本文選題：鼻咽癌　切入點：腫瘤干細胞　出處：《南方醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：鼻咽癌是我國南方地區(qū)及東南亞國家高發(fā)的惡性腫瘤之一,該類腫瘤的復發(fā)及遠處轉移是導致治療失敗及死亡的重要方式。近年來的研究發(fā)現(xiàn),腫瘤干細胞是導致包括鼻咽癌在內(nèi)的惡性腫瘤復發(fā)和耐藥的重要原因,目前已從多種惡性腫瘤中分選出腫瘤干細胞。腫瘤干細胞表現(xiàn)出一定的自身特性,如自我復制能力強,高表達端粒酶活性,具有CD133+、CD44+表面標志物等。這為進一步探討腫瘤干細胞在腫瘤發(fā)生發(fā)展及靶向腫瘤干細胞提供了新領域。本課題組曾開展了端粒酶抑制劑PinX1基因靶向鼻咽癌細胞端粒酶活性抑制鼻咽癌細胞的前期研究工作,在此基礎上,擬采用PinX1基因靶向鼻咽癌CD133+腫瘤干細胞,探討其對該腫瘤干細胞端粒酶活性下調、抑制腫瘤干細胞活性,并進一步探討端粒重復序列結合蛋白(TRF1及TRF2)的調控作用機制,闡明其在PinX1基因作用中的地位。一.CD133+陽性鼻咽癌細胞具有腫瘤干細胞特征目的:觀察以CD133+作為干細胞標志物分選CD133陽性細胞的增殖、遷移等能力。方法:鼻咽癌CNE2細胞通過免疫磁珠分選的方法獲得CD133+細胞和CD133-細胞,并通過流式細胞術對分選的效率進行驗證。然后將獲得的CD133+細胞和CD133-細胞進行干細胞球誘導及裸鼠體內(nèi)成瘤,通過QPCR法檢測CD133+細胞和CD133-細胞中干細胞標志物SOX2、Nanog的表達。其次用CCK-8觀察兩株細胞的生長情況,通過Transwell法和劃線法觀察細胞遷移情況,通過Transwell法觀察細胞侵襲情況。結果:1.以未加任何標記的CNE2細胞系為陰性對照,對常規(guī)培養(yǎng)3天的細胞表面的CD133和CD44的表達情況檢測發(fā)現(xiàn),CD133+和CD133-細胞成功分選。2.將新分選的CD133+和CD133-細胞,接種到含有bFGF、EGF、胰島素和B27無血清干細胞誘導培養(yǎng)基中,14天后,CD133+腫瘤細胞球的半徑顯著大于CD133-細胞。3.通過QPCR檢測CD133+和CD133-細胞中的Nanog和SOX2,Nanog和SOX2的mRNA在CD133+細胞中的表達量顯著高于CD133-細胞。4.使用CCK-8法考察分選的CD133+和CD133-細胞的生長情況,發(fā)現(xiàn)CD133+細胞增殖速度顯著快于CD133-細胞。5.通過劃線實驗或Transwell法檢測CD133+和CD133-細胞的遷移和侵襲能力,發(fā)現(xiàn)CD133+細胞比CD133-細胞遷移、侵襲能力強。6.該種CD133+鼻咽癌干細胞比CD133-細胞群有較強的裸鼠成瘤能力。結論:采用CD133磁珠從鼻咽癌CNE2細胞中成功分選出CD133+鼻咽癌腫瘤干細胞,同時表達Nanog和SOX2及端粒酶活性,體外成球、增殖、遷移、侵襲能力及裸鼠成瘤等腫瘤干細胞特征明顯強于CD133-鼻咽癌細胞群。二.PinX1基因靶向端粒酶抑制CD133+鼻咽癌干細胞目的:探討PinX1基因對CD133+鼻咽癌干細胞增殖和遷移等活性的影響方法:在CD133+細胞中轉染PinX1過表達質粒及相應的空載質粒,在CD133-細胞中轉染PinX1 siRNA及NC siRNA,將獲得細胞進行干細胞球誘導,用CCK-8觀察細胞的生長情況,通過Transwell法和劃線法觀察細胞遷移情況,通過Transwell法觀察細胞侵襲情況,通過流式細胞術檢測細胞凋亡情況。結果:1.PinXl質粒轉染CD133+鼻咽癌干細胞后,能明顯下調該腫瘤干細胞端粒酶活性,2.CD133+細胞中轉染PinX1過表達質粒后,細胞增殖速度低于空載組;CD133-細胞中轉染PinX1 siRNA后生長速度高于NC對照組。3.CD133+細胞轉染PinX1過表達質粒后,干細胞球半徑顯著變小,細胞成球能力減弱;而CD133-細胞中轉染siRNA后,成球半徑略有增加,細胞成球能力略有增強。4.CD133+細胞轉染PinX1過表達質粒后,細胞遷移、侵襲比例低于空載組;CD133-細胞轉染PinX1 siRNA后,細胞遷移、侵襲比例高于NC組。5.CD133+細胞轉染PinX1過表達質粒后,細胞凋亡比例高于空載組;CD133-細胞轉染PinX1 siRNA后,細胞凋亡比例低于NC組。結論:PinX1能夠通過下調腫瘤干細胞中端粒酶活性抑制CD133+鼻咽癌干細胞的增殖和遷移、增加CD133+細胞的凋亡。三.hTERT及TRFs在PinX1基因作用中的調控機制目的:進一步探討hTERT及TRFs在PinX1基因作用中調控作用機制方法:我們通過QPCR檢測hTERT的表達檢測CD133+細胞中端粒酶的活性,并通過QPCR的方法檢測PinX1的表達。在CD133+細胞中轉染PinX1過表達質粒及相應的空載質粒,在CD133-細胞中轉染PinX1 siRNA及NC siRNA,通過QPCR和WB方法觀察hTERT、PinX1、TRFs的表達。結果:1.CD133+細胞中PinX1表達量低于CD133-細胞,而hTERT的在CD133+細胞中的表達量顯著高于CD133-細胞,表明CD133+細胞端粒酶活性比CD133-強。2.我們成功構建了 PinX1過表達質粒和siRNA,并通過QPCR和WB驗證了 PinX1過表達質粒和siRNA在CNE2細胞中的成功影響了 PinX1含量。3.在CD133+細胞中轉染PinX1過表達質粒后hTERT表達量低于空載質粒,而TRF1高于空載組,在CD133-細胞中轉染PinX1 siRNA,hTERT表達高于NC組,TRF1低于NC組。但TRF2不受影響。結論:PinX1基因下調CD133+鼻咽癌腫瘤干細胞端粒酶活性,同時可上調TRF1表達,抑制該腫瘤干細胞的生物學活性,即PinX1基因可通過hTERT-TRF1發(fā)揮對鼻咽癌腫瘤干細胞抑制作用。
[Abstract]:Nasopharyngeal carcinoma is one of the southern region of China and Southeast Asian countries of high incidence of malignant tumor, recurrence and distant metastasis of the tumor is the important cause of failure and death. Recent studies have found that tumor stem cells is an important cause of malignant tumors including nasopharyngeal carcinoma, recurrence and drug resistance, has been in a variety of malignant tumors select the tumor stem cells. Tumor stem cells showed some characteristics, such as self replication ability, high telomerase activity, with CD133+, CD44+ surface markers for further study. The