PinX1基因靶向端粒酶抑制鼻咽癌干细胞及其TRFs调控机制的研究

发布时间：2018-03-04 17:34

本文选题：鼻咽癌　切入点：肿瘤干细胞　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：鼻咽癌是我国南方地区及东南亚国家高发的恶性肿瘤之一,该类肿瘤的复发及远处转移是导致治疗失败及死亡的重要方式。近年来的研究发现,肿瘤干细胞是导致包括鼻咽癌在内的恶性肿瘤复发和耐药的重要原因,目前已从多种恶性肿瘤中分选出肿瘤干细胞。肿瘤干细胞表现出一定的自身特性,如自我复制能力强,高表达端粒酶活性,具有CD133+、CD44+表面标志物等。这为进一步探讨肿瘤干细胞在肿瘤发生发展及靶向肿瘤干细胞提供了新领域。本课题组曾开展了端粒酶抑制剂PinX1基因靶向鼻咽癌细胞端粒酶活性抑制鼻咽癌细胞的前期研究工作,在此基础上,拟采用PinX1基因靶向鼻咽癌CD133+肿瘤干细胞,探讨其对该肿瘤干细胞端粒酶活性下调、抑制肿瘤干细胞活性,并进一步探讨端粒重复序列结合蛋白(TRF1及TRF2)的调控作用机制,阐明其在PinX1基因作用中的地位。一.CD133+阳性鼻咽癌细胞具有肿瘤干细胞特征目的:观察以CD133+作为干细胞标志物分选CD133阳性细胞的增殖、迁移等能力。方法:鼻咽癌CNE2细胞通过免疫磁珠分选的方法获得CD133+细胞和CD133-细胞,并通过流式细胞术对分选的效率进行验证。然后将获得的CD133+细胞和CD133-细胞进行干细胞球诱导及裸鼠体内成瘤,通过QPCR法检测CD133+细胞和CD133-细胞中干细胞标志物SOX2、Nanog的表达。其次用CCK-8观察两株细胞的生长情况,通过Transwell法和划线法观察细胞迁移情况,通过Transwell法观察细胞侵袭情况。结果:1.以未加任何标记的CNE2细胞系为阴性对照,对常规培养3天的细胞表面的CD133和CD44的表达情况检测发现,CD133+和CD133-细胞成功分选。2.将新分选的CD133+和CD133-细胞,接种到含有bFGF、EGF、胰岛素和B27无血清干细胞诱导培养基中,14天后,CD133+肿瘤细胞球的半径显著大于CD133-细胞。3.通过QPCR检测CD133+和CD133-细胞中的Nanog和SOX2,Nanog和SOX2的mRNA在CD133+细胞中的表达量显著高于CD133-细胞。4.使用CCK-8法考察分选的CD133+和CD133-细胞的生长情况,发现CD133+细胞增殖速度显著快于CD133-细胞。5.通过划线实验或Transwell法检测CD133+和CD133-细胞的迁移和侵袭能力,发现CD133+细胞比CD133-细胞迁移、侵袭能力强。6.该种CD133+鼻咽癌干细胞比CD133-细胞群有较强的裸鼠成瘤能力。结论:采用CD133磁珠从鼻咽癌CNE2细胞中成功分选出CD133+鼻咽癌肿瘤干细胞,同时表达Nanog和SOX2及端粒酶活性,体外成球、增殖、迁移、侵袭能力及裸鼠成瘤等肿瘤干细胞特征明显强于CD133-鼻咽癌细胞群。二.PinX1基因靶向端粒酶抑制CD133+鼻咽癌干细胞目的:探讨PinX1基因对CD133+鼻咽癌干细胞增殖和迁移等活性的影响方法:在CD133+细胞中转染PinX1过表达质粒及相应的空载质粒,在CD133-细胞中转染PinX1 siRNA及NC siRNA,将获得细胞进行干细胞球诱导,用CCK-8观察细胞的生长情况,通过Transwell法和划线法观察细胞迁移情况,通过Transwell法观察细胞侵袭情况,通过流式细胞术检测细胞凋亡情况。结果:1.PinXl质粒转染CD133+鼻咽癌干细胞后,能明显下调该肿瘤干细胞端粒酶活性,2.CD133+细胞中转染PinX1过表达质粒后,细胞增殖速度低于空载组;CD133-细胞中转染PinX1 siRNA后生长速度高于NC对照组。3.CD133+细胞转染PinX1过表达质粒后,干细胞球半径显著变小,细胞成球能力减弱;而CD133-细胞中转染siRNA后,成球半径略有增加,细胞成球能力略有增强。4.CD133+细胞转染PinX1过表达质粒后,细胞迁移、侵袭比例低于空载组;CD133-细胞转染PinX1 siRNA后,细胞迁移、侵袭比例高于NC组。5.CD133+细胞转染PinX1过表达质粒后,细胞凋亡比例高于空载组;CD133-细胞转染PinX1 siRNA后,细胞凋亡比例低于NC组。结论:PinX1能够通过下调肿瘤干细胞中端粒酶活性抑制CD133+鼻咽癌干细胞的增殖和迁移、增加CD133+细胞的凋亡。三.hTERT及TRFs在PinX1基因作用中的调控机制目的:进一步探讨hTERT及TRFs在PinX1基因作用中调控作用机制方法:我们通过QPCR检测hTERT的表达检测CD133+细胞中端粒酶的活性,并通过QPCR的方法检测PinX1的表达。在CD133+细胞中转染PinX1过表达质粒及相应的空载质粒,在CD133-细胞中转染PinX1 siRNA及NC siRNA,通过QPCR和WB方法观察hTERT、PinX1、TRFs的表达。结果:1.CD133+细胞中PinX1表达量低于CD133-细胞,而hTERT的在CD133+细胞中的表达量显著高于CD133-细胞,表明CD133+细胞端粒酶活性比CD133-强。2.我们成功构建了 PinX1过表达质粒和siRNA,并通过QPCR和WB验证了 PinX1过表达质粒和siRNA在CNE2细胞中的成功影响了 PinX1含量。3.在CD133+细胞中转染PinX1过表达质粒后hTERT表达量低于空载质粒,而TRF1高于空载组,在CD133-细胞中转染PinX1 siRNA,hTERT表达高于NC组,TRF1低于NC组。但TRF2不受影响。结论:PinX1基因下调CD133+鼻咽癌肿瘤干细胞端粒酶活性,同时可上调TRF1表达,抑制该肿瘤干细胞的生物学活性,即PinX1基因可通过hTERT-TRF1发挥对鼻咽癌肿瘤干细胞抑制作用。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.63

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