Obestatin对大鼠胰腺外分泌调节作用的实验研究
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摘要
Obestatin(肥胖抑制素)是最近从大鼠胃组织中发现的一种新的脑肠肽。它是一种ghrelin相关肽，与ghrelin来自同一基因。obestatin与ghrelin在调节摄食、胃肠生理等方面作用完全相反，被认为是一对“阴阳”肽。ghrelin也称为食欲刺激素，是一种由23个氨基酸组成的脑肠肽，生长激素促分泌素受体（growth hormone secretagogue receptor，GHSR）为其受体，ghrelin对胰腺外分泌也具有重要的调节作用，主要表现为促进胰腺外分泌，其机制是通过迷走-迷走神经通路发挥作用的。虽然obestatin和ghrelin来源于同一前体proghrelin，但obestatin自发现以来就充满了各种争议，2005年，Zhang等首先在胃黏膜中发现并命名了obestatin，在最初的功能研究中，腹腔和侧脑室注射人源性obestatin可以抑制大鼠的食欲，减少体重，进一步的研究发现：obestatin可以抑制渴感和焦虑、提高大鼠的记忆力、抑制前脂肪细胞的增长、控制机体的水代谢。在人体的研究当中，部分研究者发现在肥胖人群里，胃黏膜中obestatin表达量和血循环中obestatin含量均出现减少，因此他们推测obestatin在调节机体能量代谢方面发挥了重要作用，是ghrelin的拮抗剂。尽管有以上证据证明obestatin存在生物学作用，但绝大多数研究应用了大剂量范围的obestatin、不同的实验方法、不同的动物及不同来源的obestatin并未得出相同结论，因此有研究者提出应该重新命名obestatin。除了对obestatin生物学功能方面存在疑问外，对其受体也存在着争论：其发现者首次报道了G蛋白偶联受体39（GPR39）是obestatin的天然受体，结合实验也证明GPR39广泛存在于中枢神经系统、外周重要器官，而以胃肠道的含量最高。后来的三个实验室并未证明GPR39为obestatin的固有受体，因此对发现者的研究提出了质疑，对于以上质疑，2007年Zhang重复了自己的实验并得出他们实验室不能证明GPR39是obestatin受体的结论。因此对obestatin的研究还存在以下疑问：(1)在obestatin本身方面：在体内这种分子是否真正存在?如果存在到底有怎样的生理功能功能?是否能拮抗ghrelin的生理作用?它是否受到中枢及其他激素的调控?(2)在外源性分子方面：人工合成的外源性obestatin多肽分子是否具有与内源性分子相同的空间结构及生物活性? (3)在obestatin受体方面：obestatin是否通过GPR39受体发挥作用？是否还有别的作用途径?(4)在研究方法方面：利用人工合成的obestatin抗体，检测到的仅仅是obestatin，还是包括了无实际作用的preproghrelin或proghrelin？
胰腺的外分泌功能受到中枢神经和肠神经以及大量的激素和脑肠肽组成的复杂网络的调节，业已证实：胆囊收缩素（CCK）、促胰液素、胰多肽、ghrelin、血管活性多肽、蛙皮素和胃动素等激素可以刺激胰腺的外分泌；而生长抑素、胰高糖素、降钙素、肾上腺素和去甲肾上腺素等可以抑制胰腺的外分泌，由于对obestatin功能及受体研究存在着大量的争论，并且obestatin对胰腺外分泌功能的调节作用目前还不清楚。本研究拟采用大鼠离体腺泡、小叶及在体胰腺外分泌功能研究模型，应用分离大鼠离体腺泡细胞和胰腺小叶、静脉持续注射、侧脑室内微量注射等技术明确obestatin对胰腺外分泌功能的调节作用及其机制，并观察obestatin及其它脑肠肽对大鼠胰腺外分泌调节的交互作用，用胆囊收缩素作为阳性对照，从而进一步完善胰腺外分泌调节的机制和obestatin的生物学功能。
第一部分一种改良的胰腺腺泡提取方法
目的：对经典的Williams提取胰腺腺泡方法进行改良，以期探索一种简便，经济和稳定的胰腺腺泡分离纯化的新方法。
方法：雄性SD大鼠，鼠龄8-10w，体重200-250g。在无菌条件下，以3%戊巴比妥钠，按照15mg/kg剂量腹腔内注射麻醉后固定于解剖板上，然后放于75%酒精内消毒30分钟，在无菌操作台上于剑突下3cm处腹部正中，分层剪开皮肤、腹肌；循胃、十二指肠球降部及脾脏之间取出胰腺。室温下（23℃）在充过氧的Hepes液中将血迹冲洗干净，并用镊子轻轻的将胰腺周围脂肪、淋巴结以及腹膜剔除。以低浓度Ⅰ型胶原酶（0.3mg/ml）作为腺泡细胞分离液，用带有1ml注射器针头的注射器在大鼠胰腺间质内多点注射使胰腺小叶展开，5分钟后用小剪刀将其剪成1～2mm碎块放于含100%氧分离液中震荡消化50分钟，然后经过过滤，离心、再悬浮等过程成功提取大鼠胰腺腺泡。
结果：平均每只大鼠可以提取5～6×106个细胞，经HE和台盼蓝染色，细胞存活率大于90%，细胞纯化率为(70±6)％与王连才方法相比差异无显著性。
结论：我们认为本实验室采用的改进方法是一种简便，经济和稳定的腺泡分离纯化方法。
第二部分Obestatin对大鼠胰腺腺泡/小叶淀粉酶影响
目的：研究不同剂量肥胖抑制素对大鼠离体胰腺腺泡及胰腺小叶淀粉酶分泌的影响。
材料与方法：雄性SD大鼠，鼠龄8-10w，体重200-250g，36只。采用改良的Williams法体外分离大鼠胰腺腺泡（方法同第一部分），将胰腺腺泡与不同浓度obestatin（0.1、1、10、30nmol/L）在37℃培养箱中培养1h。在对照组腺泡中，加入胆囊收缩素（CCK-8，10-10mol/L）和obestatin共培养1h，测定培养液中上清测定淀粉酶水平，将剩余腺泡细胞放于超声破碎仪打碎（5分钟），离心后去上清测定淀粉酶水平。参照Scheele＆Palade法提取胰腺小叶：在无菌条件下，以3%戊巴比妥钠，按照15mg/kg剂量腹腔内注射麻醉后固定于解剖板上，放于75%的酒精内消毒30分钟，在无菌操作台上于剑突下3cm处腹部正中，分层剪开皮肤、腹肌；循胃、十二指肠球降部及脾脏之间取出胰腺。室温下（23℃）在充过氧的KHB液中将血迹冲洗干净，并用镊子将胰腺周围脂肪、淋巴结以及腹膜剔除。分离含有胰腺内神经末端、胰岛细胞及外分泌细胞的胰腺小叶（大小4mm～5mm），胰腺小叶与obestatin共培养30min，对照组中KCL（75mmol/L）和obestatin共培养30min，取上清测定淀粉酶水平，将剩余胰腺小叶组织放于组织匀浆器中打碎，离心后测定上清液中淀粉酶水平。
结果：obestatin不影响胰腺腺泡和胰腺小叶淀粉酶释放，且不随浓度的增高而改变(P >0.05)。CCK-8和KCL能分别刺激胰腺腺泡、胰腺小叶淀粉酶释放（P<0.05），而外源性obestatin对CCK-8和KCL引起的胰腺腺泡和胰腺小叶淀粉酶分泌增加无影响，差异无统计学意义（P >0.05）。
结论：体外实验中，外源性obestatin对大鼠胰腺腺泡及胰腺小叶淀粉酶分泌无影响，obestatin对活体大鼠胰腺外分泌影响需要进一步研究。
第三部分静脉注射obestatin对大鼠胰腺外分泌的影响
目的：采用静脉持续注射的方法研究不同剂量肥胖抑制素对大鼠胰腺外分泌功能的影响。
材料与方法：24只SD大鼠随机分成3组（N=8），麻醉后制作胰腺外分泌模型：所有大鼠用3%戊巴比妥钠（15mg/kg）腹腔内麻醉，麻醉成功后固定于操作板上，放于75%酒精内消毒30分钟，腹中线上逐层切开上腹部约3cm，于胃及十二指肠之间找到胰管及十二指肠乳头，经十二指肠乳头对侧肠管插入聚乙烯导管（PE-10）约0.5cm,尾静脉及右侧股动脉留置输液导管，十二指肠乳头上端插入PE-10以回输胆胰混合液（P-BJ）。模型稳定30分钟后开始实验，每15分钟经胰管插管收集一次P-BJ测定体积，并留取50ul测定蛋白含量，剩余P-BJ经由十二指肠回输。所有大鼠于实验开始后15分钟收集一次P-BJ作为基础分泌。A组和B组于实验开始15分钟后分别给予尾静脉持续微泵注射1nmolkg-1h-1obestatin和5nmolkg-1h-1 obestatin，45分钟后给予股静脉微泵注射400pmolkg-1h-1 CCK-8；C组于实验开始45分钟后给予股静脉注射400pmolkg-1h-1CCK-8作为对照。
结果：静脉注射CCK-8 15min后胰腺分泌量开始增高，并于30min后达高峰，而胰腺蛋白也呈相应升高，差异均具有显著性（P <0.05），单独静脉注射obestatin对大鼠胰腺外分泌量及胰液蛋白无影响（P >0.05），obestatin与CCK-8同时注射并不能改变由CCK-8引起的胰腺外分泌及胰液蛋白的增高。结论：外周静脉给予CCK-8可增加胰液和胰液蛋白的分泌，静脉注射obestatin对胰腺外分泌功能无影响，并且不能改变由CCK-8引起的胰液和胰液蛋白的增高。
第四部分脑室微量注射obestatin对大鼠胰腺外分泌的影响
目的采用侧脑室微量注射的方法研究肥胖抑制素对大鼠胰腺外分泌功能的影响。
