本文選題：N-氨甲酰谷氨酸 + 精氨酸　； 參考：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：N-氨甲酰谷氨酸(N-Carbamylglutamate,NCG)作為精氨酸(Arginine,Arg)內(nèi)源激活劑被廣泛應(yīng)用于動(dòng)物生產(chǎn)。研究發(fā)現(xiàn)其可顯著縮短動(dòng)物初情期、提高動(dòng)物繁殖性能,但對(duì)于其作用機(jī)理的研究不多。本試驗(yàn)以小鼠下丘腦永生GnRH神經(jīng)元離體細(xì)胞系GT1-7細(xì)胞為模型,研究NCG、Arg及雌激素受體抑制劑對(duì)GnRH合成與釋放及GnRH相關(guān)基因表達(dá)的影響。試驗(yàn)一:探討不同濃度NCG及Arg處理GT1-7細(xì)胞不同時(shí)間對(duì)GT1-7細(xì)胞增殖、GnRH分泌及GnRH分泌相關(guān)基因(GnRH、Kiss-1、GPR54、ERα、c-fos、nNOS)表達(dá)的影響。體外培養(yǎng)GT1-7細(xì)胞,處理組在培養(yǎng)液中分別添加NCG(10μM、100μM、1mM)或Arg(2mM、4mM),對(duì)照組培養(yǎng)液中分別添加NCG溶劑1M NaOH溶液或細(xì)胞培養(yǎng)液DMEM,處理12、24h后,收集細(xì)胞培養(yǎng)上清并進(jìn)行細(xì)胞計(jì)數(shù)。使用ELISA試劑盒測(cè)定細(xì)胞培養(yǎng)上清GnRH濃度,并對(duì)GnRH分泌相關(guān)基因表達(dá)進(jìn)行測(cè)定。結(jié)果表明,與對(duì)照組相比,各水平NCG及Arg處理對(duì)細(xì)胞增殖無(wú)影響(P0.05);NCG(10μM、100μM、1mM)處理12h和高濃度Arg(40mM)處理24h均能抑制GT1-7細(xì)胞Gn RH分泌(P0.05)。NCG(1m M)處理顯著抑制GnRH、Kiss-1、GPR54、nNOS mRNA表達(dá)(P0.05),Arg(4m M)處理顯著抑制GnRH、Kiss-1、GPR54、nNOS以及ERαmRNA表達(dá)(P0.05),可見(jiàn)NCG及Arg可能是通過(guò)下調(diào)Kiss-1、GPR54及nNOS基因表達(dá)抑制GnRH分泌。試驗(yàn)二:研究雌激素及雌激素受體抑制劑調(diào)控GT1-7細(xì)胞GnRH分泌的可能通路。以100pM雌激素及不同濃度雌激素受體抑制劑ICI182780(10pM、100pM、1nM)處理GT1-7細(xì)胞12h,分別研究其對(duì)GnRH分泌及相關(guān)基因表達(dá)的影響。結(jié)果顯示,100pM雌激素顯著促進(jìn)GnRH分泌(P0.05),雌激素受體抑制劑能夠抑制雌激素對(duì)GnRH的促進(jìn)作用,1nM雌激素受體抑制劑顯著抑制GnRH分泌(P0.05);雌激素受體抑制劑(1nM)顯著抑制GnRH、ERα、Kiss-1以及nNOS mRNA表達(dá)(P0.05)。表明雌激素受體抑制劑是通過(guò)下調(diào)ERα、Kiss-1以及nNOS mRNA表達(dá)抑制GnRH分泌的。綜上所述,NCG、Arg和雌激素受體抑制劑ICI182780均能抑制GnRH分泌,這種作用是通過(guò)下調(diào)ERα、Kiss-1以及nNOS mRNA實(shí)現(xiàn)的,提示除Kiss-1外,nNOS可能是GnRH合成分泌的信號(hào)通路上的調(diào)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 偽 -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 偽 mRNA in GT1-7 cell line (P0.05mM). NCG and NCG inhibited the expression of GnRHHS-1GPR5nNOS and ER 偽 mRNA of GT1-7 cell line (P0.05mM), both NCG and NCG inhibited the expression of GnRHHHGPR5nNOS mRNA and the expression of ER 偽 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 偽 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 偽 -Kiss-1 and nNOS mRNA. In conclusion, both NCGG and ICI182780 can inhibit the secretion of GnRH by down-regulating ER 偽 -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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S816

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本文編號(hào)：2045038