本文選題：膠質瘤 + 端粒��； 參考：《吉林大學》2016年博士論文

【摘要】：惡性膠質瘤是最常見的人類神經(jīng)系統(tǒng)原發(fā)性惡性腫瘤,同時也是造成兒童和青少年“腫瘤相關死亡”的第二大惡性腫瘤。膠質母細胞瘤是惡性膠質瘤中相對較常見的一種病理類型,而且還是惡性度最高,發(fā)展速度最快,侵襲性最強的惡性膠質瘤。目前的研究表明膠質母細胞瘤對于術后放化療的敏感性也是最弱的。膠質母細胞瘤是預后極差的一種惡性膠質瘤,統(tǒng)計顯示,診斷為膠質母細胞瘤的患者,經(jīng)過手術治療和聯(lián)合放化療治療后的平均生存期不足18個月。人類細胞的端粒結構位于染色體末端,由重復的TTAGGG序列構成,作用是幫助細胞分辨正常的染色體末端和雙鏈DNA損傷。人類端粒的3′懸端是由一個富G序列單鏈DNA構成。3′懸端的DNA序列有兩種形態(tài),當DNA序列打開時,能夠與端粒酶結合,進行端粒DNA的延長反應。而當單鏈DNA關閉時,形成了所謂的T-loop結構和D-loop結構,主要是為了保持端粒末端的完整性。如果能夠采用措施使端粒末端維持一種打開狀態(tài),那么就會導致端粒結構的紊亂,表現(xiàn)為染色體末端融合、后期橋和端粒末端降解,最后導致細胞走向凋亡和衰老。惡性腫瘤細胞最顯著的標志是長度永遠不縮短的端粒DNA,從而使細胞擁有無限增殖的能力。研究證明大約85%的人惡性腫瘤細胞中表達處于激活狀態(tài)的端粒酶來維持端粒長度。這些研究為制定靶向端粒酶結構的抗腫瘤治療方案的可行性提供了依據(jù)。端粒酶抑制劑能夠與端粒3'懸端單鏈DNA有效結合形成穩(wěn)定的G-quadruplex四鏈體結構,抑制端粒酶催化的端粒合成反應和帽狀結構的形成。這種有效抑制端粒延長的作用使得端粒酶抑制劑成為很有潛力的化療藥物選擇。雖然端粒酶抑制劑是一種很有潛力的抗癌治療藥物,但是傳統(tǒng)的端粒酶抑制劑的弊端在于,達到能夠誘導細胞走向凋亡和衰老的端粒DNA縮短所需要的時間過長。而且,有研究顯示,部分端粒DNA配體作用于惡性腫瘤細胞后,產(chǎn)生的端粒DNA縮短效果會誘導惡性腫瘤細胞激活體內的端粒DNA長度延長機制,進而維持惡性腫瘤細胞內的端粒長度。這也是阻礙端粒酶抑制劑成為臨床用藥的重要因素。BRACO-19是結合端粒形成G-quadruplex結構最有效的端粒DNA配體之一,研究顯示它具有顯著的抑制端粒酶活性和抗腫瘤作用。然而,G-quadruplex配體分子作用于端粒結構的研究仍處于臨床前階段,因此,確定其潛在反應機制仍是主要的挑戰(zhàn)性課題。在本論文中,我們主要應用TRAP assay,免疫熒光,western blotting,染色質免疫沉淀(Ch IP)以及端粒熒光原位雜交分析BRACO-19作用后產(chǎn)生的端粒酶的活性抑制,端粒結構紊亂及細胞增殖的抑制。本論文的主要觀點如下:1、我們發(fā)現(xiàn)BRACO-19能顯著抑制端粒酶活性,選擇性抑制膠質瘤細胞增殖,對于普通膠質細胞的抑制作用不明顯。BRACO-19可以明顯的誘導人膠質母細胞瘤細胞內端粒酶出現(xiàn)核質轉運效應。進一步研究發(fā)現(xiàn)BRACO-19誘導膠質瘤細胞出現(xiàn)的增值抑制與細胞內出現(xiàn)的DNA損傷反應有密切的聯(lián)系。而且,我們證實了進一步證實BRACO-19誘導的DNA損傷反應發(fā)生在端粒上;2、BRACO-19誘導了TRF2和POT1(端粒結合蛋白)從端粒上解離,這一結論間接證明了膠質瘤細胞內G-quadruplex形成及T-loop結構解散。進一步研究發(fā)現(xiàn),BRACO-19作用后的膠質瘤細胞出現(xiàn)了明顯的端粒DNA 3′-懸端的縮短;3、BRACO-19誘導膠質瘤細胞出現(xiàn)了細胞周期阻滯,細胞凋亡和衰老,這可能是端粒結構紊亂的結果,而且p53和p21蛋白的上調與這些生物學效應有密切的聯(lián)系;4、BRACO-19作用膠質瘤細胞后出現(xiàn)了端粒融合和后期橋形成等染色體異�，F(xiàn)象,這是端粒結構紊亂的結果。通過端粒-末端脫氧核糖核酸轉移酶(Td T)的共定位分析證實了BRACO-19作用后膠質瘤細胞出現(xiàn)的端粒去保護狀態(tài)。本文率先證明了BRACO-19結合端粒從而抑制端粒酶活性對于腦腫瘤細胞的生長抑制作用,單獨用藥或者聯(lián)合其他治療方法,比如放射性治療,可以為腦膠質母細胞瘤以至其他種類的惡性腫瘤的治療提供新的依據(jù)。
[Abstract]:Malignant glioma is the most common primary malignant tumor of the human nervous system. It is also the second major malignant tumor causing "tumor related death" in children and adolescents. Glioblastoma is a relatively common pathological type of malignant glioma, and it is also the most malignant, fast and aggressive evil. The present study shows that the sensitivity of glioblastoma to postoperative chemoradiotherapy is also the weakest. Glioblastoma is a malignant glioma with poor prognosis. Statistics show that the average survival time of patients diagnosed as glioblastoma is less than 18 months after surgical treatment and combined radiotherapy. Human cells The telomere structure is located at the end of the chromosome and consists of a repeat TTAGGG sequence that helps the cells to distinguish between the normal chromosome ends and the double strand DNA damage. The 3 'end of the human telomere is a DNA sequence consisting of a.3' suspended from a single strand DNA rich in G sequence. When the DNA sequence is opened, it can be combined with telomerase to carry out telomere D. When the single strand DNA is closed, the so-called T-loop structure and D-loop structure are formed, mainly to maintain the integrity of the telomere end. If the end of the telomere end is maintained, the telomere structure disorder, the fusion of the end of the chromosome, the end of the terminal bridge and the end of the telomere will be reduced. The most significant sign of malignant tumor cells is that the telomere DNA, which is never shortened in length, makes the cells have the ability to proliferate indefinitely. Studies have shown that about 85% of human malignant tumor cells express telomerase in active state to maintain telomere length. These studies