方法SD大鼠随机分成3组（A,B,C组），麻醉后制作胰腺外分泌模型：所有大鼠用3%戊巴比妥钠（15mg/kg）腹腔内麻醉，麻醉成功后固定于操作板上，放于75%酒精内消毒30分钟，沿着腹中线逐层切开上腹部约3cm，于胃及十二指肠之间找到胰管及十二指肠乳头，经十二指肠乳头对侧肠管插入聚乙烯导管（PE-10）约0.5cm,尾静脉留置输液导管，十二指肠乳头上端插入PE-10以回输胆胰混合液（P-BJ）。模型稳定30分钟后开始实验，每15分钟经胰管插管收集一次P-BJ测定体积，并留取50ul测定蛋白含量，剩余P-BJ经由十二指肠回输。所有大鼠于实验开始后15分钟收集一次P-BJ作为基础分泌。A组和B组于实验开始15分钟后分别给予侧脑室微量注射30nmolkg-1obestatin,C组于实验开始45分钟后给予静脉注射400pmol kg-1h-1 CCK-8作为对照。
结果：与基础分泌相比，侧脑室微量注射obestatin后大鼠胰腺外分泌量及胰液蛋白与基础相比差异无显著性（P >0.05），静脉注射CCK-8可以引起胰腺外分泌量的增加，并于30min后达高峰，差异具有显著性意义；而胰腺蛋白也呈相应升高，与大鼠基础分泌相比，差异具有显著性（P <0.05），obestatin脑室内注射并不能改变由CCK-8引起的胰腺外分泌及胰液蛋白的增高。
结论外周静脉给予CCK-8增加胰液和胰液蛋白的分泌，侧脑室微量注射obestatin对胰腺外分泌功能无影响，并且不能改变由CCK-8引起的胰液和胰液蛋白的增高。
第五部分十二指肠内灌注obestatin对大鼠胰腺外分泌影响的初步研究
目的：采用十二指肠内持续灌注的方法研究不同剂量obestatin对大鼠胰腺外分泌功能的影响
方法：SD大鼠14只，随机分成2组，麻醉后制作胰腺外分泌模型：所有大鼠用3%戊巴比妥钠（15mg/kg）腹腔内麻醉，麻醉成功后固定于操作板上，放于75%酒精内消毒30分钟，在上腹部打开腹腔，于胃及十二指肠之间找到胰管及十二指肠乳头，经十二指肠乳头对侧肠管插入聚乙烯导管（PE-10）约0.5cm，十二指肠乳头上端插入PE-10以微泵灌注obestatin及回输胆胰混合液（P-BJ）。模型稳定30分钟后开始实验，每15分钟经胰管插管收集一次P-BJ测定体积，并留取50ul测定蛋白含量，剩余P-BJ经由十二指肠回输。所有大鼠于实验开始后15分钟收集一次P-BJ作为基础分泌。B组于实验开始15分钟后分别给予十二指肠内微泵灌注5nmolkg-1h-1obestatin 105分钟, A组除了不做十二指肠灌注以外，其余处理均同B组。
结果十二指肠内持续灌注5nomlkg-1h-1obestatin各时点大鼠胰腺外分泌量及胰液蛋白与对照组相比均无显著性差异（P >0.05）结论十二指肠内持续灌注obestatin对大鼠胰腺外分泌量及胰液蛋白无影响。
全文小结：本课题通过采用①建立离体大鼠胰腺腺泡细胞及胰腺小叶，分别与不同浓度的obestatin及腺泡刺激剂(CCK-8)和胰腺小叶刺激剂（KCL）进行共培养，观察细胞内外淀粉酶水平的变化，进而分析obestatin对胰腺外分泌功能的影响；②建立活体麻醉大鼠胰腺外分泌动物模型，静脉给予不同浓度的obestatin观察其对胰腺外分泌量及胰液蛋白的影响；并与胰腺刺激剂CCK-8共同注射，观察其是否与CCK-8存在协同或拮抗作用。③建立麻醉大鼠胰腺外分泌模型，通过侧脑室微量注射obestatin，观察其是否能通过中枢神经影响大鼠胰腺外分泌功能。④通过十二指肠内灌注的方法观察obestatin对大鼠胰腺外分泌功能的影响，结果显示：体外实验中，外源性obestatin对大鼠胰腺腺泡及胰腺小叶淀粉酶分泌无影响，外周静脉给予CCK-8可增加胰液和胰液蛋白的分泌；在麻醉大鼠中，静脉给予obestatin对胰腺外分泌功能无影响，并且不能改变由CCK-8引起的胰液和胰液蛋白的增高。脑室内微量注射obestatin对胰腺外分泌功能无影响，并且不能改变由CCK-8引起的胰液和胰液蛋白的增高。单独十二指肠内灌注obestatin对大鼠胰腺外分泌功能无影响。
Obestatin is a recently identified 23-amino acid gut-derived peptide encodedby the preproghrelin gene. Although originating from the same precursor as the widely studied appetite-stimulating peptide ghrelin, obestatin was reported to bring on antithetical physiological effects with regards to food intake and body weight regulation. Obestatin and ghrelin were called yin and yang peptidefor encoded by the same gene and have opposite physiological functions in several systems. ghrelin, a 28-amino acid and ligand for the growth hormone secretagogue receptor-1a, has demonstrated to be a orexigenic peptide. As a ghrelin-associated peptide, obestatin was derived from the same proghrelin as ghrelin by different post-translational cleavage and borned with contestation. In 2005, zhang et al first identified obestatin as a 23-amino acid isolated from stomach mucosa and encoded by preproghrelin gene. The initial work showed that intraperitoneal or intracerebroventricular injection of human obestatin supressed food intake and decreased body-weight gain. Further studies indicated thatobestatin was involved in inhibiting thirst and anxiety , improving memory, affecting cell proliferation, controlling fluid homeostasis. However, controversialresults focus on its anorexigenic actions. In vivo and vitro studies, several investigators reported anorexigenic effects of peripheral obestatin in rodents. Furthermore, decreased gastric body mucosa obestatin expression and obestatin levels in plasma in obese patents indicated that obestatin may play an important role in the regulation of energy balance, apparently being able to antagonize the effects of ghrelin. In spite of those evidence, the anorexigenic effects of obestatin were not reproduced by subsequent studies using differernt dose of obestatin, experimental protocols and species or origin of the peptide. Different from ghrelin, obestatin has high affinity for GPR39 which belongs to the ghrelin receptor subfamily and hypothesized to be a congate ligand for GPR39. Binding studies and molecular approaches indicated that GPR39 was expressed ina wide range of tissues including central nevous system, peripheral organs, especially at a higher level in digestive system. Opposite to the initial report, later studies from different groups were unalble to corroboratre the binding between obestatin and GPR39. In 2007, Zhang et al. proved again that their original result of obestatin for GPR39 was unreproductive. Other receptor of obestatin was reported by Yasuda et al that Zn2+ could be a physiologically relevant agonist or modulator of GPR39.
