Fluorescence Resonance Energy Transfer Competitive Binding Assay for Secretin Receptor （Class B-GPCR）

Human secretin is responsible for carrying a number of physiological functions including energy and water homeostasis, thus making secretin receptor a promising target for drug development. For GPCRs （G protein-coupled receptors）, radioactive ligands are usually used in conventional binding assays to characterize the binding affinities of the ligands. An alternative non-hazardous fluorescence based binding assay is lucrative over the radio-ligand assays. Here, we have developed a FRET （fluorescence resonance energy transfer） competitive binding assay for human secretin receptor. The receptor gene sequence is cloned in the SNAP （single nucleotide amplified polymorphisms） tag-plasmid and expressed in CHO （chinese hamster ovary）-K1 cells. Its expression and function is confirmed with immunofluorescence localization and receptor activation. The receptor and the ligand are labeled with fluorescent donor （Tb） and acceptor （Alexa488）. FRET signals are produced when the labeled ligand is bound to the receptor and the same drop when it is displaced by the test compounds. The saturation concentration of the receptor labeling is 100 nM, and the ligand Kd value is 500 nM. At these concentrations, the IC50 of unlabeled secretin is 1.63 4- 3.55 nM. Additionally, few class-B ligands are screened and hold good correlation with traditional radio-ligand assay. Henceforth, this FRET binding assay can be efficiently used as a primary screening tool for peptide analogs.

Effect of cholecystokinin and secretin on contractile activity of isolated gastric muscle strips in guinea pigs

AIM To study the effect of cholecystokinin-octapeptide（CCK-8）and secretin on contractileactivity of isolated gastric muscle strips inguinea pigs.METHODS Each isolated gastric muscle stripwas suspended in a tissue chamber containing5 mL Krebs solution constantly warmed by waterjacked at 37℃ and supplied with a mixed gas of95% O2 and 5% CO2 After incubating for lhunder 1 g tension,varied concentrations of CCK-8 and secretin were added respectively in thetissue chamber and the contractile response wasmeasured isometrically on ink-writing recorders.RESULTS CCK-8 could increase①all regionalcircular and longitudinal muscular tension at rest（fundus LM 19.7%±2.1%,P【0.01;fundus CM16.7%±2.2%,P【0.01;gastric body LM 16.8%±2.3%,P【0.01;body CM 12.7%±2.6%,P【0.01;antrum LM 12.3%±1.3%,P【0.01;antrum CM 16.7%±4.5%,P【0.01;pylous CM12.7%±5.0%,P【0.05）;②contractilefrequencies of body LM,both LM and CM ofantrum and pylorus CM（5.1/min±0.2/min to5.6/min±0.2/min,5.9/min±0.2/min to 6.6/min±0.l/min,5.4/min±0.3/min to 6.3/min±0.4/rain,1.3/min±0.2/min to 2.3/min±0.3/min,respectively,P【0.05）;③the mean contractileamplitude of antral circular muscle（58.6%±18.4%,P【0.05）and ④the motility index ofpylorus CM（145.0%±23.8%,P【0.01）,butdecrease the mean contractile amplitude ofgastric body and antral LM（-10.3%±3.3%,-10.5%±4.6%,respectively,P【0.05）.All the CCK-8 effects were not blocked by atropine orindomethacin.Secretin had no effect on gastricsmooth muscle activity.CONCLUSION CCK-8 possessed bothexcitatory and inhibitory action on contractileactivity of different regions of stomach in guineapigs.Its action was not mediated via cholinergicM receptor and endogenous prostagiandinreceptor,

Quantification of pancreatic exocrine function of chronic pancreatitis with secretin-enhanced MRCP

