喉癌干细胞耐药基因的初步筛选

发布时间：2018-03-05 06:53

本文选题：喉鳞状细胞癌　切入点：多药耐药　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:了解喉癌干细胞与喉癌化疗耐受之间的关系,并利用转录组测序技术筛选出干细胞与普通喉癌细胞之间的差异基因,从中找到多药耐药相关基因,初步分析喉癌干细胞多药耐药的机制。方法:1.用流式细胞仪检测Hep-2、TU177细胞中CD133、CD44的表达率,并使用RTCA法检测Hep-2、TU177细胞在顺铂、5-fu、紫杉醇三种化疗药物作用下的生长曲线,计算不同时间的IC50值,比较两种细胞系中CD133、CD44的表达率与化疗耐受之间的关系。2.用免疫磁珠分选法从Hep-2、TU177细胞中分选CD133+CD44+细胞,使用qPCR法检测CD133+CD44+细胞与亲本细胞中MDR1、MRP2基因的表达,验证干细胞与化疗耐受的关系。3.将分选出的CD133+CD44+、CD133-CD44-及正常的Hep-2、TU177细胞进行转录组测序,检测干细胞与普通喉癌细胞之间的差异基因,进行耐药相关基因筛选,初步分析干细胞耐药机制。结果:1.Hep-2细胞中CD133、CD44阳性率大于TU177细胞。并且,Hep-2细胞在三种药物作用下不同时间的IC50值均高于TU177细胞,说明同为喉癌细胞,Hep-2细胞相对于TU177细胞耐药性更强,同时与CD133、CD44阳性表达率呈正相关。2.Hep-2、TU177经免疫磁珠分选后CD133+CD44+细胞比例可高达80%-90%,这表明MACS富集CD133+CD44+细胞有效。Hep-2分选后MDR1较亲本细胞升高2.68倍,MRP2较亲本细胞升高1.68倍;TU177分选后MDR1较亲本细胞升高6.72倍,MRP2较亲本细胞升高42.69倍,可以看出干细胞富集后,多药耐药相关基因表达增加,说明干细胞的表达与喉癌化疗耐受呈正相关。3.通过转录组测序找出两个喉癌细胞系共有的差异基因12个,通过对它们进行功能及表达通路分析,发现FOS、JUN和NR4A1参与耐药相关的PI3K/AKT信号通路、MAPK信号通路及Wnt信号通路,可能和喉癌多药耐药有关,推测这三种基因可能是通过促进肿瘤细胞增殖、抑制凋亡发挥抗化疗的作用。结论:喉癌干细胞与多药耐药有关,并且可能是通过FOS、JUN和NR4A1基因通过促进细胞增殖、抑制凋亡发挥作用,但具体功能及作用机制仍需进一步实验验证。
[Abstract]:Objective: to investigate the relationship between laryngeal cancer stem cells and chemotherapy tolerance, and to screen the differentially expressed genes between stem cells and common laryngeal cancer cells by transcriptome sequencing, and to find multidrug resistance-related genes. The mechanism of multidrug resistance of laryngeal cancer stem cells was preliminarily analyzed. Methods: 1. The expression rate of CD133TU177 cells was detected by flow cytometry, and the growth curve of Hep-2TU177 cells treated with cisplatin 5-ful and paclitaxel was detected by RTCA assay. To calculate the IC50 value at different time, and to compare the relationship between the expression rate of CD133hCD44 and chemotherapeutic tolerance in the two cell lines. (2) the CD133 CD44 cells were isolated from Hep-2TU177 cells by immunomagnetic bead sorting method, and the expression of MDR1- MRP2 gene in CD133 CD44 cells and parental cells was detected by qPCR method. To verify the relationship between stem cells and chemotherapeutic tolerance. The selected CD133 CD44 CD133-CD44- and normal Hep-2nTU177 cells were sequenced to detect the differentially expressed genes between stem cells and normal laryngeal cancer cells, and to screen drug-resistance-related genes. Results 1. The CD133 CD44 positive rate of Hep-2 cells was higher than that of TU177 cells, and the IC50 value of Hep-2 cells at different time was higher than that of TU177 cells. The results showed that laryngeal carcinoma cell line Hep-2 was more resistant than TU177 cell. At the same time, there was a positive correlation between CD133 and CD44 expression. 2. The percentage of CD133 CD44 cells was as high as 80-90 after immunomagnetic bead sorting. This indicated that the percentage of CD133 CD44 cells enriched in CD133 CD44 cells was 2.68 times higher than that of parent cells after the separation of Hep-2. MRP2 was 1.68 times higher than that of parent cells, and the percentage of TU177 was 1.68 times higher than that of parent cells. MDR1 was 6.72 times higher than parent cells, and MRP2 was 42.69 times higher than parent cells. It can be seen that after stem cell enrichment, the expression of multidrug resistance-related genes increased, indicating that the expression of stem cells was positively correlated with chemotherapy tolerance of laryngeal carcinoma. The 12 genes shared by two laryngeal cancer cell lines were identified by transcriptome sequencing. Through the analysis of their function and expression pathway, it was found that FOSJUN and NR4A1 were involved in PI3K/AKT signaling pathway and Wnt signal pathway, which may be related to multidrug resistance in laryngeal carcinoma. Conclusion: laryngeal cancer stem cells are associated with multidrug resistance, and may promote cell proliferation through FOSJUN and NR4A1 genes, suggesting that these three genes may play an anti-chemotherapeutic role by promoting tumor cell proliferation and inhibition of apoptosis, conclusion: laryngeal cancer stem cells are associated with multidrug resistance, and may promote cell proliferation through FOSJUN and NR4A1 genes. Inhibition of apoptosis plays a role, but the specific function and mechanism still need further experimental verification.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.65

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