大鼠下丘脑腹内侧核—伏隔核nesfatin-1通路构成及胃功能调控研究

发布时间：2018-01-13 02:34

本文关键词：大鼠下丘脑腹内侧核—伏隔核nesfatin-1通路构成及胃功能调控研究　出处：《青岛大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:Nesfatin-1是新发现的一种能够抑制摄食和胃运动的新型脑肠肽类激素,广泛分布于中枢神经系统和胃肠道等外周器官。“饱食中枢”下丘脑腹内侧核是调控摄食的重要核团;伏隔核参与获取食物和进食冲动的调控,两个核团中均有nesfatin-1的表达。目前下丘脑腹内侧核和伏隔核中nesfatin-1对摄食和胃酸分泌、胃运动、胃排空等胃功能的调控作用仍不清楚。本实验将研究集中于下丘脑腹内侧核和伏隔核:①观察胃扩张对下丘脑腹内侧核内nesfatin-1免疫阳性神经元活性的影响;研究伏隔核→下丘脑腹内侧核nesfatin-1神经通路的构成;②探讨nesfatin-1对下丘脑腹内侧核内胃扩张敏感神经元电生理活性的影响,进一步观察伏隔核对下丘脑腹内侧核内胃扩张敏感神经元的调控作用,阐明伏隔核→下丘脑腹内侧核nesfatin-1功能通路的构成;③探讨下丘脑腹内侧核nesfatin-1对摄食、胃酸分泌、胃运动和胃排空的调控作用,以及伏隔核→下丘脑腹内侧核nesfatin-1通路对腹内侧核功能的影响,补充和完善摄食和胃功能调控的中枢机制。方法:1.采用荧光免疫组织化学方法,观察nesfatin-1在下丘脑腹内侧核表达及胃扩张对nesfatin-1神经元的激活作用;采用荧光金逆行追踪结合荧光免疫组化技术,观察伏隔核→下丘脑腹内侧核nesfatin-1神经通路的构成。2.采用细胞表面微量注射药物、脑核团电刺激、神经元胞外记录等方法,观察nesfatin-1、黑皮质素3/4受体拮抗剂SHU9119以及电刺激伏隔核对下丘脑腹内侧核胃扩张敏感神经元电生理活动的影响。3.采用核团微量注射药物、脑核团电刺激、摄食量记录、胃酸滴定测量、在体胃运动和胃排空酚红定量测定等方法,观察下丘脑腹内侧核微量注射nesfatin-1以及电刺激伏隔核对清醒大鼠摄食量、胃酸分泌、胃运动和胃排空的影响。结果:1.大鼠下丘脑腹内侧核内存在nesfatin-1免疫阳性神经元;胃扩张刺激腹内侧核nesfatin-1免疫阳性神经元中c-Fos表达增多,细胞活性增强。2.下丘脑腹内侧核中注射荧光金可逆行至伏隔核,且存在荧光金与nesfatin-1免疫阳性神经元共染,提示伏隔核与下丘脑腹内侧核间存在向下丘脑腹内侧核投射的神经纤维,且部分投射纤维由nesfatin-1免疫阳性神经元发出延伸至下丘脑腹内侧核。3.腹内侧核共记录到64个胃扩张敏感神经元,其中35个神经元(54.70%)为胃扩张兴奋神经元(GD-E),29个神经元(45.3%)为胃扩张抑制神经元(GD-I),提示下丘脑腹内侧核可接受来自胃部的传入信息。4.腹内侧核微量注射nesfatin-1,24.14%的GD-I神经元兴奋,放电频率从2.45 ± 0.37 Hz升高到3.82 ±0.56 Hz(p0.05);22.86%的GD-E神经元被抑制,放电频率从 2.77 ±0.39 降低到 1.67± 0.37 Hz(p0.05)。Nesfatin-1 对胃扩张敏感神经元的影响可被腹内侧核预先注射黑皮质素3/4受体拮抗剂SHU9119部分阻断(p0.05)。5.电刺激伏隔核,腹内侧核胃扩张敏感神经元中的59.26%GD-I神经元兴奋,放电频率从2.33 ± 0.57 Hz升高到3.45 ± 0.34 Hz(p0.05);此作用可被腹内侧核预先注射nesfatin-1抗体部分阻断(p0.05);电刺激伏隔核,53.06%的GD-E神经元兴奋,放电频率从2.74 ±0.71 Hz增加到5.08 ±1.15 Hz(p0.05);腹内侧核中预先注射nesfatin-1抗体可进一步增强GD-E神经元兴奋性,放电频率升至6.81 ± 1.13 Hz(p0.05)。腹内侧核单独注射nesfatin-1抗体或生理盐水GD神经元放电活动无显著改变(p0.05)。6.腹内侧核注射低剂量nesfatin-1,大鼠2h内摄食量显著减少,注射高剂量的nesfatin-1,摄食量的减少可持续约4-6h;腹内侧核注射nesfatin-1,由皮下注射2-脱氧-D-葡萄糖引起的胃酸分泌也受到显著抑制(48.63 ± 10.21%);注射nesfatin-1后约4 min,大鼠胃运动开始下降,15 min达最低点,下降(35.2 ± 8.9%,p0.05);腹内侧核注射nesfatin-1,大鼠胃排空显著减弱,排空率降至50.15 ±15.6%(p0.05);腹内侧核预先给予黑皮质素3/4受体拮抗剂SHU9119,可部分阻断腹内侧核注射nesfatin-1对摄食和胃功能的抑制效应(p0.05)。7.20μA低电流刺激大鼠伏隔核,大鼠摄食无显著改变(P0.05),但采用50μA高电流刺激伏隔核,2-6h间大鼠的累积摄食量均低于假电刺激组(p0.05),且2-脱氧-D-葡萄糖诱导的胃酸分泌增多也受到显著抑制(9.16 ±1.53降至5.52± 0.88 μEq/15min,p0.05);50 μA高电流刺激伏隔核,大鼠胃运动MI%值降至假电刺激对照组的72.25 ±9.82%(p0.05);胃排空率也减少为假电刺激对照组的68.36 ±8.79%(0.05)。下丘脑腹内侧核预先注射nesfatin-1抗体,可消弱电刺激伏隔核抑制大鼠摄食、胃酸分泌和胃动力效应(P0.05)。下丘脑腹内侧核单独注射nesfatin-1抗体或NS对大鼠摄食量、胃酸分泌量和胃动力无显著影响(0.05)。结论:1.下丘脑腹内侧核有nesfatin-1免疫阳性神经元表达,且胃扩张可改变部分nesfatin-1神经元活性;2.伏隔核内nesfatin-1免疫阳性神经元可发出神经纤维投射至下丘脑腹内侧核,形成伏隔核-下丘脑腹内侧核nesfatin-1神经通路;3.下丘脑腹内侧核可接受胃来源的信号,nesfatin-1可影响这些胃来源信号的传入,并可通过改变传出冲动参与食物摄取、胃酸分泌、胃运动以及冒排空等功能的调控;4.Nesfatin-1在伏隔核-下丘脑腹内侧核通路可调控大鼠摄食、胃酸分泌、胃运动以及胃排空,这提示,伏隔核和下丘脑腹内侧核间有nesfatin-1功能通路存在。
[Abstract]:Objective: Nesfatin-1 is capable of inhibiting food intake and gastric motility model of brain gut peptide hormone newly discovered, widely distributed in the central nervous system and peripheral organs such as gastrointestinal tract. "Satiety center" in the ventromedial hypothalamic nucleus is an important nucleus in the regulation of food intake; the nucleus accumbens in obtaining food and eating impulse control, expression nesfatin-1 has two nuclei. The ventromedial hypothalamic nucleus and nucleus accumbens nesfatin-1 on food intake and gastric acid secretion, gastric motility, gastric emptying and gastric function regulation remains unclear. This study will focus on the ventromedial hypothalamic nucleus and nucleus accumbens: To observe gastric dilatation effect on the ventral hypothalamus the medial nucleus of nesfatin-1 immunoreactive neuronal