胞膜电位大幅度降低,凋亡细胞比例增加;Real-time PCR检测结果显示:Nanog基因沉默后,Ep CAM+CD44+HCT-116中抗凋亡基因Bcl-2的m RNA表达水平显著降低(P<0.05),而促凋亡基因Bax及Caspase-3的m RNA表达水平均显著升高(P<0.01);Western blot结果显示:Nanog基因沉默后,Ep CAM+CD44+HCT-116中cleaved-caspase3蛋白表达量显著增加(P<0.05)。Transwell小室结果显示:沉默Nanog基因可显著抑制Ep CAM+CD44+HCT-116的侵袭(P<0.01);同时Real-time PCR结果显示:Nanog基因沉默后,Ep CAM+CD44+HCT-116中促进侵袭基因MMP-2及MMP-9的m RNA表达水平显著降低(P<0.05,P<0.01),而抑制侵袭基因Timp-1的m RNA表达水平显著升高(P<0.01)。动物实验结果显示,Nanog si RNA组肿瘤体积及瘤重显著低于对照组及空白组(P<0.05),并且Nanog si RNA组荷瘤鼠的生存期也显著长于对照组及空白组(P<0.05)。综上所述,Nanog沉默能够抑制结肠癌干细胞的增殖和侵袭,促进结肠癌干细胞的凋亡,并降低结肠癌干细胞的成瘤能力,这为针对结肠癌干细胞的靶向治疗提供了实验依据。更多还原

【Abstract】 Colorectal cancer is one of the most common malignant tumors of digestive tract in the world,with mortality rate at the third place in tumor-related death.The incidence and mortality rate of colorectal cancer have increased in our country year by year trend.So far,surgical resection combined with radiotherapy and chemotherapy remains the main treatment for colorectal cancer,but patients still appear postoperative tumor recurrence and metastasis,seriously endangering the health and life of patients.Therefore,it is urgently required to develop more suitable and effective method for treatment of colorectal cancer.In recent years,with the deep research of malignant tumors and the putting forward of cancer stem cells(CSC)theory,it has been found that there are very few cancer stem cells with "stem cell properties" like self-renewal and multiple differentiation potential,which are closely related to tumor development,metastasis and recurrence.The CSC theory proposes that CSCs are the initiating cells of tumors,which may be the origin of malignant tumors and only targeting treatment of CSCs may eventually cure the tumor.Stemness factors play an important role in self-renewal and differentiation of stem cells,in which the stem cell transcription factor Nanog not only plays a vital role in maintaining the pluripotency and self-renewal of embryonic stem cells,but also expresses abnormally in multiple tumors.Existing studies show that the high expression of Nanog is closely related to the poor prognosis of tumors,suggesting that Nanog may promote the development of tumor by maintaining the stemness properties of cancer stem cells.Therefore,in order to explore the biological effect of Nanog on colorectal cancer stem cells,in this study,Ep CAM and CD44 were used as markers to screen human colorectal cancer stem cells(CCSC)of Ep CAM+CD44+ HCT-116 from human colorectal cancer cell line HCT-116 by immunomagnetic beads.RNA interference(RNAi)was used to silence the expression of Nanog in colorectal cancer stem cells and we explored its effect on the biological behavior of colorectal cancer stem cells.Results show that Ep CAM+CD44+ HCT116 were suspended and cultured in serum-freeDMEM/F12 medium,and formed tumor microspheres from single cells gradually,which then re-adhered and differentiate after serum was added to the medium.The Real-time PCR analysis shows that the expression of Nanog in Ep CAM+CD44+ HCT-116 cells was significantly higher than that of unsorted HCT-116 cells(P<0.01);after transfection of Nanog si RNA for 48 h,the expression of Nanog was significantly decreased at both m RNA level and protein level(P<0.01,P<0.05).The MTS proliferation assays show that Nanog silencing could significantly inhibit the proliferation of Ep CAM+CD44+ HCT-116(P<0.01);the Annexin V-FITC/PI double staining method distinguishes the early and late apoptotic cells from the perspective of the integrity of the cell membrane,results show that the percentage of early and late apoptotic cells increased after Nanog gene silencing in Ep CAM+CD44+ HCT-116;the JC-1 method reflects the apoptosis from the point of view of cell membrane potential,results show that the cell membrane potential was significantly decreased after Nanog gene silencing and the proportion of apoptotic cells increased;the Real-time PCR analysis shows that the m RNA expression level of anti-apoptotic gene Bax in Ep CAM+CD44+ HCT-116 was significantly decreased(P<0.05)after Nanog gene silencing,while the m RNA expression level of pro-apoptotic genes Bax and Caspase-3 was significantly increased(P<0.01);and the western blot results show that the expression of cleaved-caspase3 protein in Ep CAM+CD44+ HCT-116 was significantly increased after Nanog gene silencing(P<0.05).The transwell chamber results show that silencing Nanog gene significantly inhibited the invasion of Ep CAM+CD44+ HCT-116(P<0.01);meanwhile,the Real-time PCR analysis shows that the m RNA expression level of pro-invasion genes MMP-2 and MMP-9 were significantly decreased after Nanog gene silencing(P<0.05,P<0.01),while the m RNA expression level of anti-invasive gene Timp-1 was significantly increased(P<0.01).The tumor xenograft assays show that,the tumor size and tumor weight of Nanog si RNA group were significantly lower than those of NC si RNA and mock groups(P<0.05),and the mice survival time of Nanog si RNA group was significantly longer than those of NC si RNA and mock groups(P <0.05).Taken together,our studies reveal that Nanog silencing can suppress the proliferation and invasion of colorectal cancer stem cells,and induce apoptosis,as well as reduce the tumorigenic capacity of colorectal cancer stem cells,which provide experimental basis fortargeted therapy of colorectal cancer stem cells.更多还原