occurrence and development of tumor stem cells in tumor target provides a new field of cancer stem cells. The research group has carried out preliminary studies of telomerase inhibitor PinX1 gene targeting telomerase activity in nasopharyngeal carcinoma cells on nasopharyngeal carcinoma cells, on this basis, adopts PinX1 gene targeting nasopharyngeal carcinoma C D133+ cancer stem cells, on the regulation of telomerase activity of tumor stem cells, inhibition of tumor stem cells, and to further explore the telomeric repeat binding protein (TRF1 and TRF2) of the regulation mechanism, clarify its position in the function of PinX1 gene in nasopharyngeal carcinoma cells..CD133+ positive tumor stem cells Objective: To observe the characteristics of CD133+ as a stem cell marker sorting of CD133 positive cell proliferation and migration ability of human nasopharyngeal carcinoma cell line CNE2. Methods: CD133+ cells and CD133- cells by immunomagnetic separation method, which was verified by flow cytometry on separation efficiency. CD133+ cells and CD133- cells and then obtained stem cells in vitro and in vivo the tumor induced by stem cell marker SOX2 QPCR assay of CD133+ cells and CD133- cells, the expression of Nanog. Then to observe the growth of cells with CCK-8 two Condition, by Transwell method and scoring method to observe cell migration, cell invasion was measured by Transwell. Results: 1. CNE2 cell lines by without any labeled as negative control, the expression of the surface of conventional culture after 3 days of CD133 and CD44 detection showed that CD133+ and CD133- cells sorting.2. new separation of CD133+ and CD133- cells were inoculated into bFGF containing EGF, B27, insulin and serum free stem cells inducing culture medium, 14 days later, CD133+ tumor cells was significantly greater than the radius of the ball CD133-.3. cells was detected by QPCR CD133+ and CD133- cells in Nanog and SOX2, Nanog and SOX2 mRNA expression in CD133+ cells the amount of cell growth was significantly higher than that of CD133-.4. CD133+ and CD133- on cell sorting using CCK-8 method, CD133+ cell proliferation rate was significantly faster than CD133-.5. cells by Transwell assay or crossed experiment The migration and invasion of CD133+ cells and CD133- cells, CD133+ cells than CD133- cell migration, invasion ability of the.6. CD133+ nasopharyngeal carcinoma stem cells have a stronger tumorigenicity ability than CD133- cells. Conclusion: from nasopharyngeal carcinoma CNE2 cells successfully isolated CD133+ nasopharyngeal cancer stem cells using CD133 beads, and the expression of Nanog and SOX2 and telomerase activity in vitro proliferation, migration, ball, invasion and tumorigenicity of tumor stem cells characteristics of nasopharyngeal carcinoma cells was stronger than that of CD133- group. Two.PinX1 gene targeting telomerase inhibition of CD133+ nasopharyngeal carcinoma stem cells Objective: To study the effect of PinX1 gene of stem cell proliferation and migration activity of CD133+ in nasopharyngeal carcinoma: CD133+ cells were transfected with PinX1 expression plasmid and the corresponding plasmid, in CD133- cells transfected with PinX1 siRNA and NC siRNA, will