【作者】 王东；

【导师】 马景鑑；

【作者基本信息】 天津医科大学 ， 外科学， 2011， 硕士

【摘要】 目的本实验通过应用小分子干扰RNA (siRNA)技术、化学合成反义核酸分子、构建质粒,干扰垂体瘤细胞的NPY表达及调节miR-15a/-16-1,为探讨NPY和miRNA-15a及miRNA-16-1在人垂体瘤细胞中的表达及其在垂体瘤发生、发展过程中可能存在的相互调节关系,探寻治疗垂体瘤的新靶点,拓展一条治疗垂体腺瘤的新方法。方法细胞实验部分：垂体瘤细胞分组：A组、空白对照,B组、NPY增强剂,C组、NPY拮抗剂,D组、NPY特异性siRNA转染垂体瘤细胞,E组、化学合成针对miRNA-15a反义核酸分子转染垂体瘤细胞,F组、携带miRNA-15a质粒转染垂体瘤细胞,G组、化学合成针对miRNA-16-1反义核酸分子转染垂体瘤细胞,H组、携带miRNA-16-1质粒转染垂体瘤细胞。应用神经肽Y拮抗剂、增强剂,同时设计NPY特异性小分子干扰RNA转染垂体瘤细胞,细胞培养48小时后,分别应用实时荧光定量PCR检测垂体瘤细胞中NPY mRNA水平和western blot方法检测细胞内蛋白表达量的变化,MTT法检测细胞增殖活性,流式细胞技术检测垂体瘤细胞周期和凋亡。通过化学合成反义核酸分子及质粒转染垂体瘤细胞,细胞培养48小时后,分别应用荧光定量PCR方法检测细胞核中niR-15a/-16-1 mRNA表达水平,MTT法检测细胞增殖活性,流式细胞技术测定垂体瘤细胞周期分布。干扰垂体瘤细胞中NPY,同时分别检测miR-15a/-16-1变化,通过上调及下调垂体瘤细胞中miR-15a/-16-1后,检测NPY的变化,并对NPY及miR-15a/-16-1进行相关性统计学分析,探讨二者之间可能存在的相关关系。垂体瘤组织实验部分：收集天津医科大学总医院神经外科垂体瘤病例30例,术前已通过临床、内分泌检验、影像学确诊,并经手术及术后病理证实。标本共分两组,不同病理类型垂体瘤组织的标本30例为A组,其中包括生长激素腺瘤、泌乳素腺瘤、促肾上腺皮质激素腺瘤、无功能垂体腺瘤。垂体组织(垂体瘤周边的残余正常垂体)30例为B组。分别进行NPY、miR-15a/-16-1的检测,比较正常垂体组织与垂体瘤组织中NPY及miR-15a/-16-1表达的差异。结果垂体瘤细胞实验结果：各实验组垂体瘤细胞在给予神经肽Y拮抗剂、增强剂、同时设计NPY特异性小分子干扰RNA转染垂体瘤细胞、应用质粒和反义核酸,上调、下调：niRNA-15a/-16-1,等相应处理48小时后,进行实时荧光定量PCR和Western blot、MTT、细胞生长曲线及流式细胞术等方法进行检测。给予上述不同因素处理48小时后,采用实时荧光定量PCR检测各组垂体瘤细胞中的NPY基因表达如下：C、D、F、H组分别与A组相比较,NPY mRNA基因表达降低,2-ΔΔCT值<1,其差异有统计学意义,分别P<0.05；B、E、G组分别与A组相比较,NPY mRNA基因表达升高,2-ΔΔCT值<1,其差异均有统计学意义,分别P＜0.05。Western blot检测垂体瘤细胞中NPY蛋白的表达,各实验组垂体瘤细胞中的NPY蛋白表达如下：C、D、F、H组分别与A组相比较NPY蛋白表达降低,其差异均有统计学意义,分别P＜0.05; B、E、G组与A组相比NPY蛋白表达升高,其差异均有统计学意义,分别P<0.05。细胞生长曲线图显示各组垂体瘤细胞的增殖能力：C、D、F、H组分别与A组相比较明显降低,其差异均有统计学意义,分别P<0.05；B、E、G组分别与A组相比细胞增殖能力增高,其差异均有统计学意义,分别P<0.05。流式细胞技术检测结果显示：C、D、F、H组分别与A组相比S期细胞明显降低,GO/G1期细胞数明显升高,各组间差异均有统计学意义,分别P<0.05,M期细胞无明显变化；B、E、G组分别与A组相比S期细胞明显升高,GO/G1期细胞数明显降低,各组间差异均有统计学意义,分别P<0.05,M期细胞无明显变化。垂体瘤组织实验结果：RT-PCR检测NPY、miR-15a/-16-1在各垂体瘤组织分组中的基因表达,NPY在垂体瘤组织中较正常垂体组织中呈高表达,垂体腺瘤中miRNA-15a和miRNA-16-1的表达与正常垂体组织相比明显下调。两组间差异有统计学意义,分别P<0.05。结论使用NPY拮抗剂及siRNA沉默NPY的基因表达后,垂体瘤细胞生长速度缓慢,且部分细胞出现凋亡；使用NPY增强剂后,增强了垂体瘤细胞内NPY表达,垂体瘤细胞增殖也明显。调节人垂体瘤细胞miR-15a/-16-1表达后,垂体瘤的细胞生长受到影响,下调miRNA-15a/-16-1表达,垂体瘤细胞增殖明显；上调miRNA-15a/-16-1的表达,垂体瘤细胞生长受到抑制。给予人垂体瘤细胞增强和拮抗NPY后,检测niRNA-15a/-16-1的表达,结果显示增强NPY表达时,miRNA-15a/-16-1表达水平下调；拮抗NPY表达时,miRNA-15a/-16-1水平上调,NPY与miRNA-15a/-16-1呈负相关关系。通过调节人垂体瘤细胞miRNA-15a/-16-1后,检测NPY的表达,当上调miRNA-15a/-16-1时,NPY表达降低；下调miRNA-15a/-16-1时,NPY表达增强。两者NPY与miRNA-15a/-16-1呈负相关关系。垂体瘤组织中检测NPY及miR-15a/-16-1,与正常垂体组织中表达不同。垂体瘤组织中NPY表达较正常垂体组织中NPY表达增强。垂体瘤组织中miR-15a/-16-1表达较正常垂体组织中miR-15a/-16-1表达降低。本实验为临床基因治疗垂体瘤疾病提供实验依据和新的治疗思路。更多还原

