神经肽Y和miRNA在垂体瘤细胞中的作用及相关性
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。
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.
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。
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.
引文
[1]Ogasawara H, Aso H, Nagai Y, et al. Presence of neuropeptide Y in somatotrophs of cattle. Domestic Animal Endocrinology,2008,35 (2):274-280.
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[3]甄自刚,马景鑑,阎学江.垂体腺瘤患者血浆神经肽Y与垂体激素水平相关性研究.中华神经外科杂志,2005,21(10)：612-615.
[4]郭常利,马景鑑,杨树源,等.人体血浆和垂体肿瘤组织神经肽Y含量及其临床意义.中国临床神经科学,2001,9(2)：148—150.
[5]陈来照,马景鑑.神经肽Y及其受体在实验大鼠垂体腺瘤中的表达.中华神经外科杂志,2007,23(12)：949-952.
[6]Bottoni A,Piccin D.miR-15a and miR-16-1 down-regulation in pituitary adenomas.Cell Physiol,2005,204(1):280-285.
[7]Fire A,Xu S,Mello cl.et al.Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans.Nature,1998:391(6669):806-11.
[8]Randolph L,Suzanne E,Hao Yin,et al.The functional consequence of RhoA knockdown by RNA interference in rat cerebral arteries.American Journal Of Physiology-Heart and Circulatory Physiology,2007,293(1),440-447.
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[12]Treip CS.The regression of oestradiol-induced pituitary tumours in the rat. pathol,1983,141(1)29-40.
[13]Kamath J, Yarbrough G, Prange A.The thyrotropin-releasing hormone (TRH)-immune system homeostatic hypothesis.Pharmacology & Therapeutics.2009,121, (1): 20-28.
[14]Diaz-Cabiale Z, Parrado C, Narvaez M, et al.Galanin receptor/Neuropeptide Y receptor interactions in the dorsal raphe nucleus of the rat. Neuropharmacology, 2011,105,(9):17-20.
[15]Shimizu H, Ohtani K, Kato Y, et al. Withdrawal of estrogen increases hypothalamic neuropeptide Y (NPY) mRNA expression in ovariectomized obese rat. Neurosci Lett,1997,227(2):143.
[16]Korner M, Waser B, Thalmann GN, et al.High expression of NPY receptors in the human tissue. Mol Cell Endocrinol,2011,337(1-2):62-70.
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[20]Funabashi T, Kleopoulos SP, Brooks PJ, et al. Changes in estrogenic regulation of estrogen receptor alpha mRNA and progesterone receptor mRNA in the female rat hypothalamus during aging:an in situ hybridization study. Neurosci Res,2000,38(1):85-92.
[21]Conde-Sieira M, Agulleiro MJ, Aguilar AJ,et al. Effect of different glycaemic conditions on gene expression of neuropeptides involved in control of food intake in rainbow trout; interaction with stress.Exp Biol,2010,213(22):3858-65.
[22]Nilsen KE,Walker MC,Cock HR.Characterization of the tetanus toxin model of refractory focal neocortical epilepsy in the rat. Epilepsia,2009,46(2):179-187.
[23]Stanic D, Mulder J, Watanabe M,et al.Characterization of NPY Y2 receptor protein expression in the mouse brain. Ⅱ. Coexistence with NPY, the Yl receptor, and other neurotransmitter-related molecules.Comparative Neurology,2011, 519(7):1219-57.
[24]Grouzmann E, Deruaz JP, Gomez F, et al. Immunolocalization of neuropeptide Y in human pituitary tumours.Regul Pept,1998,75-76:89-92.
[25]Chabot JG,Enjalbert A,Pelletier G,et al.evidence for a direct action of neuropeptideY in the rat pituitary gland.Neuroendocrinology,1988,47(6):511-517.
[26]Chen J, Zhang Y,.Shen P. A protein kinase C activity localzed to neuropepide Y-like neurons mediates ethanol intoxication in drosophila melanogaster. Neuroscience,2008,156(1):42-47.