activity; V septum, a study of the ventromedial hypothalamic nucleus nesfatin-1 neural pathway; the effects of nesfatin-1 on hypothalamic ventromedial nucleus in the stomach by the expansion of Aman Chang's sense of God Effect of electrophysiological activity, further observe the regulation of nucleus accumbens ventromedial hypothalamic nucleus on gastric distension sensitive neurons, which clarify the nucleus accumbens, ventromedial hypothalamus nesfatin-1 pathway; to explore the ventromedial hypothalamic nucleus nesfatin-1 on gastric acid secretion, feeding, regulation of gastric motility and gastric emptying, and V septal nucleus, ventromedial hypothalamic nucleus nesfatin-1 pathway influence on the function of the ventromedial nucleus, supplement and perfect the central mechanism of feeding and regulation of gastric function. Methods: 1. using immunohistochemistry method to observe the effect of nesfatin-1 on activation of nesfatin-1 neurons in the ventromedial hypothalamic nucleus expression and gastric dilatation; using Fluorogold retrograde tracing combined with fluorescence immunohistochemistry observation of nucleus accumbens, ventromedial hypothalamic nucleus nesfatin-1 neural pathway composed of.2. cell surface by microinjection of drugs, brain nuclei Group of electrical stimulation, extracellular neural recording method, observation of nesfatin-1,.3. of melanocortin 3/4 receptor antagonist SHU9119 and stimulation of nucleus accumbens ventromedial hypothalamic nucleus on gastric distension sensitive neurons electrophysiological activity by nucleus microinjection of drugs, brain stimulation, food intake records, acid titration measurements in vivo gastric motility and gastric emptying by quantitative analysis, observation of the ventromedial hypothalamic nucleus microinjection of nesfatin-1 and stimulation of nucleus accumbens in conscious rats food intake, gastric acid secretion, affect gastric motility and gastric emptying. Results: 1. rat ventromedial hypothalamic nucleus nesfatin-1 immunoreactive neurons; gastric distension stimulation increased the expression of c-Fos the ventromedial nucleus of nesfatin-1 immunoreactive neurons, enhance.2. cell activity in the ventromedial hypothalamic nucleus injection of fluorogold reversible to the nucleus accumbens, and the presence of fluorescent gold and nesfatin-1 Immunoreactive neurons were stained, suggesting that the nucleus accumbens and the ventromedial hypothalamic nucleus between the hypothalamic ventromedial nucleus projecting to the nerve fibers, and the portion of the projection fibers by nesfatin-1 immunoreactive neurons by extending to the ventromedial hypothalamic ventromedial nucleus of 64.3. neurons were recorded in the expansion of Zhang Mingan's stomach, of which 35 neurons (54.70%) for gastric dilatation (GD-E) neurons, 29 neurons (45.3%) neurons for dilatation of the stomach (GD-I), suggesting that the ventromedial hypothalamic nucleus can receive afferent signals from the ventromedial nucleus of.4. microinjection of nesfatin-1,24.14% from stomach GD-I neurons, discharge frequency from 2.45 + 0.37 Hz + 0.56 Hz (up to 3.82 P0.05 22.86%); the GD-E neurons were inhibited, the discharge frequency is reduced to 1.67 from 2.77 + 0.39 + 0.37 Hz (P0.05) effect of.Nesfatin-1 on gastric distension sensitive neurons in the ventromedial nucleus can be pre injection Melanocortin 3/4 receptor antagonist SHU9119 partially blocked (P0.05).5. electrical stimulation of nucleus accumbens, ventral medial nucleus of gastric distension sensitive neurons in 