receive the cells of stem cells induced by ball. CCK-8 observed the growth of cells by Transwell method and scoring method was used to observe the cell migration, observed by Transwell cell invasion, cell apoptosis by flow cytometry. Results: 1.PinXl CD133+ plasmid was transfected into nasopharyngeal carcinoma stem cells, can obviously lower the cancer stem cell telomerase activity in 2.CD133+ cells transfected with PinX1. The expression plasmid, cell proliferation rate is lower than the no-load group; CD133- cell growth rate higher than that of NC control group.3.CD133+ cells transfected with PinX1 expression plasmid was transfected into PinX1 siRNA after stem cell radius decreases, weakening the ability of cells into the ball; and in CD133- cells after transfection of siRNA, a radius slightly increased, cell migration and cell a ball slightly enhanced in.4.CD133+ cells transfected with PinX1 expression plasmid, after the invasion was lower than that of empty vector group; cell migration of CD133- cells transfected with PinX1 siRNA, after the invasion Hit ratio is higher than that of NC group.5.CD133+ cells transfected with PinX1 expression plasmid, the apoptosis ratio higher than the control group; CD133- cells transfected with PinX1 siRNA, the apoptosis ratio is lower than the NC group. Conclusion: PinX1 can through down-regulation of tumor stem cells in inhibiting telomerase activity in nasopharyngeal carcinoma CD133+ stem cell proliferation and migration, increase the apoptosis of CD133+ cells. The regulation mechanism of.HTERT three and TRFs in the function of PinX1 gene in hTERT and TRFs: to further investigate the effect of PinX1 gene in the regulation mechanism of methods: We used CD133+ cell telomerase detection to detect the expression of QPCR in hTERT activity, and detected by QPCR. The expression of PinX1 in CD133+ cells transfected with PinX1 expression plasmid and the corresponding the empty plasmid in CD133- cells transfected with PinX1 siRNA and NC siRNA, by QPCR and WB method to observe the hTERT, PinX1, TRFs expression. Results: 1.CD133+ cells In the expression of PinX1 was lower than that of CD133- cells, and its expression in CD133+ cells of hTERT was significantly higher than that of CD133- cells showed that the telomerase activity of CD133+ cells was stronger than that of CD133-.2., we successfully constructed PinX1 expression plasmid and siRNA, and through QPCR and WB to verify the expression of PinX1 and siRNA plasmid in CNE2 cells successfully influence the content of PinX1.3. in CD133+ cells transfected with PinX1 hTERT expression plasmid was lower than the empty plasmid, TRF1 higher than the control group, in CD133- cells transfected with PinX1 siRNA, the expression of hTERT was higher than group NC, TRF1 lower than NC group. But TRF2 is not affected. Conclusion: the down-regulation of PinX1 gene in nasopharyngeal carcinoma CD133+ cancer stem cell telomerase activity. At the same time can upregulate the expression of TRF1, inhibit the biological activity of tumor stem cells, PinX1 gene may play an inhibitory effect of stem cells of nasopharyngeal carcinoma by hTERT-TRF1.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.63

