中枢注射CCK对大鼠摄食行为以及相关神经元功能的影响

发布时间：2018-05-01 01:27

本文选题：胆囊收缩素 + 下丘脑　；参考：《浙江大学》2007年博士论文

【摘要】： 胆囊收缩素(cholecystokinin，CCK)是胃肠激素，主要分泌于十二指肠和空肠，除了在外周发挥多种调节胃肠功能的作用，也见于脑内，在脑内作为神经传递介质发挥作用，因此，CCK也是一种脑肠肽。1973年，Gibbs实验室首次发现大鼠腹腔注射CCK-8导致大鼠摄食量明显减小，反应呈剂量依赖性，外周循环中CCK抑制食欲的作用已在不同种类的动物和人的研究中证实，外周注射CCK抑制食欲的作用是短暂的，使进食量减少但代偿性的进食次数增多，重复或长期应用CCK并不使体重减轻，CCK对食欲的作用通过CCK-1受体介导。然而，脑内CCK对摄食的作用及其机理尚不清楚。Blevin et al曾对大鼠脑内多部位直接注射CCK-8，诱导出短时间的摄食抑制，并证明作用部位主要为DMH和Arc。OLETF大鼠(Otsuka Long-Evans Tokushima fatty rats)的CCK-1受体基因先天性缺失，表现为贪食，逐渐转为肥胖，最后出现2型糖尿病，对于OLETF大鼠的研究提示，CCK无论在外周还是在脑内的作用均对大鼠摄食控制中起抑制作用，进一步对于OLETF大鼠的研究发现，成年OLETF大鼠DMHNPY(神经肽Y)明显增高，CCK1受体缺失导致DMH NPY基因表达失调可能对OLETF大鼠肥胖和糖尿病的形成起作用。随之，免疫组化显示大鼠DMH的NPY神经元上具有CCK-1受体，提示下丘脑内CCK可能通过CCK-1受体介导作用于DMH的NPY神经元起作用，推测“DMH CCK—NPY”信号通路可能参与摄食控制。尽管如此，其确切的机理仍不明。本研究通过观察DMH核团内注射CCK对大鼠摄食的影响以及时间过程特点，检测DMH NPY基因、Arc NPY基因、Arc POMC基因和PVN CRF基因表达以及观察DMH核团内注射CCK后丘脑和脑干神经元激活的部位，探讨DMH注射CCK抑制摄食的特征以及相应的神经元活动的特征。方法：以成年Sprague-Dawley雄性大鼠(250～300g)为材料，置于20℃恒温环境，12h∶12h(明∶暗)的灯光周期中分笼饲养。 1．清醒大鼠摄食实验：12只大鼠，实验组n=7，对照组n=6。大鼠麻醉后，按Paxinos-Watson图谱在下丘脑背内侧区(dorsal medial hypothalamus，DMH)插入套管，坐标为前囟后3.1 mm，旁开0.4 mm，颅骨表面下8.1 mm。一周后大鼠恢复良好，可进入实验。大鼠于手术恢复期时使其建立饮食规律：关灯前2小时禁食，随之22小时予以常规颗粒饲料。动物可自由饮水。实验组DMH核团微量注射CCK-8 500nmol／0.3ul，对照组DMH核团微量注射人工脑脊液(aCSF)0.3ul，人工脑脊液组成：(147mM Na~+，2.7mM K~+，1.2mMC Ca~(++)，0.85mMMg~(++) and 153.8mMCl~-)，注射于关灯前实施，注射后立即关灯，并给予食物，然后分别于注射后30min、1h、2h、4h、22h记录进食量。7天后，给予第二次DMH核团注射CCK-8和aCSF，实验组对照组交叉，注射时间和剂量以及进食量的记录同第一次。 2．下丘脑NPY，CRF和POMC基因表达分析：摄食实验后，13只大鼠重新分组，实验组n=7，对照组n=6。DMH核团内注射CCK和aCSF步骤同前，注射后关灯但继续禁食，3小时后断头取脑，急速置于-80℃保存，待组织学检查套管位置和检测DMH NPY、Arc NPY、Arc POMC和PVN CRF的mRNA表达。大鼠前脑中部作14μm系列冠状切片贴于玻片上，以4％多聚甲醛固定。挑取PVN、DMH、Arc的切片，应用RNA原位核酸杂交，分别检测DMH NPY mRNA、Arc NPY mRNA和POMC mRNA、PVN CRF mRNA的表达。~(35)S-cRNA探针以POMC、NPY和CRF cDNA为模板体外转录。切片以醋酸酐处理，酒精脱水后加杂交缓冲液(含~(35)S-cRNA 6~*10~8 cpm／μl)55℃过夜，杂交后清洗、脱水、干燥、曝光显影。放射自显影图象以NIH Scion Image软件进行定量分析。 3．下丘脑和小脑c-FOS表达：28只雄性大鼠检测DMH注射CCK后下丘脑和小脑与摄食相关神经核中c-FOS细胞。大鼠分二组，每组14只，清醒状态下分别注射CCK-8和aCSF，剂量和方法同前。注射后立即禁食，90分钟后以戊巴比妥麻醉后，经心脏以PBS和4％多聚甲醛灌流，然后取脑置于4％多聚甲醛／25％蔗糖溶液中浸泡，4℃保存1-2天，在前脑中部和后脑作40μm系列冠状切片，包括下列部位：室旁核(paraventrical nucleus PVN)、视上核(supraoptic nucleus SON)、视交叉上suprachiasmatic neucleus SCh)、后交叉区retrochiasmatic area(RCh)、外侧下丘脑(lateral hypothalamus LH)、丘脑背内侧核(dorsomedial hypothalamic hypothalamic nucleus DMH)、丘脑腹内侧核(ventromedial hypothalamic nucleus VMH)、弓状核(arcuate nucleusArc)、后脑杏仁核(amygadala nucleus，CeA)、最后区(area postrema AP)、孤束核(nucleus of the solitary tract NTS)。c-FOS以免疫组织化学法检测，采用漂浮法，0.3％过氧化氢1h，羊血清包被1h，1∶10，000兔c-FOS抗体(Oncogene Science，SanDiego，CA)孵化过夜，生物素-羊抗兔血清1h，ABC复合试剂(Elite Vectastain Kit，Vector Labs，Burlingame，CA)1h，以二氨基联苯(DAB)显色。终止反应后，将组织切片贴到玻片上，干燥，酒精脱水后，显微镜下观察切片内套管轨迹和c-FOS表达，套管位置不正确者弃去。c-FOS阳性细胞定量以自动图象分析软件处理(IpLab，Scanalytics，Fairfax，VA)，除DMH分别计数注射侧和注射对侧，其余部位均计数脑二侧，在每个部位均读取2～3张切片，取平均值，神经解剖学定位参照Paxinos-Watson图谱。 4．统计学检验：结果采用均数±标准误，均数t检验统计学处理，P＜0.05说明有统计学意义。结果 1．