Pancreatic exocrine secretion is known to be modulated by a number of brain-gut peptides directly or through brain-pancreas anxis. It has been reported that cholecystokinin(CCK), secretin , pancreatic polypeptide(PP), ghrelin and amphetamine-regulated transcript peptide(CART) stimulate pancreatic secretion, whereas neuropeptide Y and somatostatin serve as inhibitors. Interestingly, like ghrelin, obestatin was found to stimulate the secretation of pancreatic juice enzymes through a vagal pathway in anaesthetized rats. Saleem and co-workers reported that obestatin inhibited the secretion of both somatostatin and PP in ratislets. These results need a further insight into the influence of obestatin on pancreatic secretion. Since previous study has investigated the inhibition effectsof exogenous ghrelin on pancreatic secretion through a vagal pathway. However，effect of exogenous obestatin on exotrine of pancreas await further confirmation. Therefore, the aim of the present study was to analyse the effect of amidated obestatin in rats on pancreatic secretion. CCK-8 was used as a positivecontrol for stimulation of pancreatic secretion. In order to verify the internalvalidity of our study design. Inaddition, the in vitro effects of obestatin was determined.
FirsFirst part: A simple modified method for isolating and purifying the rat
pancreatic acinar cells
Objective：To develop a simple and economical modified method for isolatingand purifying pancreatic acinar cells in rat．
Methods：SD rats were starved for 12h with water given. Male SD rats(8-10weeks) weighting 200-250g were anesthetized by intraperitoneally injection ofsodium 3% pentobarbital and fixed on the operating plat. After sterilzed in 75% alcohol for 30minutes, the rat was made a midline incision, pancreas between duodenum and spleen was moved out. The pancreatic tissues were washedwith Hepes after intraabdominal anesthesia, then the pancreas is trimmed freeof fat and mesentery at room temperature(23℃), and 5ml of the collagenase(0.3 mg per ml in Krebs-Ringer bicarbonate solution) is injected into the interstitium of the tissue so as to distend the gland and rapidly expose the majority of its lobules to the enzymes. After 5 minutes, the tissue is cutted into small pieces about 2-4mm. The gland and excess enzyme solution is transferred to a 10ml erlenmeyer flask, equilibrated with 95%02-5% C02, and incubated at 37℃for 50 min with agitation at 60 oscillations per min. After the tissue structure collapse and loosens. Acinar cells are lightly liberated by sequential passage through pipets with tip diameters of 1.3 mm and 0.9 mm (5 times in each). The digestion was stopped by Krebs-Ringer bicarbonate solution containing 4% bovine-plasma albumin. Finally,acinar cells were harvested in conical centrifuge tubes, and centrifuged at 50g for 5 min to form pellets of packed cells and places them in a protective environment. After two further washes ofthe pooled cells in 8 ml of the above solution, they are suspended in Krebs-Ringer bicarbonate buffer containing 1% bovine-plasma albumin. All subsequent incubations are performed at 37℃.
ResultsResults: The cell count(5-6×106)and purification (70±6)％of the pancreatic acinar cels harvested by the modified method had no statistical diference compared with Wang's method. The cell activity (>90％)of modified method group was higher than the control method group．
Conclusion：Modified method is a simple and effective method for isolating and purifying the pancreatic acinar cells．
Second part: Effect of different doses of obestatin on amylase of pancreatic acinar/lobules in rats
Objective: To investigate the effects of different doses of obestatin on exocrine of pancreatic acinar/lobules in rats.
