【作者】 陈洁；

【导师】 赵正言；

【作者基本信息】 浙江大学 ， 儿科学， 2007， 博士

【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。³⁵S-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³⁵S-cRNA 6^*10⁸ 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。更多还原

【Abstract】 Cholecystokinin （CCK） is a brain-gut peptide that plays an important role in the control of food intake. Peripheral CCK acts as a satiety signal to limit meal size. CCK is released from the duodenum and jejunum in response to the intra-luminal presence of nutrient-digestive products. Peripheral CCK administration reduces food intake in a dose-related manner across a range of experimental situations and in a variety of species, and the actions of CCK in food intake are specific to a reduction in meal size. The feeding inhibitory effects of exogenously administered CCK appear to mimic a physiological role for endogenous CCK. Administration of CCK receptor-specific antagonists results in an increase in food intake, and this increase is manifested as an increase in meal size. The feeding inhibitory actions of both endogenously released and exogenously administered CCK are mediated through their interaction with CCK1 receptors. In contrast to the well characterized satiety actions of peripheral CCK, the role for brain CCK in the control of food intake has not yet been known. Blevins et al. demonstrated that infusing smaller doses of CCK-8 into specific brain sites resulted in site-specific feeding inhibitory actions in the rat, and this anorexic dose of CCK-8 did not increase plasma CCK-8 levelssufficiently to suppress feeding via a peripheral mechanism. Data from Otsuka Long-Evans Tokushima fatty （OLETF） rats, which have congenital CCK1 receptor deficiency and become hyperphagic and obese, have suggested that both peripheral and brain CCK take roles in the controls of food intake. Analysis of hypothalamic gene expression in OLETF rats have suggested that the dysregulation of dorsomedial hypothalamus neuropeptide Y （DMH NPY） gene expression resulting from CCK1 receptor deficiency may play an etiological role in the hyperphagia and obesity of OLETF rats. Subsequently, Immunohistochemical studies have revealed that CCK1 receptors and NPY were co-localized in DMH neurons. Although we have proposed a role for DMH CCK-NPY signaling in the control of food intake, we have yet to identify the pathways underlying this action. In the present study, we aimed to characterize the feeding inhibition and patterns of brain neuronal activation produced by injection of CCK into the DMH. Firstly,we examined the time course of feeding inhibitory effects of DMH CCK administration. Secondly we also examined whether DMH CCK administration resulted in alterations in hypothalamic corticotrophin-releasing factor （CRF）, Pro-opiomelanocortin （POMC） and NPY gene expression. Finally, we assessed brain patterns of c-Fos activation induced by DMH CCK administration to identify candidate brain sites that might mediate the actions of the DMH CCK-NPY signaling system.Methods: Male Sprague-Dawley rats weighing 250-300 g purchased from Charles River Laboratories, Inc. （Wilmington, MA） served as subjects. Rats were individually housed in hanging wire mesh cages and maintained on a 12:12-h light-dark cycle in a temperature-controlled environment （22℃） with ad libitum access to water and feeding schedules as described in each experiment.DMH CCK-8 injection and food intake. Thirteen male Sprague-Dawley rats were implanted with unilateral indwelling DMH cannulae and were randomly divided into two groups. Just before lights off, one group of 6 animals was injectedwith 0.3 μl of artificial cerebral-spinal fluid (aCSF: 147 mM Na⁺, 2.7 mM K⁺, 1.2 mM Ca²⁺, 0.85 mM Mg²⁺ and 153.8 mM Cl^-) and the other group of 7 animals was injected with 0.5 nmol of CCK in 0.3 μl aCSF. Pelleted chow was returned to the cages immediately after the injection. Food intakes were measured at 30 min, 1 h, 2 h, 4 h, and 22 h later. After 7-day recovery, all rats were given a second DMH injection with aCSF or CCK-8 （0.5 nmol）, i.e., the