DMH注射CCK对大鼠摄食的影响 500nmol CCK-8直接注射到DMH后，大鼠在注射后的0.5h，1h，2h，4h，22h内的累计摄食量均较对照组显著减少，比较0～0.5h，0.5-1h，1～2h，2～4h，4～22h各个时间段的摄食量，发现在0.5h内和2～4h时间段实验组较对照组摄食量显著减少，其余无显著差异。 2．DMH CCK注射后神经肽表达的变化实验组动物在DMH的NPYmRNA表达较对照组降低27％，Arc的NPYmRNA表达较对照组降低24％；实验组PVN的CRFmRNA表达增高38％；而POMCmRNA在Arc表达二组未见显著差异。 3．DMH CCK注射对下丘脑和小脑尾部与摄食控制相关部位c-FOS蛋白激活的影响实验组在下丘脑DMH、Arc、PVN、SCh、RCh上c-Fos表达明显高于对照组，在SON、LH、VMH、ME上二组无显著差异，在脑干NTS、AP上未见c-Fos表达。注射侧的DMH无论实验组还是对照组可见非常强烈的c-FOS表达，但二者比较未见显著性差异，比较二组注射对侧DMH的c-FOS阳性细胞数，实验组明显较对照组增高。ArC上c-FOS激活较对照组明显，，主要见于内侧Arc。PVN上c-FOS表达显著增加主要见于小细胞PVN上。结论 DMH CCK具有摄食抑制作用，与外周CCK作用短暂不同，DMH CCK作用持续时间较长；DMH CCK作用于NPY神经元抑制NPY基因表达而发挥摄食抑制的作用，并上调PVN CRF基因表达，DMH CCK抑制Arc NPY基因表达，但不影响Arc POMC基因表达；DMH CCK增加可激活下丘多个神经元如PVN，Arc，cDMH，RCh，SCh等，与外周CCK不同，DMH CCK不引起NTS和AP的神经元活动。上述结果表明，DMH CCK-NPY信号系统在控制摄食和能量代谢平衡中发挥重要作用，下丘脑多条依赖PVN CRF和Arc NPY的神经信号途径介导其作用。
[Abstract]:Cholecystokinin (CCK) is a gastrointestinal hormone, which is mainly secreted in the duodenum and jejunum. It is also seen in the brain as a neurotransmitter in the brain, as well as in the brain as a neurotransmitter. Therefore, CCK is also a kind of brain gut peptide.1973. Gibbs laboratory was first found to be intraperitoneal injection of CCK-8 guide in rats. The intake of food in rats was significantly reduced and the response was dose-dependent. The effect of CCK on appetite suppression in the peripheral circulation has been confirmed in different kinds of animal and human studies. The effect of peripheral injection of CCK to inhibit appetite is transient, reducing intake of feed but increasing the number of compensatory feeding times, repeated or long-term application of CCK does not reduce weight, CCK The effect on appetite was mediated by CCK-1 receptor. However, the effect and mechanism of CCK on feeding in the brain was not yet clear that.Blevin et al had injected CCK-8 directly into the brain of the rat, induced short time feeding inhibition, and proved that the main site of action was the DMH and Arc.OLETF rats (Otsuka Long-Evans Tokushima fatty). Gene congenital absence, manifested as gluttony, gradually turned into obesity, and finally appeared in type 2 diabetes. For OLETF rats, the effect of CCK both in the peripheral and in the brain inhibited the feeding control of rats. Further to the study of OLETF rats, it was found that the DMHNPY (neuropeptide Y) of adult OLETF rats was significantly higher, CCK1 received by CCK1. The maladjustment of DMH NPY gene expression may play a role in the formation of obesity and diabetes in OLETF rats. Subsequently, immunohistochemical staining shows that the NPY neurons of the rat DMH have CCK-1 receptors, suggesting that CCK in the hypothalamus may be used as a CCK-1 receptor mediated to DMH NPY neurons, and that the "DMH CCK --" signaling pathway may be possible. In this study, the effects of CCK on the feeding of rats in the DMH nucleus and the characteristics of the time process were observed. The DMH NPY gene, the Arc NPY gene, the Arc POMC gene and the PVN CRF gene were detected and the activation sites of the neurons in the thalamus and brainstem neurons after CCK were observed in the DMH nucleus. Objective to investigate the characteristics of inhibition of ingestion by DMH injection of CCK and the characteristics of corresponding neuronal activity.
