本文選題：腫瘤干細(xì)胞 + 結(jié)腸癌　； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：結(jié)腸癌(Colorectal cancer)是世界上最常見(jiàn)的消化道惡性腫瘤之一,其死亡率居腫瘤相關(guān)死亡率的第三位。在我國(guó),結(jié)腸癌的發(fā)病率及死亡率也呈逐年上升的趨勢(shì)。目前針對(duì)結(jié)腸癌的治療手段仍以外科手術(shù)切除聯(lián)合放化療為主,但是患者仍會(huì)出現(xiàn)術(shù)后腫瘤復(fù)發(fā)和轉(zhuǎn)移,嚴(yán)重危害患者的健康與生命。因此,急需尋找更合適及更有效的治療結(jié)腸癌的方法。近年來(lái),隨著對(duì)惡性腫瘤的深入研究及腫瘤干細(xì)胞理論的提出,人們發(fā)現(xiàn),腫瘤中存在極少數(shù)具有“干性”,能夠自我更新并具有多向分化潛能的腫瘤干細(xì)胞(Cancer stem cell,CSC),與腫瘤發(fā)生發(fā)展、轉(zhuǎn)移及復(fù)發(fā)密切相關(guān)。腫瘤干細(xì)胞理論認(rèn)為,腫瘤干細(xì)胞是腫瘤的啟動(dòng)細(xì)胞,可能是惡性腫瘤形成的根源,只有靶向治療腫瘤干細(xì)胞,才能最終治愈腫瘤。干性基因在干細(xì)胞的自我更新和分化中發(fā)揮關(guān)鍵作用,其中干細(xì)胞轉(zhuǎn)錄因子Nanog不僅在維持胚胎干細(xì)胞多能性和自我更新方面發(fā)揮重要作用,而且在多種腫瘤中異常表達(dá)。研究發(fā)現(xiàn),Nanog的高表達(dá)與腫瘤不良預(yù)后密切相關(guān),提示Nanog可能通過(guò)維持腫瘤干細(xì)胞的干性促進(jìn)腫瘤的發(fā)生發(fā)展。因此,為了探究Nanog對(duì)結(jié)腸癌干細(xì)胞的生物學(xué)影響,本實(shí)驗(yàn)以Ep CAM和CD44為腫瘤干細(xì)胞篩選標(biāo)志,通過(guò)免疫磁珠法從人結(jié)腸癌細(xì)胞系HCT-116中篩選出Ep CAM+CD44+HCT-116的人結(jié)腸癌干細(xì)胞(Colorectal cancer stem cell,CCSC),利用RNA干擾(RNA interference,RNAi)沉默結(jié)腸癌干細(xì)胞中Nanog的表達(dá),探究其對(duì)結(jié)腸癌干細(xì)胞生物學(xué)行為的影響。結(jié)果顯示,篩選獲得的Ep CAM+CD44+HCT-116細(xì)胞在無(wú)血清DMEM/F12培養(yǎng)基中懸浮生長(zhǎng),由單個(gè)細(xì)胞逐漸長(zhǎng)成腫瘤微球;經(jīng)含血清培養(yǎng)基誘導(dǎo)后,細(xì)胞微球呈現(xiàn)貼壁分化狀態(tài)。Real-time PCR檢測(cè)結(jié)果顯示:Ep CAM+CD44+HCT-116細(xì)胞中Nanog的m RNA表達(dá)顯著高于未分選的HCT-116細(xì)胞(P0.01);Ep CAM+CD44+HCT-116經(jīng)Nanog si RNA作用48h之后,細(xì)胞中Nanog基因的m RNA和蛋白表達(dá)水平均顯著下降(P0.01,P0.05)。MTS增殖實(shí)驗(yàn)顯示Nanog基因沉默可顯著抑制Ep CAM+CD44+HCT-116增殖(P0.01)。Annexin V-FITC/PI雙染法從細(xì)胞膜的完整程度上區(qū)分早、晚期凋亡細(xì)胞,結(jié)果顯示:Ep CAM+CD44+HCT-116中Nanog基因沉默后,早、晚期凋亡細(xì)胞比例增加;JC-1法通過(guò)細(xì)胞膜電位的變化反映細(xì)胞凋亡情況,結(jié)果顯示:Nanog基因沉默后細(xì)胞膜電位大幅度降低,凋亡細(xì)胞比例增加;Real-time PCR檢測(cè)結(jié)果顯示:Nanog基因沉默后,Ep CAM+CD44+HCT-116中抗凋亡基因Bcl-2的m RNA表達(dá)水平顯著降低(P0.05),而促凋亡基因Bax及Caspase-3的m RNA表達(dá)水平均顯著升高(P0.01);Western blot結(jié)果顯示:Nanog基因沉默后,Ep CAM+CD44+HCT-116中cleaved-caspase3蛋白表達(dá)量顯著增加(P0.05)。Transwell小室結(jié)果顯示:沉默Nanog基因可顯著抑制Ep CAM+CD44+HCT-116的侵襲(P0.01);同時(shí)Real-time PCR結(jié)果顯示:Nanog基因沉默后,Ep CAM+CD44+HCT-116中促進(jìn)侵襲基因MMP-2及MMP-9的m RNA表達(dá)水平顯著降低(P0.05,P0.01),而抑制侵襲基因Timp-1的m RNA表達(dá)水平顯著升高(P0.01)。動(dòng)物實(shí)驗(yàn)結(jié)果顯示,Nanog si RNA組腫瘤體積及瘤重顯著低于對(duì)照組及空白組(P0.05),并且Nanog si RNA組荷瘤鼠的生存期也顯著長(zhǎng)于對(duì)照組及空白組(P0.05)。綜上所述,Nanog沉默能夠抑制結(jié)腸癌干細(xì)胞的增殖和侵襲,促進(jìn)結(jié)腸癌干細(xì)胞的凋亡,并降低結(jié)腸癌干細(xì)胞的成瘤能力,這為針對(duì)結(jié)腸癌干細(xì)胞的靶向治療提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:Colon cancer (Colorectal cancer) is one of the most common malignant tumor of digestive tract, the mortality rate was third. The tumor related mortality in our country, the incidence and mortality of colorectal cancer also showed an increasing trend. At present for colon cancer treatment is still outside the surgery resection combined with radiotherapy and chemotherapy, but there would still be in patients with postoperative tumor recurrence and metastasis, patients with serious harm to health and life. Therefore, the urgent need to find a more suitable method and treatment of colon cancer is more effective. In recent years, with the proposed cell theory on malignant tumor research and tumor stem were found, with a very small number of "dry" the tumor, capable of self-renewal and multi-directional differentiation of cancer stem cells (Cancer stem cell, CSC), and the occurrence and development of tumor, metastasis and recurrence are closely related. The cancer stem cell theory, tumor stem Cells are tumor initiating cells, may be the root cause of the formation of malignant tumors, only targeted therapy of cancer stem cells to cure the tumor. Dry gene plays a key role