[27]Hideki Ogasawara, Hisashi Aso, Yasuhiro Nagai, et al. Presence of neuropeptide Y in somatotrophs of cattle Domestic Animal.Endocrinology,2008,35 274-280.
[28]Freeman ME. Neuropeptide Y:a unique member of the constellation of gonadotropin-releasing hormones. Endocrinology,1993,133(6):2411-2412.
[29]Veenstra JA. Neuropeptide evolution:Neurohormones and neuropeptides predicted from the genomes of Capitella teleta and Helobdella robusta. Gen Comp Endocrinol,2011,(2):160-75.
[30]Chiba A. Neuropeptide Y-immunoreactive (NPY-ir) structures in the brain of the gar Lepisosteus oculatus (Lepisosteiformes, Osteichthyes) with special regard to their anatomical relations to gonadotropin-releasing hormone (GnRH)-ir structures in the hypothalamus and the terminal nerve. Gen Comp Endocrinol, 2005,142(3):336-346.
[31]Veenstra JA. Neuropeptide evolution:Neurohormones and neuropeptides predicted from the genomes of Capitella teleta and Helobdella robusta. Gen Comp Endocrinol,2011,(2):160-75.
[32]Fahrenkrug J, Hannibal J.Localisation of the neuropeptide PACAP and its receptors in the rat parathyroid and thyroid glands. Gen Comp Endocrinol, 2011, 171(1):105-13.
[33]Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function. Cell, 2004,116(2):281-297.
[34]Gao FB.Posttranscriptional control of neuronal development by microRNA networks.Trends Neurosci.2008,31(1):20-26.
[35]Filipowicz W,Bhattacharyya SN.Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight?Nat Rev Genet.2008,9(2):102-114.
[36]Cowland JB,Hother C,Gronbaek K.MicroRNAs and cancer.APMIS.2007,1 15(10):1090-110.
[37]CimminoA,Calin GA, Fabbri M, et a.l miR-15 and miR-16 induce apoptosis by targetingBCL2. ProcNatlAcad SciUSA,2005,102(39):13944-13949.
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[39]Calin GA, Sevignani C, Dumitru CD, et al.Human microRNA gene sate frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA,2004,101:2999-3004.
[40]RI Aqeilan,GA Calin and CM Croce:miR-15a and miR-16-1 in cancer:discovery, functionand future perspectives. Cell Death and Differentiation,2010,17: 215-220.
[41].Ramon Martinez, Manel Esteller:The DNA methylome of glioblastoma multiforme.Neurobiology of Disease,2010,10:1016.
[42].Lagerstrom MC, Fredriksson R, Bjarnadottir TK, et al. Origin of the prolactin-releasing hormone (PRLH) receptors:evidence of coevolution between PRLH and a redundant neuropeptide Y receptor during vertebrate evolution. Genomics,2005,85(6):688-703.
[43]Hernadi I,Pirger Z,Kiss T,et al.The presence and distribution of pituitary adenylate cyclase activating polypeptide and its receptor in the snail Helix pomatia.Neuroscience,2008,155:387-402.
[44]Corthals SL, Jongen-Lavrencic M, de Knegt Y,et al.Micro-RNA-15a and micro-RNA-16 expression and chromosome 13 deletions in multiple myeloma. Leuk Res,2010,34(5):677-81.
[45]Fernandez R,Aguilar E,Tena-Sempere M,et al.Effects of polypeptide YY(3-36) upon luteinzing hormone-releasing hormone and gonadotropin secretion in prepubertal rats:in vivo and in vitro studies.Endocrinology,2005,146:1403-1410.
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[1]Tatemoto K, Carlquist M, Mutt V, et al. Neuropeptide Y-a novel brain peptide with structural similarities to peptide Y and pancreatic polypeptide[J]. Nature,1982, 296(15):659-660.