Methods: adult Sprague-Dawley male rats (250 ~ 300g) were fed in a constant temperature environment at 20 degrees centigrade and divided into cages in the light cycle of 12h: 12h (Ming: Dark).
1. conscious rats feeding experiment: 12 rats, experimental group n=7, control group n=6. rats after anesthesia, according to the Paxinos-Watson map in the medial dorsalis of the hypothalamus (dorsal medial hypothalamus, DMH) into the sleeve, the coordinates of the anterior fontanelle, 3.1 mm, 0.4 mm next to the skull surface 8.1 mm. one week after the recovery of rats, can enter the experiment. Rats in the operation restorer The diet rule was set up at the time of recovery: 2 hours before the light was fasted and the conventional granule feed was given for 22 hours. The animals were free of drinking water. The DMH nucleus of the experimental group was injected with CCK-8 500nmol / 0.3ul, and the DMH nucleus of the control group injected the artificial cerebrospinal fluid (aCSF) 0.3ul, and the artificial cerebrospinal fluid was composed of 147mM Na~+, 2.7mM K~+, 1.2mMC 2.7mM (+ +). (+ + + +) and 153.8mMCl~-), put into practice before the injection of light, turn off the light immediately after the injection, and give food, and then after the injection of 30min, 1H, 2h, 4h, 22h record food intake.7 after.7, give second DMH nucleus injection CCK-8 and aCSF, the experimental group cross the control group, injection time and dose as well as the amount of food intake records the same first time.
2. analysis of NPY, CRF and POMC gene expression in the hypothalamus: after the feeding experiment, 13 rats were regrouped and the experimental group was n=7. The control group was injected with CCK and aCSF in the n=6.DMH nucleus before the same step. After the injection, the light was shut down and the brain was kept at the speed of 3 hours, and then stored at -80 C at a rapid speed, then the casing position of the group was examined and the DMH NPY, Arc NPY was detected. And the expression of mRNA in PVN CRF. The central part of the rat's forebrain was placed on the slide of 14 mu m series on the slide and fixed with 4% polyformaldehyde. The slices of PVN, DMH, Arc were selected and the DMH NPY mRNA was detected by RNA in situ hybridization. The section was treated with acetic anhydride with acetic anhydride, after dehydration of alcohol and hybrids (containing ~ (35) S-cRNA 6~*10~8 CPM / L) at 55 degrees centigrade for the night. After hybridization, it was cleaned, dehydrated, dried and exposed. The autoradiographic image was quantified by NIH Scion Image software.