Methods: Preparation of dispersed pancreatic acini was performing according to improved method of Williams et al. The isolated pancreatic acini were incubated with different doses of obeatatin（0.1、1、10、30nmol/L）with or without CCK-8（10-10mol/L）for 1 hour. The acinar cells contained within the pellet were lysed with a sonicator for 5 minutes. Amylase content in the supernatants and pellet was detected using the Phadebas reagent and expressed as asa percentage of total amylase content. Pancreatic lobules, containing intrapancreatic nerve terminals and islets in addition to exocrine cells,were prepared bythe method of Scheele＆Palade. Male SD rats (8-10weeks) weighting 200-250gwere anesthetized by intraperitoneally injection of sodium 3% pentobarbital and fixed on the operating plat. Ater sterilzed in 75% alcohol for 30minutes, the rat was made a midline incision, pancreas between duodenum and spleenwas moved out. The pancreatic tissues were washed with Hepes after intraabdominal anesthesia, then the pancreas is trimmed free of fat and mesentery at r oom temperature(23℃),The pancreatic tissue was divided into 4mm×5mm lobular unites; Pancreatic lobules were incubated with different concentrations of obestatin for 30min at 37℃with or without KCL. The medium was then aspirated, diluted 1:10 and assayed for amylase.To the remaining tissue, 1ml KHB was added and the tissue was homogenized. Amylase in medium and tissue homogenates were assayed and expressed as the percentage of total tissue content.
ResultResults: Graded contrations of obestatin alone produced no significant changein basal amylase release and has no dose-dependent manner（P >0.05）. Co-incubation of CCK-8 with various concentrations of obestatin produced no alteration on amylase release compared to that observed with CCK-8 alone（P >0.05）.Incubated with potassium significantly increased amylase relase in pancreatic lobules（P <0.05）. Obeatatin produced no change on amylase release withpotassium in pancreatic lobules（P >0.05）.
Conclusions: Our results demonstrate that exogenous obestatin has no effectson pancreatic exocrine of rats in vitro study.
Third part: Effect of intravenous injection of obestatin on pancreatic exocrine secretion in rats
Objective: To evaluated the effects of intravenous injection of obestatin on pancreatic exocrine secretion in rats.
Methods: Sprague-Dawley weighting 250-300g rats were randomly divided into three groups(group A,B,C). The pancreatic exocrine model was made beforeexperiments. After anesthetized with intraperitoneal injection of 3%sodium pentobarbital. The rats were fixed on an operating plat and sterilzed by 75% alcohol for 30minutes,then the rat was made a midline incision,Two polyethylenecatheter (PE-10) were respectively placed into the femoral vein and tail veinfor infusion of obestatin and other drugs using a syringe-driven pump. The third polyethylene cannula (PE-10) was inserted into the common bile-pancreatic duct at Oddi’s sphincter. To permit the return of pancreatic-bile juice (P-BJ),the forth cannula was advanced into the duodenum slightly above the ampulla. Combined P-BJ was collected every 15 minutes after 30-min. The volumewas measured, and an aliquot was taken and diluted with distilled water for protein determination.The remainder of the undiluted P-BJ was pumped back into the rat via the duodenal cannula during the next collection period. Protein i n the P-BJ was measured spectro-photometrically by a BCA protein assay. Ingroup A and B，two obestatin boluses (1nmol kg-1h-1 and 5nmol kg-1h-1) weregiven intravenously. After 45-min, CCK-8 were given from femoral vein at adose of 400pmol kg-1h-1. In group C, CCK-8 was given alone as control.
ResultsResults: Intravenous infusion of CCK-8 evoked a significant increase in P-BJand pancreatic protein secretion (from 23.7±2.5 to 47.6±2.0 mg/15 min, P< 0.05). Contrast to the baseline levels, the volume of P-BJ and the protein output have no signifigant change in group A and B(15min, 30min; P > 0.05).The signifigant increase of protein secretion stimulated by CCK-8 was not change after injection of obestatin.
Conclusions: Intravenous injection of obestatin has no impacts on the volumeof P-BJ and the protein output. Combined injection of obestatin did not affect CCK-8–induced pancreatic secretion.
Forth part Effect of central administration of obestatin on pancreatic secretion
Objective：The present study was set up to bring more clarity into the contested effects of central administration of obestatin on pancreatic secretion .
Methods：Sprague-Dawley rats were randomly divided into three groups (group A,B,C). The pancreatic exocrine model was made before experiments. The rats were anesthetized with intraperitoneal injection of sodium pentobarbital. Apolyethylene catheter (PE-10) was placed into the tail vein for infusion of CCK using a syringe-driven pump. The second polyethylene cannula (PE-10) wasinserted into the common bile-pancreatic duct at Oddi’s sphincter. To permitthe return of pancreatic-bile juice (P-BJ), the forth cannula was advanced intothe duodenum slightly above the ampulla. Combined P-BJ was collected every 15 minutes after 30-min. The volume was measured, and an aliquot was taken and diluted with distilled water for protein determination. Protein in the PBJwas measured spectro-photometrically by a BCA protein assay. The remainder of the undiluted P-BJ was pumped back into the rat via the duodenal cannula during the next collection period. After that, an incision was made in thescalp, and a small hole was drilled in the skull using a dental drill. A micropipettes was positioned 1.0 mm lateral and 0.46 mm posterior to bregma, and2.20mm ventral from the skull surface. After surgery, animals were allowed to recover 30min prior to experiment. The micropipettes were filled with obest n the P-BJ was measured spectro-photometrically by a BCA protein assay. Ingroup A and B，two obestatin boluses (1nmol kg-1h-1 and 5nmol kg-1h-1) weregiven intravenously. After 45-min, CCK-8 were given from femoral vein at adose of 400pmol kg-1h-1. In group C, CCK-8 was given alone as control.
ResultsResults: Intravenous infusion of CCK-8 evoked a significant increase in P-BJand pancreatic protein secretion (from 23.7±2.5 to 47.6±2.0 mg/15 min, P< 0.05). Contrast to the baseline levels, the volume of P-BJ and the protein output have no signifigant change in group A and B(15min, 30min; P > 0.05).The signifigant increase of protein secretion stimulated by CCK-8 was not change after injection of obestatin.
Conclusions: Intravenous injection of obestatin has no impacts on the volumeof P-BJ and the protein output. Combined injection of obestatin did not affect CCK-8–induced pancreatic secretion.
Forth part Effect of central administration of obestatin on pancreatic secretion
Objective：The present study was set up to bring more clarity into the contested effects of central administration of obestatin on pancreatic secretion .
