喉癌多药耐药相关基因的筛查及中药逆转机制的研究

发布时间：2018-02-24 16:02

本文关键词： 喉癌多药耐药 Hep-2/v细胞系 miRNA 基因芯片基因表达谱粉防己碱川芎嗪　出处：《吉林大学》2010年博士论文　论文类型：学位论文

【摘要】： 喉癌是头颈部较为常见的恶性肿瘤之一,但在其综合治疗中,化疗的疗效欠佳,其主要原因是由于多药耐药性的产生。因此研究喉癌多药耐药相关基因的表达和调控机制以及筛选具有高效、低毒及多靶点性的中药逆转剂,对于提高喉癌的疗效,尤其是远期疗效,有重要的意义。本文以长春新碱为诱导剂,采用药物浓度梯度递增法体外诱导建立了喉癌多药耐药Hep-2/v细胞模型。在此基础上应用基因芯片技术研究喉癌耐药细胞株Hep-2/v与其亲本细胞Hep-2之间的多药耐药相关miRNA及mRNA表达谱,筛选出差异表达的miRNA及mRNA,并利用Target数据库初步预测了miRNA的靶基因。MTT、流式细胞术及RT-PCR、Western Blot等技术研究中药对喉癌细胞的逆转特性及多药耐药相关基因mRNA的表达变化。统计学方法对实验结果进行了分析。实验结果如下: (1)以药物浓度梯度递增法成功构建了喉癌多药耐药细胞模型。 (2)AFFX miRNA芯片筛查出差异表达的多药耐药相关miRNA共7个,其中表达上调2个,表达下调5个。 (3)全基因组寡核酸微阵列芯片筛查出差异表达的多药耐药相关基因共有605个,其中表达上调的基因270个,表达水平下调的基因335个。 (4)结合基因芯片筛查结果,初步预测miRNA-210的靶基因为HTRA1及NUPR1;miRNA-923的靶基因为NUPR1;miRNA-93的靶基因为RGS10。 (5)粉防己碱及川芎嗪对Hep-2/v细胞的逆转倍数分别为2.22倍和1.88倍。RT-PCR及Western Blot检测显示粉防己碱能够逆转Hep-2/v细胞内HTRA1、RGS10及MDR1基因的表达,但对NUPR1基因的表达无逆转作用。本实验对喉癌多药耐药相关基因的表达及调控机制进行了研究,表明miRNA可通过调控其靶基因的表达参与喉癌的多药耐药性。同时对中药的逆转机制进行了研究,并筛选出对喉癌耐药细胞有明显逆转作用的中药逆转剂,为进一步研究奠定了基础。
[Abstract]:Laryngeal carcinoma is one of the most common malignant tumors in head and neck. The main reason is the production of multidrug resistance. Therefore, to study the mechanism of expression and regulation of multidrug resistance-related genes in laryngeal cancer and to screen Chinese medicine reversal agents with high efficiency, low toxicity and multiple targets can improve the curative effect of laryngeal cancer. Especially the long-term effect, has the important significance. In this paper, vincristine was used as inducer. The multidrug resistant (Hep-2/v) cell model of laryngeal carcinoma was established by the method of drug concentration gradient increasing in vitro. The multidrug resistance-related miRNA and mRNA expression profiles between the drug resistant laryngeal cancer cell line Hep-2/v and their parent cell Hep-2 were studied by gene chip technique. The differentially expressed miRNA and mRNAs were screened, and the target gene. MTT, flow cytometry and RT-PCR- Western Blot of miRNA were preliminarily predicted by Target database to study the reverse characteristics of Chinese herbal medicine on laryngeal cancer cells and the expression of multidrug resistance-associated gene mRNA. The experimental results were analyzed by statistical method. The results are as follows:. 1) A multidrug resistant cell model of laryngeal carcinoma was successfully constructed by increasing drug concentration gradient. A total of 7 differentially expressed multidrug resistance-related miRNA were screened by AFFX miRNA microarray, of which 2 were up-regulated and 5 down-regulated. A total of 605 differentially expressed multidrug resistance-related genes were screened by genomic oligonucleic acid microarray, among which 270 genes were up-regulated and 335 genes were down-regulated. Combined with the results of gene microarray screening, it was preliminarily predicted that the target of miRNA-210 was HTRA1 and NUPR1 miRNA-923 because of NUPR1 miRNA-93. The reverse times of Tetrandrine and tetramethylpyrazine on Hep-2/v cells were 2.22 and 1.88 times respectively. RT-PCR and Western Blot analysis showed that Tetrandrine could reverse the expression of RGS10 and MDR1 genes in Hep-2/v cells, but had no reverse effect on NUPR1 gene expression. In this study, the expression and regulatory mechanism of multidrug resistance-associated genes in laryngeal carcinoma were studied. The results showed that miRNA could participate in the multidrug resistance of laryngeal carcinoma by regulating the expression of its target genes, and the reversal mechanism of traditional Chinese medicine was also studied. The Chinese medicine reversal agents which can reverse the drug resistance of laryngeal cancer cells were screened, which laid a foundation for further study.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.65;R285.5

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