[2]Balasubramaniam A.Clinical potentials of neuropeptide Y family of hormones [J].Am J Surg,2002,183(4):430-434.
[3]Tu C,Zhao D,Lin X,el al.Levels of neuropeptide-Y in the plasm a and skin tissue tluids of patients with vitiligo [J]. Dermatol Sci,2001,27 (3):178-182.
[4]Parker RM, Herzog H,Depaulis A, et al.Comparison of Y-receptor subtype expression in the rat hippocampus[J]. Regul Pept,2008,75-76(25):109-115.
[5]Nilsen KE,Walker MC,Cock HR.Characterization of the tetanus toxin model of refractory focalneocorticalepilepsy in the rat [J]. Epilepsia,2005,46(2):179-187.
[6]Tasan RO,Nguyen NK,Weger S,et al.The central and basolateral amygdala are critical sites of neuropep tide Y/Y2 receptor mediated regulation of anxiety and depression.Neurosei,2010,30 (18):628262-628290.
[7]Lee NJ,Herzog H.NPY regulation of bone remode ling [J].Neuropeptides,2009,12 (6):457-463.
[8]Yang Kaiping,Guan Haiyan,Arany E,et al. Neuropeptide Y is produced in visceral adipose tissue and promotes proliferation of adipocyte precursor cells via the Yl receptor [J].FASEB,2008,22 (7):2452-2464.
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[3]甄自刚,马景鑑,阎学江.垂体腺瘤患者血浆神经肽Y与垂体激素水平相关性研究.中华神经外科杂志,2005,21(10)：612-615.
[4]郭常利,马景鑑,杨树源,等.人体血浆和垂体肿瘤组织神经肽Y含量及其临床意义.中国临床神经科学,2001,9(2)：148—150.
[5]陈来照,马景鑑.神经肽Y及其受体在实验大鼠垂体腺瘤中的表达.中华神经外科杂志,2007,23(12)：949-952.
[6]Bottoni A,Piccin D.miR-15a and miR-16-1 down-regulation in pituitary adenomas.Cell Physiol,2005,204(1):280-285.
[7]Fire A,Xu S,Mello cl.et al.Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans.Nature,1998:391(6669):806-11.
[8]Randolph L,Suzanne E,Hao Yin,et al.The functional consequence of RhoA knockdown by RNA interference in rat cerebral arteries.American Journal Of Physiology-Heart and Circulatory Physiology,2007,293(1),440-447.
[9]卢圣栋.现代分子生物学实验技术[M].第二版.北京：中国协和医科大学出版社.1999,p88.
[10]Tse C, Capeau J. Real time PCR methodology for quantification of nucleic acids. Ann Biol Clin (Paris),2003,61(3):279-293.
[11]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods Diego, Calif.2001; 25:402-408.
[12]Treip CS.The regression of oestradiol-induced pituitary tumours in the rat. pathol,1983,141(1)29-40.
[13]Kamath J, Yarbrough G, Prange A.The thyrotropin-releasing hormone (TRH)-immune system homeostatic hypothesis.Pharmacology & Therapeutics.2009,121, (1): 20-28.
[14]Diaz-Cabiale Z, Parrado C, Narvaez M, et al.Galanin receptor/Neuropeptide Y receptor interactions in the dorsal raphe nucleus of the rat. Neuropharmacology, 2011,105,(9):17-20.
[15]Shimizu H, Ohtani K, Kato Y, et al. Withdrawal of estrogen increases hypothalamic neuropeptide Y (NPY) mRNA expression in ovariectomized obese rat. Neurosci Lett,1997,227(2):143.
[16]Korner M, Waser B, Thalmann GN, et al.High expression of NPY receptors in the human tissue. Mol Cell Endocrinol,2011,337(1-2):62-70.
[17]Schwartz MW, Sipols AJ, Marks JL, et al. Inhibition of hypothalamic neuropeptide Y gene expression by insulin. Endocrinology,1992,130(6): 3608-3618.