Methods：Sprague-Dawley rats were randomly divided into three groups (group A,B,C). The pancreatic exocrine model was made before experiments. The rats were anesthetized with intraperitoneal injection of sodium pentobarbital. Apolyethylene catheter (PE-10) was placed into the tail vein for infusion of CCK using a syringe-driven pump. The second polyethylene cannula (PE-10) wasinserted into the common bile-pancreatic duct at Oddi’s sphincter. To permitthe return of pancreatic-bile juice (P-BJ), the forth cannula was advanced intothe duodenum slightly above the ampulla. Combined P-BJ was collected every 15 minutes after 30-min. The volume was measured, and an aliquot was taken and diluted with distilled water for protein determination. Protein in the PBJwas measured spectro-photometrically by a BCA protein assay. The remainder of the undiluted P-BJ was pumped back into the rat via the duodenal cannula during the next collection period. After that, an incision was made in thescalp, and a small hole was drilled in the skull using a dental drill. A micropipettes was positioned 1.0 mm lateral and 0.46 mm posterior to bregma, and2.20mm ventral from the skull surface. After surgery, animals were allowed to recover 30min prior to experiment. The micropipettes were filled with obest period. In group B, intestinal perfusion of obestatin was performed at a speedof 2ml/h for 105min. The control group had no intestinal infusion.
ResultsResults: After the intraduodenal infusion of obestatin, the volume of combinedbile-pancreatic secretions and the protein output was not changed contrast tocontrol group( P > 0.05).
Conclusions: Intraduodenal infusion of obestatin has no effects on the volumeand the protein output of combined bile-pancreatic secretions
胰腺的外分泌功能受到中枢神经和肠神经以及大量的激素和脑肠肽组成的复杂网络的调节，业已证实：胆囊收缩素（CCK）、促胰液素、胰多肽、ghrelin、血管活性多肽、蛙皮素和胃动素等激素可以刺激胰腺的外分泌；而生长抑素、胰高糖素、降钙素、肾上腺素和去甲肾上腺素等可以抑制胰腺的外分泌，由于对obestatin功能及受体研究存在着大量的争论，并且obestatin对胰腺外分泌功能的调节作用目前还不清楚。本研究拟采用大鼠离体腺泡、小叶及在体胰腺外分泌功能研究模型，应用分离大鼠离体腺泡细胞和胰腺小叶、静脉持续注射、侧脑室内微量注射等技术明确obestatin对胰腺外分泌功能的调节作用及其机制，并观察obestatin及其它脑肠肽对大鼠胰腺外分泌调节的交互作用，用胆囊收缩素作为阳性对照，从而进一步完善胰腺外分泌调节的机制和obestatin的生物学功能。
第一部分一种改良的胰腺腺泡提取方法
目的：对经典的Williams提取胰腺腺泡方法进行改良，以期探索一种简便，经济和稳定的胰腺腺泡分离纯化的新方法。
方法：雄性SD大鼠，鼠龄8-10w，体重200-250g。在无菌条件下，以3%戊巴比妥钠，按照15mg/kg剂量腹腔内注射麻醉后固定于解剖板上，然后放于75%酒精内消毒30分钟，在无菌操作台上于剑突下3cm处腹部正中，分层剪开皮肤、腹肌；循胃、十二指肠球降部及脾脏之间取出胰腺。室温下（23℃）在充过氧的Hepes液中将血迹冲洗干净，并用镊子轻轻的将胰腺周围脂肪、淋巴结以及腹膜剔除。以低浓度Ⅰ型胶原酶（0.3mg/ml）作为腺泡细胞分离液，用带有1ml注射器针头的注射器在大鼠胰腺间质内多点注射使胰腺小叶展开，5分钟后用小剪刀将其剪成1～2mm碎块放于含100%氧分离液中震荡消化50分钟，然后经过过滤，离心、再悬浮等过程成功提取大鼠胰腺腺泡。
结果：平均每只大鼠可以提取5～6×106个细胞，经HE和台盼蓝染色，细胞存活率大于90%，细胞纯化率为(70±6)％与王连才方法相比差异无显著性。
结论：我们认为本实验室采用的改进方法是一种简便，经济和稳定的腺泡分离纯化方法。
第二部分Obestatin对大鼠胰腺腺泡/小叶淀粉酶影响
目的：研究不同剂量肥胖抑制素对大鼠离体胰腺腺泡及胰腺小叶淀粉酶分泌的影响。
材料与方法：雄性SD大鼠，鼠龄8-10w，体重200-250g，36只。采用改良的Williams法体外分离大鼠胰腺腺泡（方法同第一部分），将胰腺腺泡与不同浓度obestatin（0.1、1、10、30nmol/L）在37℃培养箱中培养1h。在对照组腺泡中，加入胆囊收缩素（CCK-8，10-10mol/L）和obestatin共培养1h，测定培养液中上清测定淀粉酶水平，将剩余腺泡细胞放于超声破碎仪打碎（5分钟），离心后去上清测定淀粉酶水平。参照Scheele＆Palade法提取胰腺小叶：在无菌条件下，以3%戊巴比妥钠，按照15mg/kg剂量腹腔内注射麻醉后固定于解剖板上，放于75%的酒精内消毒30分钟，在无菌操作台上于剑突下3cm处腹部正中，分层剪开皮肤、腹肌；循胃、十二指肠球降部及脾脏之间取出胰腺。室温下（23℃）在充过氧的KHB液中将血迹冲洗干净，并用镊子将胰腺周围脂肪、淋巴结以及腹膜剔除。分离含有胰腺内神经末端、胰岛细胞及外分泌细胞的胰腺小叶（大小4mm～5mm），胰腺小叶与obestatin共培养30min，对照组中KCL（75mmol/L）和obestatin共培养30min，取上清测定淀粉酶水平，将剩余胰腺小叶组织放于组织匀浆器中打碎，离心后测定上清液中淀粉酶水平。
结果：obestatin不影响胰腺腺泡和胰腺小叶淀粉酶释放，且不随浓度的增高而改变(P >0.05)。CCK-8和KCL能分别刺激胰腺腺泡、胰腺小叶淀粉酶释放（P<0.05），而外源性obestatin对CCK-8和KCL引起的胰腺腺泡和胰腺小叶淀粉酶分泌增加无影响，差异无统计学意义（P >0.05）。
结论：体外实验中，外源性obestatin对大鼠胰腺腺泡及胰腺小叶淀粉酶分泌无影响，obestatin对活体大鼠胰腺外分泌影响需要进一步研究。
第三部分静脉注射obestatin对大鼠胰腺外分泌的影响
目的：采用静脉持续注射的方法研究不同剂量肥胖抑制素对大鼠胰腺外分泌功能的影响。
材料与方法：24只SD大鼠随机分成3组（N=8），麻醉后制作胰腺外分泌模型：所有大鼠用3%戊巴比妥钠（15mg/kg）腹腔内麻醉，麻醉成功后固定于操作板上，放于75%酒精内消毒30分钟，腹中线上逐层切开上腹部约3cm，于胃及十二指肠之间找到胰管及十二指肠乳头，经十二指肠乳头对侧肠管插入聚乙烯导管（PE-10）约0.5cm,尾静脉及右侧股动脉留置输液导管，十二指肠乳头上端插入PE-10以回输胆胰混合液（P-BJ）。模型稳定30分钟后开始实验，每15分钟经胰管插管收集一次P-BJ测定体积，并留取50ul测定蛋白含量，剩余P-BJ经由十二指肠回输。所有大鼠于实验开始后15分钟收集一次P-BJ作为基础分泌。A组和B组于实验开始15分钟后分别给予尾静脉持续微泵注射1nmolkg-1h-1obestatin和5nmolkg-1h-1 obestatin，45分钟后给予股静脉微泵注射400pmolkg-1h-1 CCK-8；C组于实验开始45分钟后给予股静脉注射400pmolkg-1h-1CCK-8作为对照。
结果：静脉注射CCK-8 15min后胰腺分泌量开始增高，并于30min后达高峰，而胰腺蛋白也呈相应升高，差异均具有显著性（P <0.05），单独静脉注射obestatin对大鼠胰腺外分泌量及胰液蛋白无影响（P >0.05），obestatin与CCK-8同时注射并不能改变由CCK-8引起的胰腺外分泌及胰液蛋白的增高。结论：外周静脉给予CCK-8可增加胰液和胰液蛋白的分泌，静脉注射obestatin对胰腺外分泌功能无影响，并且不能改变由CCK-8引起的胰液和胰液蛋白的增高。
第四部分脑室微量注射obestatin对大鼠胰腺外分泌的影响
目的采用侧脑室微量注射的方法研究肥胖抑制素对大鼠胰腺外分泌功能的影响。