rats that had previously received CCK-8 administration were given aCSF injection at this time and vice versa. Food intakes were measured as following the first injections.Analyses of hypothalamic NPY, CRF and POMC gene expression. After feeding tests, 13 DMH cannulated rats were weight matched and randomly divided into two groups: aCSF control （n = 6） and CCK-8 treatment （n = 7）, for assessing whether DMH CCK-8 injection affected hypothalamic NPY, CRF and POMC mRNA expression. Animals were maintained on the same feeding schedule as above in which regular chow was removed from the cages 2 hours before lights off and returned to the cages just before dark onset and with access to water ad libitum. Again, rats received either aCSF or CCK-8 injections as described above but food was not returned to the cages. Three hours following injections, rats were sacrificed with an overdose of sodium pentobarbital, and brains were removed rapidly and frozen at -80℃ for subsequent analyses of hypothalamic NPY, CRF and POMC gene expression. 14-μm coronal brain sections ranging from 1.8-3.4 mm caudal to bregma were cut with a cryostat, mounted on superfrost/plus slides and fixed with 4% paraformaldehyde. ³⁵S-labeled NPY, POMC and CRF antisense riboprobes were transcribed by using in vitro transcription systems and purified. Sections for Arc POMC mRNA Arc NPY mRNA, determination, DMH NPY mRNA and for CRF mRNA in the paraventricular nucleus （PVN） were taken. Sections were treated with acetic anhydride and incubated in hybridization buffer containing 500 (J.g/ml yeast tRNA and 10⁸ cpm/ml of ³⁵S-UTP at 55℃ overnight. After hybridization, sectionswere washed, dehydrated, air-dried and exposed with BMR-2 film for 1-3 days. Quantitative analysis of the in situ hybridization data was done with NIH Scion image software （National Institutes of Health）. Autoradiographic images were first scanned by EPSON Professional Scanner （EPSON） and saved via a computer for subsequent analyses with Scion image software using autoradiographic ¹⁴C micro scales （Amersham） as a standard. Arc POMC or PVN CRF mRNA levels were determined by a mean of the product of hybridization area x density （background density was subtracted） for each rat. Data from each group were normalized to vehicle aCSF treated controls as 100%, and all data were presented as mean ± SEM.DMH CCK injection and c-Fos immunohistochemistry. Twenty-eight male Sprague-Dawley rats were implanted with unilateral indwelling DMH cannulae as described above. After postoperative recovery and habituation to the injection procedure, rats were randomly divided into two groups （n=14）: one group was injected with 0.3 μl of aCSF and the other with 0.5 nmol of CCK-8 in 0.3 μl aCSF. All DMH injections were performed as described above, but rats were not allowed to access to chow food after DMH injection. Ninety minutes following injections, rats were anesthesized with Euthasol （pentobarbital sodium and phenytoin, Delmarva Laboratories, Midlothian, VA） and perfused transcardially with phosphate buffered saline （PBS, pH 7.4） followed by 4% paraformaldehyde in PBS. Brains were removed and stored in 25% sucrose containing 4% paraformaldehyde at 4℃ for subsequent c-Fos immunoreactivity determinations. In the initial step, 3 animals per group were examined for determination of the regions where c-Fos activation was potentially induced by DMH CCK injection. Since the initial c-Fos immunoreactivity determination revealed that DMH CCK-induced c-Fos positive cells were exclusively localized to hypothalamic areas, subsequent quantitative c-Fos immunoreactivity was only determined in brain regions over the hypothalamus in the remaining animals （11 rats per group）. Forty μm coronal sections extending from0.48 mm anterior to bregma to 4.36 mm posterior to bregma were cut, and every other section was collected in PBS for c-Fos immunoreactivity determination. The number of c-Fos positive cells was counted in the following areas: the suprachiasmatic neucleus （SCh）, the retrochiasmatic area （RCh）, the supraoptic nucleus （SON）, the PVN, the DMH, the Arc, the medial eminence （ME）, the ventromedial hypothalamus （VMH）, and the lateral hypothalamus （LH）, as well as the central neucleus of amygdala （CeA）. Images of sections were captured by digital camera attached to Zeiss Axio Imager. The area of interest was outlined based on cellular morphology and c-Fos positive cells were automatically counted by the imaging program （IPLab, Scanalytics, Fairfax, VA） by setting minimum and maximum optical density levels. Cell counts of c-Fos immunoreactivity were made separately in the ipsilateral （iDMH） and contralateral （cDMH） to the site of DMH CCK injection. Data for c-Fos activation in all other areas were bilaterally assessed, and were presented as the total number of c-Fos positive cells per section.Results: i. Effects of DMH CCK injection on food intake. Parenchymal injection of CCK into the DMH decreased food intake during the entire 22 hour observation period. The magnitude of feeding inhibitory effect was time dependent. DMH CCK administration resulted in a 51% reduction in the first 30 min as compared to vehicle treated rats. In contrast to the time course of the effect of peripherally administered CCK, the feeding inhibition produced by DMH CCK injection was long lasting with a 38.4% suppression maintained at 4 hours. Compensation for this reduction did not occurred within the next 18 hours. Food intake of DMH CCK administered rats remained significantly reduced at 22 hours as compared to the vehicle controls. ii. Effects of DMH CCK injection on Arc NPY,DMH NPY,PVN CRF and Arc POMC gene expression. Relative to vehicle-treated rats, DMH CCK administration elevated PVN CRF gene expression with a 38% increase in mRNA levels, down-regulated DMH NPY gene expressionwith a 27% decrease in mRNA levels and Arc NPY gene expression a with 24% decrease in m RNA levels. DMH CCK administration did not affect Arc POMC mRNA levels as compared to vehicle treatment iii. Characterization of brain c-Fos activation induced by DMH CCK administration. Examination of c-Fos immunoreactivity throughout the entire brain revealed that DMH CCK-induced c-Fos activation was exclusively localized to the hypothalamus. The positive sites included the SCh, RCh, PVN, cDMH, and Arc, but not the SON, VMH, LH and ME. DMH CCK injection resulted in a 5-fold increase in the number of c-Fos positive cells in the cDMH as compared to that of vehicle treated rats. This c-Fos immunoreactivity was detected in all three subregions, i.e., the dorsal, ventral and compact part of the cDMH. Within the PVN, DMH CCK-induced c-Fos activation was primarily located in the medial parvicellular part of the PVN,and with a 4-fold increase as compared to the vehicle treatment, whereas very few c-Fos positive neurons were detected in the lateral magnocellular part of the PVN. DMH CCK injection also significantly increased c-Fos immunoreactivity in the Arc, with a majority of c-Fos positive cells in the medial part DMH CCK administration increased the number of c-Fos positive cells by 3.8 folds in the SCh and 5.3 folds in the RCh relative to vehicle treatment. DMH CCK injection did not induce c-Fos activation in the CeA, NTS, and AP.Conclusion: Injection of CCK into the DMH results in a rapid decrease in food intake, and this feeding inhibition maintained at least 22 hours. In response to DMH CCK injection, PVN CRF gene expression is significantly elevated, DMH NPY and Arc NPY gene expression is significantly reduced, while Arc POMC gene expression is not affected. In response to DMH CCK administration, c-Fos is activated in various hypothalamic areas including the cDMH, PVN, Arc, SCh and RCh, but not in the SON, VMH, LH and ME or in the CeA and the brain stem NTS and AP. In all, these data suggest that multiple hypothalamic signaling pathwaysmay underlie the actions of DMH CCK. DMH CCK-NPY signaling system plays an important role in the control of food intake and energy balance. Its actions seem to be mediated through multiple hypothalamic pathways, which depend upon PVN CRF and Arc NPY.更多还原