N-氨甲酰谷氨酸、精氨酸及雌激素受体抑制剂对GT1-7细胞GnRH分泌及相关基因表达的影响

发布时间：2018-06-20 17:22

  本文选题：N-氨甲酰谷氨酸 + 精氨酸　； 参考：《甘肃农业大学》2017年硕士论文

【摘要】：N-氨甲酰谷氨酸(N-Carbamylglutamate,NCG)作为精氨酸(Arginine,Arg)内源激活剂被广泛应用于动物生产。研究发现其可显著缩短动物初情期、提高动物繁殖性能,但对于其作用机理的研究不多。本试验以小鼠下丘脑永生GnRH神经元离体细胞系GT1-7细胞为模型,研究NCG、Arg及雌激素受体抑制剂对GnRH合成与释放及GnRH相关基因表达的影响。试验一:探讨不同浓度NCG及Arg处理GT1-7细胞不同时间对GT1-7细胞增殖、GnRH分泌及GnRH分泌相关基因(GnRH、Kiss-1、GPR54、ERα、c-fos、nNOS)表达的影响。体外培养GT1-7细胞,处理组在培养液中分别添加NCG(10μM、100μM、1mM)或Arg(2mM、4mM),对照组培养液中分别添加NCG溶剂1M NaOH溶液或细胞培养液DMEM,处理12、24h后,收集细胞培养上清并进行细胞计数。使用ELISA试剂盒测定细胞培养上清GnRH浓度,并对GnRH分泌相关基因表达进行测定。结果表明,与对照组相比,各水平NCG及Arg处理对细胞增殖无影响(P0.05);NCG(10μM、100μM、1mM)处理12h和高浓度Arg(40mM)处理24h均能抑制GT1-7细胞Gn RH分泌(P0.05)。NCG(1m M)处理显著抑制GnRH、Kiss-1、GPR54、nNOS mRNA表达(P0.05),Arg(4m M)处理显著抑制GnRH、Kiss-1、GPR54、nNOS以及ERαmRNA表达(P0.05),可见NCG及Arg可能是通过下调Kiss-1、GPR54及nNOS基因表达抑制GnRH分泌。试验二:研究雌激素及雌激素受体抑制剂调控GT1-7细胞GnRH分泌的可能通路。以100pM雌激素及不同浓度雌激素受体抑制剂ICI182780(10pM、100pM、1nM)处理GT1-7细胞12h,分别研究其对GnRH分泌及相关基因表达的影响。结果显示,100pM雌激素显著促进GnRH分泌(P0.05),雌激素受体抑制剂能够抑制雌激素对GnRH的促进作用,1nM雌激素受体抑制剂显著抑制GnRH分泌(P0.05);雌激素受体抑制剂(1nM)显著抑制GnRH、ERα、Kiss-1以及nNOS mRNA表达(P0.05)。表明雌激素受体抑制剂是通过下调ERα、Kiss-1以及nNOS mRNA表达抑制GnRH分泌的。综上所述,NCG、Arg和雌激素受体抑制剂ICI182780均能抑制GnRH分泌,这种作用是通过下调ERα、Kiss-1以及nNOS mRNA实现的,提示除Kiss-1外,nNOS可能是GnRH合成分泌的信号通路上的调控因子。
[Abstract]:N-Carbamylglutamate (N-CarbamylglutamateNCGN) is widely used in animal production as arginine (Arg) endogenous activator. It is found that it can significantly shorten the period of initial estrus and improve the reproductive performance of animals, but there are few studies on the mechanism of its action. In this study, the effects of NCGG Arg and estrogen receptor inhibitor on the synthesis and release of GnRH and the expression of GnRH related genes were studied in a mouse hypothalamic immortalized GnRH neuron cell line GT1-7. Experiment 1: the effects of different concentrations of NCG and Arg on GnRH secretion of GT1-7 cells and the expression of GnRH secretion-related gene GnRH1 Kiss-1GPR54ER 伪 -fossil-nNOSs were studied in GT1-7 cells at different time. GT1-7 cells were cultured in vitro. The cells in the treatment group were treated with NCGN 10 渭 M ~ (10 渭 M) 100 渭 M ~ (-1) or Arg ~ (2) m ~ (2) M ~ (-1) M ~ (-1) respectively. The control group was treated with NCG solvent (1 M NaOH) or cell culture medium (DMEM) for 24 h. The supernatant of cell culture was collected and the cell count was carried out after being treated with NCG solvent (1 M NaOH) or cell culture medium (DMEM) for 24 h. The concentration of GnRH in the supernatant of cell culture was determined by Elisa kit, and the expression of GnRH secreting genes was determined. The results showed that, compared with the control group, All levels of NCG and Arg did not affect the proliferation of GT1-7 cells. (P0.05) treatment with 10 渭 MN (100 渭 M-1) for 12 h and at high concentration (40 mm) for 24 h inhibited GnRH secretion of GT1-7 cell line GnRH (P0.05N). NCGG (1m M) significantly inhibited the expression of GnRHHHP1GPR5NNOS mRNA and the expression of GnRHHKiss-1GPR5nNOS and ER 伪 mRNA in GT1-7 cell line (P0.05mM). NCG and NCG inhibited the expression of GnRHHS-1GPR5nNOS and ER 伪 mRNA of GT1-7 cell line (P0.05mM), both NCG and NCG inhibited the expression of GnRHHHGPR5nNOS mRNA and the expression of ER 伪 mRNA in GT1-7 cells. Arg may inhibit the secretion of GnRH by down-regulating the expression of GPR54 and nNOS. Experiment 2: the possible pathway of estrogen and estrogen receptor inhibitor regulating GnRH secretion in GT1-7 cells was studied. GT1-7 cells were treated with 100pM estrogen and different concentrations of estrogen receptor inhibitor ICI182780 (10pMNM) for 12 h to study the effects of 100pM estrogen and ICI182780 inhibitor on GnRH secretion and related gene expression. The results showed that 100pM estrogen significantly promoted the secretion of GnRH, estrogen receptor inhibitor inhibited the effect of estrogen on GnRH, estrogen receptor inhibitor inhibited the secretion of GnRH significantly, estrogen receptor inhibitor 1 nM significantly inhibited GnRH 伪 Kiss-1 and nNOS mRNA expression. These results suggest that estrogen receptor inhibitors inhibit the secretion of GnRH by down-regulating the expression of ER 伪 -Kiss-1 and nNOS mRNA. In conclusion, both NCGG and ICI182780 can inhibit the secretion of GnRH by down-regulating ER 伪 -Kiss-1 and nNOS mRNA, suggesting that nNOS may be a regulatory factor in the signal pathway of GnRH synthesis and secretion except Kiss-1.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816

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