3. the expression of c-FOS in the hypothalamus and cerebellum: 28 male rats detected the c-FOS cells in the hypothalamus and cerebellum and the cerebellum and the feeding related nucleus after the injection of CCK. The rats were divided into two groups, 14 rats in each group, and CCK-8 and aCSF were injected in the sober state. The dose and the method were the same. After the injection, after the injection of pentobarbital, after the injection of pentobarbital, the heart was PBS and 4 after the 90 clock. % POM was perfused, and then the brain was soaked in 4% polyformaldehyde / 25% sucrose solution for 1-2 days and 40 mu m series of coronary slices were made in the middle and posterior brain of the forebrain, including the following parts: the paraventricular nucleus (paraventrical nucleus PVN), the supraventricular nucleus (supraoptic nucleus SON), the suprachiasmatic Neucleus SCh in the optic chiasma, and the posterior cross. The retrochiasmatic area (RCh), the lateral hypothalamus (lateral hypothalamus LH), the dorsomedial nucleus of the thalamus (dorsomedial hypothalamic hypothalamic nucleus DMH), the ventral nucleus of the thalamus (ventromedial), the arcuate nucleus, the posterior brain amygdala, the final region, the soliton Nucleus of the solitary tract NTS.C-FOS was detected by immunohistochemical method, using floating method, 0.3% hydrogen peroxide 1H, and the sheep blood albumin package was hatched by 1H, 1: 10000 rabbit c-FOS antibody. Two amino biphenyl (DAB) showed color. After the termination of the reaction, the tissue section was attached to the slide. After drying and alcohol dehydration, the path of the cannula and the c-FOS expression were observed under the microscope. The cases with incorrect casing position were treated with the automatic image analysis software (IpLab, Scanalytics, Fairfax, VA), and the.C-FOS positive cells were discarded by automatic image analysis software (IpLab, Scanalytics, Fairfax, VA), and the injection side and the injection side were counted respectively except DMH. On the contralateral side of the injection, the rest of the brain was counted on two sides. 2~3 slices were taken from each part, and the average value was obtained. The Paxinos-Watson map was used to locate the nerve anatomy.
4. statistical test: the results were corrected by mean + standard error, and the mean t test was statistically analyzed. P < 0.05 showed statistical significance.
Result
The effect of 1.DMH injection of CCK on the feeding of rats
After the direct injection of 500nmol CCK-8 to DMH, the total intake of food intake in 0.5h, 1H, 2h, 4h, 22h after injection was significantly lower than that of the control group. Compared with 0 to 0.5h, 0.5-1h, 1 to 2h, 2 ~ 4h, and 4 to each time, the food intake of the experimental group was significantly lower than that in the control group, and the rest had no significant difference.
Changes of neuropeptide expression after 2.DMH CCK injection
The NPYmRNA expression of DMH in the experimental group was 27% lower than that in the control group, and the expression of NPYmRNA in Arc decreased by 24% compared with the control group; the CRFmRNA expression of PVN in the experimental group increased by 38%, but there was no significant difference between the POMCmRNA in the Arc expression two groups.
Effects of 3.DMH CCK injection on activation of c-FOS protein in the hypothalamus and cerebellar caudal portion of food control related parts
The expression of c-Fos in DMH, Arc, PVN, SCh, RCh in the hypothalamus was significantly higher than that in the control group. There was no significant difference between the two groups on SON, LH, VMH and ME. There was no significant expression in the NTS on the brain stem and on the AP, but there was no significant difference between the experimental group and the control group, but there was no significant difference between the two and the two groups. The number of -FOS positive cells was significantly higher in the experimental group than in the control group. The activation of c-FOS on the.ArC was significantly higher than that in the control group. It was mainly seen in the significant increase in the expression of c-FOS on the medial Arc.PVN, mainly on the small cell PVN.
conclusion
DMH CCK has a feeding inhibition effect, which is briefly different from the action of peripheral CCK, and the effect of DMH CCK is longer, and DMH CCK acts on the inhibition of NPY gene expression by NPY neurons, and the expression of PVN CRF gene is up-regulated, but DMH inhibits the expression of the gene, but does not affect the expression of the gene. Many mound neurons such as PVN, Arc, cDMH, RCh, SCh and so on. Unlike peripheral CCK, DMH CCK does not cause neuronal activity of NTS and AP. The above results show that DMH CCK-NPY signal system plays an important role in controlling feeding and energy metabolism balance.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R33

【共引文献】