[18]Okabe T, Sato C, Sakamoto A,et al. Changes in neuropeptide Y gene expression in the spinal cord of chronic constrictive injury model rats after electroconvulsive stimulation. Biomed Res,2010,31(5):287-92.
[19]Dumont Y, Martel JC, Fournier A, et al. Neuropeptide Y and neuropeptide Y receptor sub-types in brain and peripheral tissues. Prog Neurobiol,1992,38(2): 125-167.
[20]Funabashi T, Kleopoulos SP, Brooks PJ, et al. Changes in estrogenic regulation of estrogen receptor alpha mRNA and progesterone receptor mRNA in the female rat hypothalamus during aging:an in situ hybridization study. Neurosci Res,2000,38(1):85-92.
[21]Conde-Sieira M, Agulleiro MJ, Aguilar AJ,et al. Effect of different glycaemic conditions on gene expression of neuropeptides involved in control of food intake in rainbow trout; interaction with stress.Exp Biol,2010,213(22):3858-65.
[22]Nilsen KE,Walker MC,Cock HR.Characterization of the tetanus toxin model of refractory focal neocortical epilepsy in the rat. Epilepsia,2009,46(2):179-187.
[23]Stanic D, Mulder J, Watanabe M,et al.Characterization of NPY Y2 receptor protein expression in the mouse brain. Ⅱ. Coexistence with NPY, the Yl receptor, and other neurotransmitter-related molecules.Comparative Neurology,2011, 519(7):1219-57.
[24]Grouzmann E, Deruaz JP, Gomez F, et al. Immunolocalization of neuropeptide Y in human pituitary tumours.Regul Pept,1998,75-76:89-92.
[25]Chabot JG,Enjalbert A,Pelletier G,et al.evidence for a direct action of neuropeptideY in the rat pituitary gland.Neuroendocrinology,1988,47(6):511-517.
[26]Chen J, Zhang Y,.Shen P. A protein kinase C activity localzed to neuropepide Y-like neurons mediates ethanol intoxication in drosophila melanogaster. Neuroscience,2008,156(1):42-47.
[27]Hideki Ogasawara, Hisashi Aso, Yasuhiro Nagai, et al. Presence of neuropeptide Y in somatotrophs of cattle Domestic Animal.Endocrinology,2008,35 274-280.
[28]Freeman ME. Neuropeptide Y:a unique member of the constellation of gonadotropin-releasing hormones. Endocrinology,1993,133(6):2411-2412.
[29]Veenstra JA. Neuropeptide evolution:Neurohormones and neuropeptides predicted from the genomes of Capitella teleta and Helobdella robusta. Gen Comp Endocrinol,2011,(2):160-75.
[30]Chiba A. Neuropeptide Y-immunoreactive (NPY-ir) structures in the brain of the gar Lepisosteus oculatus (Lepisosteiformes, Osteichthyes) with special regard to their anatomical relations to gonadotropin-releasing hormone (GnRH)-ir structures in the hypothalamus and the terminal nerve. Gen Comp Endocrinol, 2005,142(3):336-346.
[31]Veenstra JA. Neuropeptide evolution:Neurohormones and neuropeptides predicted from the genomes of Capitella teleta and Helobdella robusta. Gen Comp Endocrinol,2011,(2):160-75.
[32]Fahrenkrug J, Hannibal J.Localisation of the neuropeptide PACAP and its receptors in the rat parathyroid and thyroid glands. Gen Comp Endocrinol, 2011, 171(1):105-13.
[33]Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function. Cell, 2004,116(2):281-297.
[34]Gao FB.Posttranscriptional control of neuronal development by microRNA networks.Trends Neurosci.2008,31(1):20-26.
[35]Filipowicz W,Bhattacharyya SN.Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight?Nat Rev Genet.2008,9(2):102-114.
[36]Cowland JB,Hother C,Gronbaek K.MicroRNAs and cancer.APMIS.2007,1 15(10):1090-110.
