TXR1通过调控TSP1、CD47及DUSP1参与鼻咽癌紫杉醇耐药的研究

发布时间：2018-12-17 12:12

【摘要】：研究目的：紫杉醇是一种光谱抗肿瘤药,广泛应用于多种恶性肿瘤,但是近年来紫杉醇耐药现象的出现使化疗效果大打折扣,紫杉醇耐药机制的研究成为研究热点,但是诸多机制均不能系统阐明其耐药机制。近年来发现了TXR1(紫杉醇耐药基因1)为肿瘤研究提供新思路,但其功能及相关细胞内信号通路仍不明确,本研究旨在前期研究基础上进一步探究TXR1基因的表达在鼻咽癌肿瘤细胞紫杉醇耐药的功能。研究方法：1.以人鼻咽癌紫杉醇耐药细胞株CNE-l/Taxol为研究对象,用本课题组先前筛选的TXR1-22转染该株细胞系,用RT-PCR及Western Blot方法确定TXR1基因是否被沉默； 2.采用MTT试验检测耐药细胞TXR1基因被沉默后,肿瘤耐药能否逆转； 3.TXR1-22转染永生化正常鼻咽粘膜细胞NP69及鼻咽癌亲本细胞CN E-1/亲,采用MTT法检测NP69及CNE-1/亲生长是否受抑制。 4.通过生物信息学技术筛选TSP1、 CD47、 DUSP1三种基因,在转录水平验证其与TXR1的关系研究结果：1.RT-PCR及Western Blot结果显示耐药细胞株CNE-l/Taxol、中TXR1的表达明显受抑制； 2.通过MTT法检测了TXR1基因受抑制后肿瘤耐药被部分逆转,且有统计学意义； 3.MTT法发现转染前后的NP69及CNE-1/亲生长未受明显抑制。 4.RT-PCR结果显示TXR1基因沉默后TSP1、 CD47、 DUSP1三种基因转录水平表达明显上调,具有统计学意义研究结论：1.通过RNA干扰技术同样可以实现头颈肿瘤相关基因的沉默； 2.在鼻咽癌耐药细胞系的紫杉醇耐药基因TXR1被沉默后,其耐药性明显降低； 3.TXR1基因可以作为逆转紫杉醇耐药的可能性靶点,并且其对非靶向细胞无明显毒性,为之后临床研制新药及安全性研究提供了体外实验依据； 4.TSP1、 CD47、 DUSP1为TXR1诱发紫杉醇耐药的可能信号因子
[Abstract]:Objective: paclitaxel is a kind of spectral antitumor drug, which is widely used in many malignant tumors. However, the appearance of paclitaxel resistance in recent years has greatly reduced the effect of chemotherapy, and the mechanism of paclitaxel resistance has become a hot research topic. However, many mechanisms can not systematically elucidate the mechanism of drug resistance. In recent years, TXR1 (paclitaxel resistance gene 1) has been found to provide a new idea for tumor research, but its function and related intracellular signaling pathway are still unclear. The purpose of this study was to further explore the role of TXR1 gene expression in paclitaxel resistance in nasopharyngeal carcinoma (NPC) tumor cells. Methods: 1. The taxol resistant human nasopharyngeal carcinoma (NPC) cell line CNE-l/Taxol was transfected with previously screened TXR1-22. RT-PCR and Western Blot methods were used to determine whether the TXR1 gene was silenced. 2. MTT test was used to detect whether the drug resistance of tumor cells could be reversed after the TXR1 gene was silenced. 3.TXR1-22 was transfected into immortalized normal nasopharyngeal mucosal cells (NP69) and nasopharyngeal carcinoma parent cells (CN E-1 / parent). The growth inhibition of NP69 and CNE-1/ was detected by MTT assay. 4. Three genes of TSP1, CD47, DUSP1 were screened by bioinformatics, and the relationship between them and TXR1 was verified at the transcriptional level. The results of 1.RT-PCR and Western Blot showed that the expression of TXR1 in CNE-l/Taxol, was significantly inhibited. 2. MTT assay was used to detect the partial reversal of tumor resistance after TXR1 gene was inhibited, and 3.MTT method showed that the NP69 and CNE-1/ parental growth were not significantly inhibited before and after transfection. 4.RT-PCR results showed that the transcription level of three TSP1, CD47, DUSP1 genes was significantly up-regulated after TXR1 gene silencing, which was statistically significant: 1. The silencing of head and neck tumor-related genes can also be achieved by RNA interference technique. 2. The drug resistance of paclitaxel resistant gene TXR1 in nasopharyngeal carcinoma cell line was significantly decreased after the silencing of paclitaxel resistance gene. 3.TXR1 gene can be used as a possible target to reverse paclitaxel resistance, and it has no obvious toxicity to non-target cells, which provides in vitro experimental basis for clinical research of new drugs and safety. 4. TSP1, CD47, DUSP1 is the possible signal factor of paclitaxel resistance induced by TXR1.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.63

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