黑皮质素4型受体在绵羊下丘脑区域的定位
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摘要
黑皮质素4型受体(melanocortin 4 receptor,MC4R)作为下丘脑参与能量调控过程中的关键性受体,在动物能量代谢调节中被广泛关注。目前,关于该受体在绵羊下丘脑中分布区域的相关报道较少。针对试验过程中MC4R免疫荧光结果不稳定,分布区域不明显的特点,本研究在改进现有MC4R免疫荧光试验方法基础上,对MC4R在绵羊下丘脑的分布区域进行了观察。结果显示,分别经过4%多聚甲醛长时间固定(6 d),Triton X-100(pH≈8.68)长时间通透(2 d),以及3%过氧化氢和0.2 mol/L甘氨酸+0.3%SDS+20%甲醇的预处理后,可以获得稳定且饱满的MC4R免疫荧光结果。MC4R主要分布在绵羊下丘脑的室旁核(arcuate nucleus,ARC)区域,但在弓状核区域并无类似分布。
Melancholic 4 receptor(MC4 R) is a key receptor involved in energy regulation in the hypothalamus and is widely concerned in bodily energy metabolism.At present,the distribution of this receptor in sheep hypothalamus is unclear.Owing to MC4 R immunofluorescences were unstable and there is not sufficient evidence to prove the distribution area.Here,based on the improvement of the MC4 R immunofluorescence assay,we identified the distribution of MC4 R in the hypothalamus of sheep,through using,long-time external fixation(6 days),TritonX-100(pH≈8.68) for 4 days,and progressing the pretreatment with 3% hydrogen peroxide and 0.2 mol/L glycine + 0.3% SDS + 20% methanol.MC4 R immunofluorescence results can be stably obtained,and is mainly distributed in the paraventricular nucleus of hypothalamus(PVH) of sheep and does not show a concentrated distribution in the arcuate nucleus(ARC) neucleus.
Melancholic 4 receptor(MC4 R) is a key receptor involved in energy regulation in the hypothalamus and is widely concerned in bodily energy metabolism.At present,the distribution of this receptor in sheep hypothalamus is unclear.Owing to MC4 R immunofluorescences were unstable and there is not sufficient evidence to prove the distribution area.Here,based on the improvement of the MC4 R immunofluorescence assay,we identified the distribution of MC4 R in the hypothalamus of sheep,through using,long-time external fixation(6 days),TritonX-100(pH≈8.68) for 4 days,and progressing the pretreatment with 3% hydrogen peroxide and 0.2 mol/L glycine + 0.3% SDS + 20% methanol.MC4 R immunofluorescence results can be stably obtained,and is mainly distributed in the paraventricular nucleus of hypothalamus(PVH) of sheep and does not show a concentrated distribution in the arcuate nucleus(ARC) neucleus.
引文
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[12] RODRIGUEZ E M,BLAZQUEZ J L,GUERRA M.The design of barriers in the hypothalamus allows the median eminence and the arcuate nucleus to enjoy private milieus:the former opens to the portal blood and the latter to the cerebrospinal fluid[J].Peptides,2010,31(4):757-776.
[2] BALTHASAR N,DALGAARD L T,LEE C E,et al.Divergence of melanocortin pathways in the control of food intake and energy expenditure[J].Cell,2005,123(3):493-505.
[3] HUSZAR D,LYNCH C A,FAIRCHILD-HUNTRESS V,et al.Targeted disruption of the melanocortin-4 receptor results in obesity in mice[J].Cell,1997,88(1):131-141.
[4] KISHI T,ASCHKENASI C J,LEE C E,et al.Expression of melanocortin 4 receptor mRNA in the central nervous system of the rat[J].J Comp Neurol,2003,457(3):213-235.
[5] KASK A,SCHIOTH H B.Tonic inhibition of food intake during inactive phase is reversed by the injection of the melanocortin receptor antagonist into the paraventricular nucleus of the hypothalamus and central amygdala of the rat[J].Brain Res,2000,887(2):460-464.
[6] SHAH B P,VONG L,OLSON D P,et al.MC4R-expressing glutamatergic neurons in the paraventricular hypothalamus regulate feeding and are synaptically connected to the parabrachial nucleus[J].Proc Natl Acad Sci,2014,111(36):13193-13198.
[7] TOLSON K P,GEMELLI T,GAUTRON L,et al.Postnatal Sim1 deficiency causes hyperphagic obesity and reduced MC4R and oxytocin expression[J].J Neurosci,2010,30(10):3803-3812.
[8] SHETTY A K,RAO M S,HATTIANGADY B.Behavior of hippocampal stem/progenitor cells following grafting into the injured aged hippocampus[J].J Neurosci Res,2008,86(14):3062-3074.
[9] YAMASAKI T R,BLURTON-JONES M,MORRISSETTE D A,et al.Neural stem cells improve memory in an inducible mouse model of neuronal loss[J].J Neurosci,2007,27(44):11925-11933.
[10] SPITZER N,SAMMONS G S,PRICE E M.Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies[J].J Neurosci Methods,2011,197(1):48-55.
[11] BURKE L K,DARWISH T,CAVANAUGH A R,et al.mTORC1 in AGRP neurons integrates exteroceptive and interoceptive food-related cues in the modulation of adaptive energy expenditure in mice[J].Elife,2017,6:e22848.
[12] RODRIGUEZ E M,BLAZQUEZ J L,GUERRA M.The design of barriers in the hypothalamus allows the median eminence and the arcuate nucleus to enjoy private milieus:the former opens to the portal blood and the latter to the cerebrospinal fluid[J].Peptides,2010,31(4):757-776.