方法SD大鼠随机分成3组（A,B,C组），麻醉后制作胰腺外分泌模型：所有大鼠用3%戊巴比妥钠（15mg/kg）腹腔内麻醉，麻醉成功后固定于操作板上，放于75%酒精内消毒30分钟，沿着腹中线逐层切开上腹部约3cm，于胃及十二指肠之间找到胰管及十二指肠乳头，经十二指肠乳头对侧肠管插入聚乙烯导管（PE-10）约0.5cm,尾静脉留置输液导管，十二指肠乳头上端插入PE-10以回输胆胰混合液（P-BJ）。模型稳定30分钟后开始实验，每15分钟经胰管插管收集一次P-BJ测定体积，并留取50ul测定蛋白含量，剩余P-BJ经由十二指肠回输。所有大鼠于实验开始后15分钟收集一次P-BJ作为基础分泌。A组和B组于实验开始15分钟后分别给予侧脑室微量注射30nmolkg-1obestatin,C组于实验开始45分钟后给予静脉注射400pmol kg-1h-1 CCK-8作为对照。
结果：与基础分泌相比，侧脑室微量注射obestatin后大鼠胰腺外分泌量及胰液蛋白与基础相比差异无显著性（P >0.05），静脉注射CCK-8可以引起胰腺外分泌量的增加，并于30min后达高峰，差异具有显著性意义；而胰腺蛋白也呈相应升高，与大鼠基础分泌相比，差异具有显著性（P <0.05），obestatin脑室内注射并不能改变由CCK-8引起的胰腺外分泌及胰液蛋白的增高。
结论外周静脉给予CCK-8增加胰液和胰液蛋白的分泌，侧脑室微量注射obestatin对胰腺外分泌功能无影响，并且不能改变由CCK-8引起的胰液和胰液蛋白的增高。
第五部分十二指肠内灌注obestatin对大鼠胰腺外分泌影响的初步研究
目的：采用十二指肠内持续灌注的方法研究不同剂量obestatin对大鼠胰腺外分泌功能的影响
方法：SD大鼠14只，随机分成2组，麻醉后制作胰腺外分泌模型：所有大鼠用3%戊巴比妥钠（15mg/kg）腹腔内麻醉，麻醉成功后固定于操作板上，放于75%酒精内消毒30分钟，在上腹部打开腹腔，于胃及十二指肠之间找到胰管及十二指肠乳头，经十二指肠乳头对侧肠管插入聚乙烯导管（PE-10）约0.5cm，十二指肠乳头上端插入PE-10以微泵灌注obestatin及回输胆胰混合液（P-BJ）。模型稳定30分钟后开始实验，每15分钟经胰管插管收集一次P-BJ测定体积，并留取50ul测定蛋白含量，剩余P-BJ经由十二指肠回输。所有大鼠于实验开始后15分钟收集一次P-BJ作为基础分泌。B组于实验开始15分钟后分别给予十二指肠内微泵灌注5nmolkg-1h-1obestatin 105分钟, A组除了不做十二指肠灌注以外，其余处理均同B组。
结果十二指肠内持续灌注5nomlkg-1h-1obestatin各时点大鼠胰腺外分泌量及胰液蛋白与对照组相比均无显著性差异（P >0.05）结论十二指肠内持续灌注obestatin对大鼠胰腺外分泌量及胰液蛋白无影响。
全文小结：本课题通过采用①建立离体大鼠胰腺腺泡细胞及胰腺小叶，分别与不同浓度的obestatin及腺泡刺激剂(CCK-8)和胰腺小叶刺激剂（KCL）进行共培养，观察细胞内外淀粉酶水平的变化，进而分析obestatin对胰腺外分泌功能的影响；②建立活体麻醉大鼠胰腺外分泌动物模型，静脉给予不同浓度的obestatin观察其对胰腺外分泌量及胰液蛋白的影响；并与胰腺刺激剂CCK-8共同注射，观察其是否与CCK-8存在协同或拮抗作用。③建立麻醉大鼠胰腺外分泌模型，通过侧脑室微量注射obestatin，观察其是否能通过中枢神经影响大鼠胰腺外分泌功能。④通过十二指肠内灌注的方法观察obestatin对大鼠胰腺外分泌功能的影响，结果显示：体外实验中，外源性obestatin对大鼠胰腺腺泡及胰腺小叶淀粉酶分泌无影响，外周静脉给予CCK-8可增加胰液和胰液蛋白的分泌；在麻醉大鼠中，静脉给予obestatin对胰腺外分泌功能无影响，并且不能改变由CCK-8引起的胰液和胰液蛋白的增高。脑室内微量注射obestatin对胰腺外分泌功能无影响，并且不能改变由CCK-8引起的胰液和胰液蛋白的增高。单独十二指肠内灌注obestatin对大鼠胰腺外分泌功能无影响。
Obestatin is a recently identified 23-amino acid gut-derived peptide encodedby the preproghrelin gene. Although originating from the same precursor as the widely studied appetite-stimulating peptide ghrelin, obestatin was reported to bring on antithetical physiological effects with regards to food intake and body weight regulation. Obestatin and ghrelin were called yin and yang peptidefor encoded by the same gene and have opposite physiological functions in several systems. ghrelin, a 28-amino acid and ligand for the growth hormone secretagogue receptor-1a, has demonstrated to be a orexigenic peptide. As a ghrelin-associated peptide, obestatin was derived from the same proghrelin as ghrelin by different post-translational cleavage and borned with contestation. In 2005, zhang et al first identified obestatin as a 23-amino acid isolated from stomach mucosa and encoded by preproghrelin gene. The initial work showed that intraperitoneal or intracerebroventricular injection of human obestatin supressed food intake and decreased body-weight gain. Further studies indicated thatobestatin was involved in inhibiting thirst and anxiety , improving memory, affecting cell proliferation, controlling fluid homeostasis. However, controversialresults focus on its anorexigenic actions. In vivo and vitro studies, several investigators reported anorexigenic effects of peripheral obestatin in rodents. Furthermore, decreased gastric body mucosa obestatin expression and obestatin levels in plasma in obese patents indicated that obestatin may play an important role in the regulation of energy balance, apparently being able to antagonize the effects of ghrelin. In spite of those evidence, the anorexigenic effects of obestatin were not reproduced by subsequent studies using differernt dose of obestatin, experimental protocols and species or origin of the peptide. Different from ghrelin, obestatin has high affinity for GPR39 which belongs to the ghrelin receptor subfamily and hypothesized to be a congate ligand for GPR39. Binding studies and molecular approaches indicated that GPR39 was expressed ina wide range of tissues including central nevous system, peripheral organs, especially at a higher level in digestive system. Opposite to the initial report, later studies from different groups were unalble to corroboratre the binding between obestatin and GPR39. In 2007, Zhang et al. proved again that their original result of obestatin for GPR39 was unreproductive. Other receptor of obestatin was reported by Yasuda et al that Zn2+ could be a physiologically relevant agonist or modulator of GPR39.