【參考文獻】

相關期刊論文 前10條

1 Maximilian Stahl;Tae Kon Kim;Amer M Zeidan;;Update on acute myeloid leukemia stem cells:New discoveries and therapeutic opportunities[J];World Journal of Stem Cells;2016年10期

2 錢東;丁小鳳;程菁菁;袁智勇;;靶向端粒 端粒酶的抗腫瘤治療研究進展[J];中國腫瘤臨床;2016年15期

3 李賢斌;吳海麗;;鼻咽癌患者長期生存期的危險因素分析[J];西南國防醫(yī)藥;2016年06期

4 趙盼;劉真;管靜芝;;端粒、端粒酶及人端粒酶反轉錄酶在腫瘤中的研究進展[J];腫瘤研究與臨床;2016年04期

5 營孫陽;熊加秀;麥洪旭;林佳佳;姜麗娜;程龍;葉棋濃;;端粒酶調控研究進展[J];遺傳;2016年04期

6 林馮杰;張瑜;;鼻咽癌患者血清乳酸脫氫酶檢查的臨床意義[J];中外醫(yī)學研究;2014年28期

7 付汐;盧國英;李志輝;梁杰昌;;腫瘤干細胞標志物ALDH1及CD133在鼻咽癌中的表達及其臨床意義[J];臨床與病理雜志;2014年03期

8 楊柯柯;申聰香;文忠;關小芳;賴肖芬;錢宇虹;;hTERTp/TK/pGL3靶向抑制端粒酶活性及其對鼻咽癌干細胞的殺傷作用[J];吉林大學學報(醫(yī)學版);2013年03期

9 Fei Ren;Wei-Qi Sheng;Xiang Du;;CD133:A cancer stem cells marker, is used in colorectal cancers[J];World Journal of Gastroenterology;2013年17期

10 易俊林;高黎;黃曉東;羅京偉;肖建平;李素艷;王凱;張世平;曲媛;徐國鎮(zhèn);;416例鼻咽癌調強放療遠期生存與影響因素分析[J];中華放射腫瘤學雜志;2012年03期

，

本文編號：1566636