本文選題：膽囊收縮素 + 下丘腦　； 參考：《浙江大學(xué)》2007年博士論文

【摘要】： 膽囊收縮素(cholecystokinin，CCK)是胃腸激素，主要分泌于十二指腸和空腸，除了在外周發(fā)揮多種調(diào)節(jié)胃腸功能的作用，也見于腦內(nèi)，在腦內(nèi)作為神經(jīng)傳遞介質(zhì)發(fā)揮作用，因此，CCK也是一種腦腸肽。1973年，Gibbs實驗室首次發(fā)現(xiàn)大鼠腹腔注射CCK-8導(dǎo)致大鼠攝食量明顯減小，反應(yīng)呈劑量依賴性，外周循環(huán)中CCK抑制食欲的作用已在不同種類的動物和人的研究中證實，外周注射CCK抑制食欲的作用是短暫的，使進食量減少但代償性的進食次數(shù)增多，重復(fù)或長期應(yīng)用CCK并不使體重減輕，CCK對食欲的作用通過CCK-1受體介導(dǎo)。然而，腦內(nèi)CCK對攝食的作用及其機理尚不清楚。Blevin et al曾對大鼠腦內(nèi)多部位直接注射CCK-8，誘導(dǎo)出短時間的攝食抑制，并證明作用部位主要為DMH和Arc。OLETF大鼠(Otsuka Long-Evans Tokushima fatty rats)的CCK-1受體基因先天性缺失，表現(xiàn)為貪食，逐漸轉(zhuǎn)為肥胖，最后出現(xiàn)2型糖尿病，對于OLETF大鼠的研究提示，CCK無論在外周還是在腦內(nèi)的作用均對大鼠攝食控制中起抑制作用，進一步對于OLETF大鼠的研究發(fā)現(xiàn)，成年OLETF大鼠DMHNPY(神經(jīng)肽Y)明顯增高，CCK1受體缺失導(dǎo)致DMH NPY基因表達失調(diào)可能對OLETF大鼠肥胖和糖尿病的形成起作用。隨之，免疫組化顯示大鼠DMH的NPY神經(jīng)元上具有CCK-1受體，提示下丘腦內(nèi)CCK可能通過CCK-1受體介導(dǎo)作用于DMH的NPY神經(jīng)元起作用，推測“DMH CCK—NPY”信號通路可能參與攝食控制。盡管如此，其確切的機理仍不明。本研究通過觀察DMH核團內(nèi)注射CCK對大鼠攝食的影響以及時間過程特點，檢測DMH NPY基因、Arc NPY基因、Arc POMC基因和PVN CRF基因表達以及觀察DMH核團內(nèi)注射CCK后丘腦和腦干神經(jīng)元激活的部位，探討DMH注射CCK抑制攝食的特征以及相應(yīng)的神經(jīng)元活動的特征。 方法：以成年Sprague-Dawley雄性大鼠(250～300g)為材料，置于20℃恒溫環(huán)境，12h∶12h(明∶暗)的燈光周期中分籠飼養(yǎng)。 1．清醒大鼠攝食實驗：12只大鼠，實驗組n=7，對照組n=6。大鼠麻醉后，按Paxinos-Watson圖譜在下丘腦背內(nèi)側(cè)區(qū)(dorsal medial hypothalamus，DMH)插入套管，坐標為前囟后3.1 mm，旁開0.4 mm，顱骨表面下8.1 mm。一周后大鼠恢復(fù)良好，可進入實驗。大鼠于手術(shù)恢復(fù)期時使其建立飲食規(guī)律：關(guān)燈前2小時禁食，隨之22小時予以常規(guī)顆粒飼料。動物可自由飲水。實驗組DMH核團微量注射CCK-8 500nmol／0.3ul，對照組DMH核團微量注射人工腦脊液(aCSF)0.3ul，人工腦脊液組成：(147mM Na~+，2.7mM K~+，1.2mMC Ca~(++)，0.85mMMg~(++) and 153.8mMCl~-)，注射于關(guān)燈前實施，注射后立即關(guān)燈，并給予食物，然后分別于注射后30min、1h、2h、4h、22h記錄進食量。7天后，給予第二次DMH核團注射CCK-8和aCSF，實驗組對照組交叉，注射時間和劑量以及進食量的記錄同第一次。 2．下丘腦NPY，CRF和POMC基因表達分析：攝食實驗后，13只大鼠重新分組，實驗組n=7，對照組n=6。DMH核團內(nèi)注射CCK和aCSF步驟同前，注射后關(guān)燈但繼續(xù)禁食，3小時后斷頭取腦，急速置于-80℃保存，待組織學(xué)檢查套管位置和檢測DMH NPY、Arc NPY、Arc POMC和PVN CRF的mRNA表達。大鼠前腦中部作14μm系列冠狀切片貼于玻片上，以4％多聚甲醛固定。挑取PVN、DMH、Arc的切片，應(yīng)用RNA原位核酸雜交，分別檢測DMH NPY mRNA、Arc NPY mRNA和POMC mRNA、PVN CRF mRNA的表達。~(35)S-cRNA探針以POMC、NPY和CRF cDNA為模板體外轉(zhuǎn)錄。切片以醋酸酐處理，酒精脫水后加雜交緩沖液(含~(35)S-cRNA 6~*10~8 cpm／μl)55℃過夜，雜交后清洗、脫水、干燥、曝光顯影。放射自顯影圖象以NIH Scion Image軟件進行定量分析。 3．下丘腦和小腦c-FOS表達：28只雄性大鼠檢測DMH注射CCK后下丘腦和小腦與攝食相關(guān)神經(jīng)核中c-FOS細胞。大鼠分二組，每組14只，清醒狀態(tài)下分別注射CCK-8和aCSF，劑量和方法同前。