Pancreatic exocrine secretion is known to be modulated by a number of brain-gut peptides directly or through brain-pancreas anxis. It has been reported that cholecystokinin(CCK), secretin , pancreatic polypeptide(PP), ghrelin and amphetamine-regulated transcript peptide(CART) stimulate pancreatic secretion, whereas neuropeptide Y and somatostatin serve as inhibitors. Interestingly, like ghrelin, obestatin was found to stimulate the secretation of pancreatic juice enzymes through a vagal pathway in anaesthetized rats. Saleem and co-workers reported that obestatin inhibited the secretion of both somatostatin and PP in ratislets. These results need a further insight into the influence of obestatin on pancreatic secretion. Since previous study has investigated the inhibition effectsof exogenous ghrelin on pancreatic secretion through a vagal pathway. However，effect of exogenous obestatin on exotrine of pancreas await further confirmation. Therefore, the aim of the present study was to analyse the effect of amidated obestatin in rats on pancreatic secretion. CCK-8 was used as a positivecontrol for stimulation of pancreatic secretion. In order to verify the internalvalidity of our study design. Inaddition, the in vitro effects of obestatin was determined.
FirsFirst part: A simple modified method for isolating and purifying the rat
pancreatic acinar cells
Objective：To develop a simple and economical modified method for isolatingand purifying pancreatic acinar cells in rat．
Methods：SD rats were starved for 12h with water given. Male SD rats(8-10weeks) weighting 200-250g were anesthetized by intraperitoneally injection ofsodium 3% pentobarbital and fixed on the operating plat. After sterilzed in 75% alcohol for 30minutes, the rat was made a midline incision, pancreas between duodenum and spleen was moved out. The pancreatic tissues were washedwith Hepes after intraabdominal anesthesia, then the pancreas is trimmed freeof fat and mesentery at room temperature(23℃), and 5ml of the collagenase(0.3 mg per ml in Krebs-Ringer bicarbonate solution) is injected into the interstitium of the tissue so as to distend the gland and rapidly expose the majority of its lobules to the enzymes. After 5 minutes, the tissue is cutted into small pieces about 2-4mm. The gland and excess enzyme solution is transferred to a 10ml erlenmeyer flask, equilibrated with 95%02-5% C02, and incubated at 37℃for 50 min with agitation at 60 oscillations per min. After the tissue structure collapse and loosens. Acinar cells are lightly liberated by sequential passage through pipets with tip diameters of 1.3 mm and 0.9 mm (5 times in each). The digestion was stopped by Krebs-Ringer bicarbonate solution containing 4% bovine-plasma albumin. Finally,acinar cells were harvested in conical centrifuge tubes, and centrifuged at 50g for 5 min to form pellets of packed cells and places them in a protective environment. After two further washes ofthe pooled cells in 8 ml of the above solution, they are suspended in Krebs-Ringer bicarbonate buffer containing 1% bovine-plasma albumin. All subsequent incubations are performed at 37℃.
ResultsResults: The cell count(5-6×106)and purification (70±6)％of the pancreatic acinar cels harvested by the modified method had no statistical diference compared with Wang's method. The cell activity (>90％)of modified method group was higher than the control method group．
Conclusion：Modified method is a simple and effective method for isolating and purifying the pancreatic acinar cells．
Second part: Effect of different doses of obestatin on amylase of pancreatic acinar/lobules in rats
Objective: To investigate the effects of different doses of obestatin on exocrine of pancreatic acinar/lobules in rats.
Methods: Preparation of dispersed pancreatic acini was performing according to improved method of Williams et al. The isolated pancreatic acini were incubated with different doses of obeatatin（0.1、1、10、30nmol/L）with or without CCK-8（10-10mol/L）for 1 hour. The acinar cells contained within the pellet were lysed with a sonicator for 5 minutes. Amylase content in the supernatants and pellet was detected using the Phadebas reagent and expressed as asa percentage of total amylase content. Pancreatic lobules, containing intrapancreatic nerve terminals and islets in addition to exocrine cells,were prepared bythe method of Scheele＆Palade. Male SD rats (8-10weeks) weighting 200-250gwere anesthetized by intraperitoneally injection of sodium 3% pentobarbital and fixed on the operating plat. Ater sterilzed in 75% alcohol for 30minutes, the rat was made a midline incision, pancreas between duodenum and spleenwas moved out. The pancreatic tissues were washed with Hepes after intraabdominal anesthesia, then the pancreas is trimmed free of fat and mesentery at r oom temperature(23℃),The pancreatic tissue was divided into 4mm×5mm lobular unites; Pancreatic lobules were incubated with different concentrations of obestatin for 30min at 37℃with or without KCL. The medium was then aspirated, diluted 1:10 and assayed for amylase.To the remaining tissue, 1ml KHB was added and the tissue was homogenized. Amylase in medium and tissue homogenates were assayed and expressed as the percentage of total tissue content.