in the self-renewal and differentiation of stem cells, including stem cell transcription factor Nanog in maintenance of embryonic stem cells can play an important role and the self the update, and the abnormal expression in a variety of tumors. The study found that high expression of Nanog and tumor prognosis is closely related to the occurrence and development of Nanog may be prompted by maintaining the tumor stem cells promote tumor dry. Therefore, in order to explore the biological effects of Nanog stem cells for colon cancer, in this experiment, Ep CAM and CD44 as tumor stem cell marker screening, by immunomagnetic beads from human colon cells screened human colon cancer stem Ep CAM+CD44+HCT-116 colorectal cancer cell line HCT-116 in Colorectal (cancer stem cell, CCS C (RNA), using RNA interference interference, RNAi) silencing of colon cancer stem cells expression of Nanog, to explore its effect on colon cancer stem cells. The results showed that the Ep screened CAM+CD44+HCT-116 cells in serum-free suspension culture medium DMEM/F12 growth, from a single cell into tumor by serum containing microspheres; medium after induction, cell adherence and differentiation state of.Real-time microspheres showed PCR showed that the expression of M RNA Nanog Ep in CAM+CD44+HCT-116 cells was significantly higher than that of unsorted HCT-116 cells (P0.01); Ep CAM+CD44+ HCT-116 Nanog Si RNA by 48h, the expression level of M protein and RNA Nanog gene in the cells were significantly decreased (P0.01, P0.05.MTS) proliferation assay showed that Nanog gene silencing can significantly inhibit the proliferation of CAM+CD44+HCT-116 Ep (P0.01).Annexin V-FITC/PI double staining method from the integrity of the cell membrane to distinguish early, The results of late apoptotic cells showed that Ep in CAM+CD44+HCT-116 after Nanog gene silencing, the percentage of apoptotic cells increased early and late; JC-1 method by changing the membrane potential reflect the cell apoptosis, the results showed that Nanog gene silencing cell membrane potential decreased, apoptotic cells increased; Real-time PCR results showed that Nanog gene after the silence, the expression level of M RNA Ep anti apoptosis gene Bcl-2 in CAM+CD44+HCT-116 decreased significantly (P0.05), the expression level of M RNA and apoptosis gene Bax and Caspase-3 were significantly increased (P0.01); Western blot showed that Nanog gene silencing, cleaved-caspase3 protein expression was significantly increased in Ep CAM+CD44+HCT-116 (P0.05).Transwell chamber the results showed that silencing Nanog gene can inhibit the invasion of Ep CAM+CD44+HCT-116 (P0.01); and Real-time PCR results showed that: after Nanog gene silencing, Ep CAM+CD 44+HCT-116 m RNA MMP-2 to promote invasion gene and MMP-9 expression level were significantly decreased (P0.05, P0.01), and the inhibition of M RNA invasion gene Timp-1 expression level increased significantly (P0.01). The results of animal experiments showed that the Nanog Si RNA group tumor volume and tumor weight was significantly lower than the control group and blank control group (P0.05), and Nanog Si RNA group, the survival time of tumor bearing mice was significantly longer than that of the control group and blank control group (P0.05). In conclusion, Nanog silencing inhibits the proliferation and invasion of colon cancer stem cells and promote the apoptosis of colon cancer stem cells, and reduce colon cancer stem cell tumorigenic ability, this is for colon cancer stem cells. Targeted therapy provides an experimental basis.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.35

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