are designed to formulate target telomere. The enzyme structure provides a basis for the feasibility of antitumor therapy. Telomerase inhibitors can effectively bind to the telomere 3'suspension single strand DNA to form a stable G-quadruplex four chain body structure, inhibit telomerase catalyzed telomere synthesis and cap formation. This effective inhibition of telomere prolongation makes telomerase inhibitors Although telomerase inhibitors are a potential anticancer drug, the disadvantage of traditional telomerase inhibitors is that it takes a long time to achieve the telomere DNA shortening that can induce cell apoptosis and aging. Furthermore, some studies have shown that some telomere DNA ligands act on evil. After the tumor cells, the telomere DNA shortening can induce the telomere DNA length extension mechanism in the malignant tumor cells, and then maintain the telomere length in the malignant tumor cells. This is also an important factor hindering telomerase inhibitors from the clinical use of.BRACO-19 is the most effective end of the binding telomere to form the G-quadruplex structure. One of the grain DNA ligands, research shows that it has a significant inhibition of telomerase activity and anti-tumor effect. However, the study of G-quadruplex ligand molecules on telomere structure is still in the preclinical stage. Therefore, it is still a major challenge to determine its potential reaction mechanism. In this paper, we mainly use TRAP assay, immunofluorescence, w Estern blotting, chromatin immunoprecipitation (Ch IP) and telomere fluorescence in situ hybridization analysis of the activity inhibition of telomerase, disorder of telomere structure and inhibition of cell proliferation. The main points of this paper are as follows: 1, we found that BRACO-19 can significantly inhibit telomerase activity and selectively inhibit the proliferation of glioma cells. The inhibitory effect of common glial cells is not obvious.BRACO-19 can obviously induce the nuclear transport effect of telomerase in human glioblastoma cells. Further studies have found that the proliferation inhibition of BRACO-19 induced glioma cells is closely related to the DNA damage reaction in cells. Furthermore, we have confirmed the further evidence. The actual BRACO-19 induced DNA damage reaction occurred on the telomere; 2, BRACO-19 induced the dissociation of TRF2 and POT1 (telomere binding protein) from the telomere, which indirectly demonstrated the formation of G-quadruplex in glioma cells and the dissolution of the T-loop structure. Further studies showed that the telomere DNA 3 '- a distinct telomere of the glioma cells after BRACO-19 was used. 3, BRACO-19 induced cell cycle arrest, apoptosis and senescence in glioma cells, which may be the result of the disorder of telomere structure, and the up regulation of p53 and p21 proteins is closely related to these biological effects; and 4, after the action of BRACO-19 glioma cells, there are telomere fusion and late bridge formation of chromosomes. Common phenomenon is the result of telomere structure disorder. Through co localization analysis of telomere terminal deoxyribonucleic acid transferase (Td T), the telomere deactivation state of glioma cells after BRACO-19 action is confirmed. This article is the first to prove that BRACO-19 binding telomeres can inhibit the growth inhibition of telomerase activity to brain tumor cells. Alone or in combination with other treatments, such as radiation therapy, can provide a new basis for the treatment of glioblastoma and other types of malignant tumors.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R739.41

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本文編號：1840239