[37]CimminoA,Calin GA, Fabbri M, et a.l miR-15 and miR-16 induce apoptosis by targetingBCL2. ProcNatlAcad SciUSA,2005,102(39):13944-13949.
[38]Roccaro AM, Sacco A, Thompson B, et al.MicroRNAs 15a and 16 regulate tumor proliferation in multiple myeloma. Blood,2009,113(26):6669-80.
[39]Calin GA, Sevignani C, Dumitru CD, et al.Human microRNA gene sate frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA,2004,101:2999-3004.
[40]RI Aqeilan,GA Calin and CM Croce:miR-15a and miR-16-1 in cancer:discovery, functionand future perspectives. Cell Death and Differentiation,2010,17: 215-220.
[41].Ramon Martinez, Manel Esteller:The DNA methylome of glioblastoma multiforme.Neurobiology of Disease,2010,10:1016.
[42].Lagerstrom MC, Fredriksson R, Bjarnadottir TK, et al. Origin of the prolactin-releasing hormone (PRLH) receptors:evidence of coevolution between PRLH and a redundant neuropeptide Y receptor during vertebrate evolution. Genomics,2005,85(6):688-703.
[43]Hernadi I,Pirger Z,Kiss T,et al.The presence and distribution of pituitary adenylate cyclase activating polypeptide and its receptor in the snail Helix pomatia.Neuroscience,2008,155:387-402.
[44]Corthals SL, Jongen-Lavrencic M, de Knegt Y,et al.Micro-RNA-15a and micro-RNA-16 expression and chromosome 13 deletions in multiple myeloma. Leuk Res,2010,34(5):677-81.
[45]Fernandez R,Aguilar E,Tena-Sempere M,et al.Effects of polypeptide YY(3-36) upon luteinzing hormone-releasing hormone and gonadotropin secretion in prepubertal rats:in vivo and in vitro studies.Endocrinology,2005,146:1403-1410.
[46]Shalak V, Kaminska M,Mitnacht—Kraus R,et al. The EMAPII cytokine is released from the mammalian muftisynthetase complex afte cleavage of its p43/proEMAPII component. J Biol Chem,2010,276:23769—23776.
[1]Tatemoto K, Carlquist M, Mutt V, et al. Neuropeptide Y-a novel brain peptide with structural similarities to peptide Y and pancreatic polypeptide[J]. Nature,1982, 296(15):659-660.
[2]Balasubramaniam A.Clinical potentials of neuropeptide Y family of hormones [J].Am J Surg,2002,183(4):430-434.
[3]Tu C,Zhao D,Lin X,el al.Levels of neuropeptide-Y in the plasm a and skin tissue tluids of patients with vitiligo [J]. Dermatol Sci,2001,27 (3):178-182.
[4]Parker RM, Herzog H,Depaulis A, et al.Comparison of Y-receptor subtype expression in the rat hippocampus[J]. Regul Pept,2008,75-76(25):109-115.
[5]Nilsen KE,Walker MC,Cock HR.Characterization of the tetanus toxin model of refractory focalneocorticalepilepsy in the rat [J]. Epilepsia,2005,46(2):179-187.
[6]Tasan RO,Nguyen NK,Weger S,et al.The central and basolateral amygdala are critical sites of neuropep tide Y/Y2 receptor mediated regulation of anxiety and depression.Neurosei,2010,30 (18):628262-628290.
[7]Lee NJ,Herzog H.NPY regulation of bone remode ling [J].Neuropeptides,2009,12 (6):457-463.
[8]Yang Kaiping,Guan Haiyan,Arany E,et al. Neuropeptide Y is produced in visceral adipose tissue and promotes proliferation of adipocyte precursor cells via the Yl receptor [J].FASEB,2008,22 (7):2452-2464.
[9]Mullins DE, Guzzi M, Xia L, et al. Pharmacological characterization of the cloned neuropeptideYy(6) receptor[J]. Eur J Pharmacol,2000,395(2):87-93.
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