本文選題：喉鱗狀細(xì)胞癌　切入點：多藥耐藥　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:了解喉癌干細(xì)胞與喉癌化療耐受之間的關(guān)系,并利用轉(zhuǎn)錄組測序技術(shù)篩選出干細(xì)胞與普通喉癌細(xì)胞之間的差異基因,從中找到多藥耐藥相關(guān)基因,初步分析喉癌干細(xì)胞多藥耐藥的機(jī)制。方法:1.用流式細(xì)胞儀檢測Hep-2、TU177細(xì)胞中CD133、CD44的表達(dá)率,并使用RTCA法檢測Hep-2、TU177細(xì)胞在順鉑、5-fu、紫杉醇三種化療藥物作用下的生長曲線,計算不同時間的IC50值,比較兩種細(xì)胞系中CD133、CD44的表達(dá)率與化療耐受之間的關(guān)系。2.用免疫磁珠分選法從Hep-2、TU177細(xì)胞中分選CD133+CD44+細(xì)胞,使用qPCR法檢測CD133+CD44+細(xì)胞與親本細(xì)胞中MDR1、MRP2基因的表達(dá),驗證干細(xì)胞與化療耐受的關(guān)系。3.將分選出的CD133+CD44+、CD133-CD44-及正常的Hep-2、TU177細(xì)胞進(jìn)行轉(zhuǎn)錄組測序,檢測干細(xì)胞與普通喉癌細(xì)胞之間的差異基因,進(jìn)行耐藥相關(guān)基因篩選,初步分析干細(xì)胞耐藥機(jī)制。結(jié)果:1.Hep-2細(xì)胞中CD133、CD44陽性率大于TU177細(xì)胞。并且,Hep-2細(xì)胞在三種藥物作用下不同時間的IC50值均高于TU177細(xì)胞,說明同為喉癌細(xì)胞,Hep-2細(xì)胞相對于TU177細(xì)胞耐藥性更強(qiáng),同時與CD133、CD44陽性表達(dá)率呈正相關(guān)。2.Hep-2、TU177經(jīng)免疫磁珠分選后CD133+CD44+細(xì)胞比例可高達(dá)80%-90%,這表明MACS富集CD133+CD44+細(xì)胞有效。Hep-2分選后MDR1較親本細(xì)胞升高2.68倍,MRP2較親本細(xì)胞升高1.68倍;TU177分選后MDR1較親本細(xì)胞升高6.72倍,MRP2較親本細(xì)胞升高42.69倍,可以看出干細(xì)胞富集后,多藥耐藥相關(guān)基因表達(dá)增加,說明干細(xì)胞的表達(dá)與喉癌化療耐受呈正相關(guān)。3.通過轉(zhuǎn)錄組測序找出兩個喉癌細(xì)胞系共有的差異基因12個,通過對它們進(jìn)行功能及表達(dá)通路分析,發(fā)現(xiàn)FOS、JUN和NR4A1參與耐藥相關(guān)的PI3K/AKT信號通路、MAPK信號通路及Wnt信號通路,可能和喉癌多藥耐藥有關(guān),推測這三種基因可能是通過促進(jìn)腫瘤細(xì)胞增殖、抑制凋亡發(fā)揮抗化療的作用。結(jié)論:喉癌干細(xì)胞與多藥耐藥有關(guān),并且可能是通過FOS、JUN和NR4A1基因通過促進(jìn)細(xì)胞增殖、抑制凋亡發(fā)揮作用,但具體功能及作用機(jī)制仍需進(jìn)一步實驗驗證。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.65

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