59.26%GD-I neurons, discharge frequency from 2.33 + 0.57 to 3.45 + 0.34 Hz Hz increased (P0.05); this effect could be the ventromedial nucleus of pre injection of nesfatin-1 antibody partially blocked (P0.05); electrical stimulation of nucleus accumbens neurons, 53.06% GD-E, discharge frequency from 2.74 + 0.71 to 5.08 + 1.15 Hz Hz (P0.05); ventromedial nucleus in the pre injection of nesfatin-1 antibody can further enhance the excitability of GD-E neurons, discharge frequency to 6.81 + 1.13 Hz (P0.05). No significant change in ventromedialis nuclear single injection of nesfatin-1 antibody or normal saline GD neurons (P0.05) low dose nesfatin-1.6. nucleus ventromedialis injection in 2h of rats significantly reduced food intake, high dose of nesfatin-1 injection, the reduction of food intake to renew 4-6h; ventromedial nucleus nesfatin-1 injection induced by subcutaneous injection of 2- deoxy -D- glucose acid secretion was also significantly inhibited (48.63 + 10.21%); nesfatin-1 after injection of about 4 min, gastric motility of rats began to decline 15, the lowest level at Min, decreased (35.2 + 8.9%, P0.05); ventromedial nucleus injection of nesfatin-1. Gastric emptying in rats decreased and the emptying rate dropped to 50.15 + 15.6% (P0.05); ventromedial pre administration of melanocortin 3/4 receptor antagonist SHU9119 partially blocked the inhibitory effect of nucleus ventromedialis injection of nesfatin-1 on food intake and gastric function (P0.05).7.20 A low current stimulation of rat nucleus accumbens, feeding rats no significant change (P0.05), but by 50 A high current stimulation of the nucleus accumbens, 2-6h rats cumulative food intake were lower than the sham stimulation group (P0.05), and 2- deoxy -D- glucose induced increase in gastric acid secretion was significantly inhibited (9.16 + 1.53 to 5.52 + 0.88 Eq/15min, P0.05); 50 A high current stimulation of nucleus accumbens, gastric motility in rats MI% decreased to sham stimulation control group 72.25 + 9.82% (P0.05); gastric emptying rate also reduces the false stimulation control group 68.36 + 8.79% (0.05). The ventromedial hypothalamic nucleus pre injection of nesfatin-1 antibody. Electrical stimulation of nucleus accumbens can weaken the inhibition of feeding rats, gastric acid secretion and gastric motility effect (P0.05). The ventromedial hypothalamic nucleus in single injection of nesfatin-1 antibody or NS on food intake of rats, gastric acid secretion and gastric motility had no significant effect (0.05). Conclusion: 1. the ventromedial hypothalamic nucleus expression of nesfatin-1 immunoreactive neurons and, the stomach expansion can change part of the activity of nesfatin-1 neurons; 2. immunoreactive neurons in the nucleus accumbens nesfatin-1 can send nerve fibers to the ventromedial hypothalamic nucleus, forming the nucleus accumbens - ventromedial hypothalamus nesfatin-1 neural pathway; 3. ventromedial hypothalamic nucleus Acceptable signal gastric sources, nesfatin-1 can influence the introduction of the source of gastric signal, and by changing the efferent impulses in food intake, gastric acid secretion, gastric motility and emptying function of risk control; 4.Nesfatin-1 in the nucleus accumbens - ventromedial hypothalamic pathway regulation of rat feeding, gastric motility and gastric acid secretion. Emptying, which suggests that the nucleus accumbens and the ventromedial hypothalamic nucleus there exist the nesfatin-1 pathway.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R33

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