ResultResults: Graded contrations of obestatin alone produced no significant changein basal amylase release and has no dose-dependent manner（P >0.05）. Co-incubation of CCK-8 with various concentrations of obestatin produced no alteration on amylase release compared to that observed with CCK-8 alone（P >0.05）.Incubated with potassium significantly increased amylase relase in pancreatic lobules（P <0.05）. Obeatatin produced no change on amylase release withpotassium in pancreatic lobules（P >0.05）.
Conclusions: Our results demonstrate that exogenous obestatin has no effectson pancreatic exocrine of rats in vitro study.
Third part: Effect of intravenous injection of obestatin on pancreatic exocrine secretion in rats
Objective: To evaluated the effects of intravenous injection of obestatin on pancreatic exocrine secretion in rats.
Methods: Sprague-Dawley weighting 250-300g rats were randomly divided into three groups(group A,B,C). The pancreatic exocrine model was made beforeexperiments. After anesthetized with intraperitoneal injection of 3%sodium pentobarbital. The rats were fixed on an operating plat and sterilzed by 75% alcohol for 30minutes,then the rat was made a midline incision,Two polyethylenecatheter (PE-10) were respectively placed into the femoral vein and tail veinfor infusion of obestatin and other drugs using a syringe-driven pump. The third polyethylene cannula (PE-10) was inserted into the common bile-pancreatic duct at Oddi’s sphincter. To permit the return of pancreatic-bile juice (P-BJ),the forth cannula was advanced into the duodenum slightly above the ampulla. Combined P-BJ was collected every 15 minutes after 30-min. The volumewas measured, and an aliquot was taken and diluted with distilled water for protein determination.The remainder of the undiluted P-BJ was pumped back into the rat via the duodenal cannula during the next collection period. Protein i n the P-BJ was measured spectro-photometrically by a BCA protein assay. Ingroup A and B，two obestatin boluses (1nmol kg-1h-1 and 5nmol kg-1h-1) weregiven intravenously. After 45-min, CCK-8 were given from femoral vein at adose of 400pmol kg-1h-1. In group C, CCK-8 was given alone as control.
ResultsResults: Intravenous infusion of CCK-8 evoked a significant increase in P-BJand pancreatic protein secretion (from 23.7±2.5 to 47.6±2.0 mg/15 min, P< 0.05). Contrast to the baseline levels, the volume of P-BJ and the protein output have no signifigant change in group A and B(15min, 30min; P > 0.05).The signifigant increase of protein secretion stimulated by CCK-8 was not change after injection of obestatin.
Conclusions: Intravenous injection of obestatin has no impacts on the volumeof P-BJ and the protein output. Combined injection of obestatin did not affect CCK-8–induced pancreatic secretion.
Forth part Effect of central administration of obestatin on pancreatic secretion
Objective：The present study was set up to bring more clarity into the contested effects of central administration of obestatin on pancreatic secretion .
Methods：Sprague-Dawley rats were randomly divided into three groups (group A,B,C). The pancreatic exocrine model was made before experiments. The rats were anesthetized with intraperitoneal injection of sodium pentobarbital. Apolyethylene catheter (PE-10) was placed into the tail vein for infusion of CCK using a syringe-driven pump. The second polyethylene cannula (PE-10) wasinserted into the common bile-pancreatic duct at Oddi’s sphincter. To permitthe return of pancreatic-bile juice (P-BJ), the forth cannula was advanced intothe duodenum slightly above the ampulla. Combined P-BJ was collected every 15 minutes after 30-min. The volume was measured, and an aliquot was taken and diluted with distilled water for protein determination. Protein in the PBJwas measured spectro-photometrically by a BCA protein assay. The remainder of the undiluted P-BJ was pumped back into the rat via the duodenal cannula during the next collection period. After that, an incision was made in thescalp, and a small hole was drilled in the skull using a dental drill. A micropipettes was positioned 1.0 mm lateral and 0.46 mm posterior to bregma, and2.20mm ventral from the skull surface. After surgery, animals were allowed to recover 30min prior to experiment. The micropipettes were filled with obest n the P-BJ was measured spectro-photometrically by a BCA protein assay. Ingroup A and B，two obestatin boluses (1nmol kg-1h-1 and 5nmol kg-1h-1) weregiven intravenously. After 45-min, CCK-8 were given from femoral vein at adose of 400pmol kg-1h-1. In group C, CCK-8 was given alone as control.
ResultsResults: Intravenous infusion of CCK-8 evoked a significant increase in P-BJand pancreatic protein secretion (from 23.7±2.5 to 47.6±2.0 mg/15 min, P< 0.05). Contrast to the baseline levels, the volume of P-BJ and the protein output have no signifigant change in group A and B(15min, 30min; P > 0.05).The signifigant increase of protein secretion stimulated by CCK-8 was not change after injection of obestatin.
Conclusions: Intravenous injection of obestatin has no impacts on the volumeof P-BJ and the protein output. Combined injection of obestatin did not affect CCK-8–induced pancreatic secretion.
Forth part Effect of central administration of obestatin on pancreatic secretion
Objective：The present study was set up to bring more clarity into the contested effects of central administration of obestatin on pancreatic secretion .
Methods：Sprague-Dawley rats were randomly divided into three groups (group A,B,C). The pancreatic exocrine model was made before experiments. The rats were anesthetized with intraperitoneal injection of sodium pentobarbital. Apolyethylene catheter (PE-10) was placed into the tail vein for infusion of CCK using a syringe-driven pump. The second polyethylene cannula (PE-10) wasinserted into the common bile-pancreatic duct at Oddi’s sphincter. To permitthe return of pancreatic-bile juice (P-BJ), the forth cannula was advanced intothe duodenum slightly above the ampulla. Combined P-BJ was collected every 15 minutes after 30-min. The volume was measured, and an aliquot was taken and diluted with distilled water for protein determination. Protein in the PBJwas measured spectro-photometrically by a BCA protein assay. The remainder of the undiluted P-BJ was pumped back into the rat via the duodenal cannula during the next collection period. After that, an incision was made in thescalp, and a small hole was drilled in the skull using a dental drill. A micropipettes was positioned 1.0 mm lateral and 0.46 mm posterior to bregma, and2.20mm ventral from the skull surface. After surgery, animals were allowed to recover 30min prior to experiment. The micropipettes were filled with obest period. In group B, intestinal perfusion of obestatin was performed at a speedof 2ml/h for 105min. The control group had no intestinal infusion.
ResultsResults: After the intraduodenal infusion of obestatin, the volume of combinedbile-pancreatic secretions and the protein output was not changed contrast tocontrol group( P > 0.05).
Conclusions: Intraduodenal infusion of obestatin has no effects on the volumeand the protein output of combined bile-pancreatic secretions
引文
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1. Konturek, S.J., R. Zabielski, J.W. Konturek, and J. Czarnecki, Neuroendocrinology of thepancreas; role of brain-gut axis in pancreatic secretion. Eur J Pharmacol, 2003. 481(1): p.1-14.
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