注射后立即禁食，90分鐘后以戊巴比妥麻醉后，經(jīng)心臟以PBS和4％多聚甲醛灌流，然后取腦置于4％多聚甲醛／25％蔗糖溶液中浸泡，4℃保存1-2天，在前腦中部和后腦作40μm系列冠狀切片，包括下列部位：室旁核(paraventrical nucleus PVN)、視上核(supraoptic nucleus SON)、視交叉上suprachiasmatic neucleus SCh)、后交叉區(qū)retrochiasmatic area(RCh)、外側(cè)下丘腦(lateral hypothalamus LH)、丘腦背內(nèi)側(cè)核(dorsomedial hypothalamic hypothalamic nucleus DMH)、丘腦腹內(nèi)側(cè)核(ventromedial hypothalamic nucleus VMH)、弓狀核(arcuate nucleusArc)、后腦杏仁核(amygadala nucleus，CeA)、最后區(qū)(area postrema AP)、孤束核(nucleus of the solitary tract NTS)。c-FOS以免疫組織化學(xué)法檢測，采用漂浮法，0.3％過氧化氫1h，羊血清包被1h，1∶10，000兔c-FOS抗體(Oncogene Science，SanDiego，CA)孵化過夜，生物素-羊抗兔血清1h，ABC復(fù)合試劑(Elite Vectastain Kit，Vector Labs，Burlingame，CA)1h，以二氨基聯(lián)苯(DAB)顯色。終止反應(yīng)后，將組織切片貼到玻片上，干燥，酒精脫水后，顯微鏡下觀察切片內(nèi)套管軌跡和c-FOS表達，套管位置不正確者棄去。c-FOS陽性細胞定量以自動圖象分析軟件處理(IpLab，Scanalytics，F(xiàn)airfax，VA)，除DMH分別計數(shù)注射側(cè)和注射對側(cè)，其余部位均計數(shù)腦二側(cè)，在每個部位均讀取2～3張切片，取平均值，神經(jīng)解剖學(xué)定位參照Paxinos-Watson圖譜。 4．統(tǒng)計學(xué)檢驗：結(jié)果采用均數(shù)±標準誤，均數(shù)t檢驗統(tǒng)計學(xué)處理，P＜0.05說明有統(tǒng)計學(xué)意義。 結(jié)果 1．DMH注射CCK對大鼠攝食的影響 500nmol CCK-8直接注射到DMH后，大鼠在注射后的0.5h，1h，2h，4h，22h內(nèi)的累計攝食量均較對照組顯著減少，比較0～0.5h，0.5-1h，1～2h，2～4h，4～22h各個時間段的攝食量，發(fā)現(xiàn)在0.5h內(nèi)和2～4h時間段實驗組較對照組攝食量顯著減少，其余無顯著差異。 2．DMH CCK注射后神經(jīng)肽表達的變化 實驗組動物在DMH的NPYmRNA表達較對照組降低27％，Arc的NPYmRNA表達較對照組降低24％；實驗組PVN的CRFmRNA表達增高38％；而POMCmRNA在Arc表達二組未見顯著差異。 3．DMH CCK注射對下丘腦和小腦尾部與攝食控制相關(guān)部位c-FOS蛋白激活的影響 實驗組在下丘腦DMH、Arc、PVN、SCh、RCh上c-Fos表達明顯高于對照組，在SON、LH、VMH、ME上二組無顯著差異，在腦干NTS、AP上未見c-Fos表達。注射側(cè)的DMH無論實驗組還是對照組可見非常強烈的c-FOS表達，但二者比較未見顯著性差異，比較二組注射對側(cè)DMH的c-FOS陽性細胞數(shù)，實驗組明顯較對照組增高。ArC上c-FOS激活較對照組明顯，，主要見于內(nèi)側(cè)Arc。PVN上c-FOS表達顯著增加主要見于小細胞PVN上。 結(jié)論 DMH CCK具有攝食抑制作用，與外周CCK作用短暫不同，DMH CCK作用持續(xù)時間較長；DMH CCK作用于NPY神經(jīng)元抑制NPY基因表達而發(fā)揮攝食抑制的作用，并上調(diào)PVN CRF基因表達，DMH CCK抑制Arc NPY基因表達，但不影響Arc POMC基因表達；DMH CCK增加可激活下丘多個神經(jīng)元如PVN，Arc，cDMH，RCh，SCh等，與外周CCK不同，DMH CCK不引起NTS和AP的神經(jīng)元活動。上述結(jié)果表明，DMH CCK-NPY信號系統(tǒng)在控制攝食和能量代謝平衡中發(fā)揮重要作用，下丘腦多條依賴PVN CRF和Arc NPY的神經(jīng)信號途徑介導(dǎo)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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R33

【共引文獻】

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1 楊利波;高燕燕;;肽YY與肥胖[J];中國實用內(nèi)科雜志;2005年12期

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3 ;Effects of PYY on the interdigestive migrating myoelectric complex in the small intestine in vivo and the neural and endocrinal mechanisms of the effects[J];Academic Journal of Xi'an Jiaotong University;2009年02期

相關(guān)博士學(xué)位論文 前3條

1 楊年紅;PYY基因—飲食交互作用與肥胖易感性的關(guān)系[D];華中科技大學(xué);2006年

2 季新榮;外科手術(shù)治療肥胖和2型糖尿病機制的研究[D];第二軍醫(yī)大學(xué);2010年

3 席麗;人血漿中PYY定量檢測方法的建立[D];西北農(nóng)林科技大學(xué);2012年

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