【Abstract】 Objective:This research aim to explore the application of RNAi, chemical synthesis of nucleic acid molecules and the construction of antisense plasmid to interfere NPY and miRNA-15a/-16-1 expression of pituitary tumor cells, and to explore the relationshipe between NPY and miRNA-15a/-16-1. Search for a new method for the diagnosis and treatment of pituitary adenomas.Methods:Pituitary tumor cell groups:A:blank control group; B:NPY enhancer group; C:NPY antagonist group; D:NPY Gene silencing group; E:antisense miRNA-15a group; F:miRNA-15a plasmid transfection group; G:antisense miRNA-16-1 group H:miRNA-16-1 plasmid transfection group. To apply NPY enhancer and NPY inhibitor, design specific siRNA transfection pituitary NPY tumor cell. After the cells being cultured for 48 hours, Gene expression of NPY mRNA of cells was tested by real time quantitative PCR, and protein expression of cells was tested by western blot. Proliferative activity was tested by MTT, cycle and apoptosis of pituitary tumor was tested by flow cytometry. Through in vitro chemical synthesis of nucleic acid molecules and the antisense plasmid pituitary tumor cells, after the cells being cultured for 48 hours, Gene expression of miRNA-15a/-16-1 of cells was tested by real time quantitative PCR, Proliferative activity was tested by MTT, cycle and apoptosis of pituitary tumor was tested by flow cytometry. After regulating miR-15a/-16-1. the change of miRNA-15a/-16-1 was tested respectively. And perform statistical analyse for NPY and miRNA-15a/-16-1. The mutual regulation between NPY and miR-15a/-16-1 was discussed simultaneouesly. Pituitary tumors tissue experiment:30 patients of pituitary tumor came from Neurosurgery in Tianjin Medical University General Hospital, were confirmed diagnosised through clinical, endocrine tests, imaging diagnosis before surgery and post operative pathological examination. Pituitary tumors tissues collected during operation were divided into two groups. A group include 30 cases of different pathological types of pituitary tumors. B group.include 30 cases of normal pituitary tissue. NPY and miRNA-15a/-16-1 was tested respectively of each group.Results:Compared with the A group, genes expression of NPY mRNA decreased in C, D, F, H group,2-ΔΔCT＜1, (P＜<0.05); genes expression of NPY mRNA increased in B, E, G group,2-ΔΔCT＜1, (P＜<0.05); Protein expression of NPY of the pituitary tumor cells was tested by Western blot in the following:compared with the A group, protein expression of NPY decreased in C, D, F, H group, (P＜0.05); protein expression of NPY increased in B, E, G group, (P＜0.05). Compared with the A group, pituitary tumor cell proliferation was significantly decreased in C, D, F, H group (P＜0.05);and increased in B, E, G group (P＜0.05), and S phase cells significantly decreased in C, D, F, H group, G0/G1 phase cells increased significantly, (P＜0.05), M phase cells did not change, but S phase cells significantly increased in B, E, G group, G0/G1 phase cells decreased significantly, (P＜0.05), M phase cells did not change.Compared with the normal pituitary tissues, the gene expression of NPY and miR-15a/-16-1 of pituitary tumors tissues Increased.Compared with the normal pituitary tissues, the expression of miRNA-15a/-16-1 of pituitary tumors tissues decreased (P＜0.05).Conclusion:Inhibiting or silencing NPY gene expression of Pituitary tumor cells. the growth of pituitary tumor cells of every group decreased, and some cells apoptotic. But enhancing NPY gene expression of pituitary tumor cell, it is proliferate evidently. Up regulating miR-15a/-16-1 expression of pituitary tumor cell, pituitary tumor cell growth was inhibited, but down regulating miRNA-15a/-16-1 expression of pituitary tumor cell, pituitary tumor cell growth was Increased.When enhancing the expression of NPY in pituitary tumors, the expression of miR-15a/-16-1 was decreased. when inhbiting the expression of NPY, the expression of miR-15a/-16-1 was increased. The relations between NPY and miRNA-15a/-16-1 shows negative correlation The expression NPY and miR-15a/-16-lin pituitary tumors, are different from it in the normal pituitary tissues. Compared with normal pituitary tissue, the expression of NPY in pituitary tumors increased. Compared with normal pituitary tissue, the expression of miR-15a/-16-1 in pituitary tumors decreased. this study provide experimental evidence and new treatment ideas for clinical gene therapy to the pituitary tumor.更多还原