AIM:To obtain reference values for pancreatic flow output rate(PFR)and peak time(PT)in healthy volunteers and chronic pancreatitis(CP);to correlate quantification of secretin enhanced magnetic resonance cholangiopancreatography(SMRCP)of pancreatic fluid output following secretin with fecal elastase-1(FE-1)tests.METHODS:The present study includes 53 subjects comprised of 17 healthy individuals and 36 patients with CP from April 2011 to January 2013.The 36 patients with CP were divided into three groups of mild CP(n=14),moderate CP(n=19)and advanced CP(n=3)by M-ANNHEIM classification for CP..Fifty-three cases underwent FE-1 test and magnetic resonance imaging using 3.0 T-device(Signa EXCITE,GE Healthcare).Coronal T2-weighted single-shot turbo spin-echo,spiratory triggered,covering the papillae,duodenum and small bowel.MRCP was performed with a heavily T2-weighted fat-suppressed long TE HASTE sequence (thick slab 2D MRCP sequence),repeated every 2 min up to 11 min after 0.1 mL/kg secretin injection(Secrelux,Sanochemia,Germany).FE-1 test used sandwich enzyme-linked immunosorbent assay(ELISA)test(ScheBo.Tech,Germany).RESULTS:A good linear correlation showed between the calculated volume and the actual volume by Phantom experiments.Fifty-three paired Quantification of secretin enhanced magnetic resonance cholangiopancreatography(MRCPQ)and FE-1 data sets were analyzed.The mean FE-1 of 53 cases was 525.41±94.44μg/g for 17 healthy volunteers,464.95±136.13μg/g for mild CP,301.55±181.55μg/g for moderate CP,229.30±146.60μg/g for advanced CP.Also,there was statistically significant difference in FE-1(P=0.0001)between health and CP.The mean values of PFR and PT were 8.18±1.11 mL/min,5.76±1.71 min for normal;7.27±2.04 mL/min,7.71±2.55 min for mild CP;4.98±2.57 mL/min,9.10±3.00 min for moderate CP;4.13±1.83 mL/min,12.33±1.55 min for advanced CP.Further,statistically significant difference in PFR(P=0.0001)and PT(P=0.0001)was observed between health and CP.Besides,there was correlation(r=0.79)and consistency(K=0.6)between MRCPQ and ELISA Test.It was related between M-ANNHEIM classification and PFR(r=0.55),FE-1(r=0.57).CONCLUSION:SMRCP can provide a safe,non-invasive and efficient method to evaluate the exocrine function of the pancreas.

Diagnostic role of secretin-enhanced MRCP in patients with unsuccessful ERCP

AIM:To evaluate the value of MR cholangiopancreatography (MRCP)in patients in whom endoscopic retrograde cholangiopancreatography(ERCP)was unsuccessfully performed by experts in a tertiary center. METHODS:From January 2000 to June 2003,22 patients fulfilled the inclusion criteria.The indications for ERCP were obstructive jaundice(n=9),abnormal liver enzymes(n=8), suspected chronic pancreatitis(n=2),recurrent acute pancreatitis(n=2),or suspected pancreatic cancer(n=1). The reasons for the ERCP failure were the postsurgical anatomy(n=7),duodenal stenosis(n=3),duodenal diverticulum(n=2),and technical failure(n=10).MRCP images were evaluated before and 5 and 10 rain after i.v. administration of 0.5 IU/kg secretin. RESULTS:The MRCP images were diagnosed in all 21 patients.Five patients gave normal MR findings and required no further intervention.MRCP revealed abnormalities(primary sclerosing cholangitis,chronic pancreatitis,cholangitis,cholecystolithiasis or common bile duct dilation)in 20 patients,who were followed up clinically.Four patients subsequently underwent laparotomy(hepaticojejunostomy in consequence of common bile duct stenosis caused by unresectable pancreatic cancer;hepaticotomy+Kehr drainage because of insufficient biliary-enteric anastomosis;choledochoj- ejunostomy,gastrojejunostomy and cysto-Wirsungo gastrostomy because of chronic pancreatitis,or choledochojejunostomy because of common bile duct stenosis caused by chronic pancreatitis).Three patients participated in therapeutic percutaneous transhepatic drainage.The indications were choledocholithiasis with choledochojejunostomy,insufficient biliary-enteric anastomosis,or cholangiocarcinoma. CONCLUSION:MRCP can assist the diagnosis and management of patients in whom ERCP is not possible.

阿尔茨海默病患者血清Ghrelin的表达水平及临床意义

目的:探讨阿尔茨海默病(AD)患者血清生长激素促泌素(Ghrelin)的表达水平及临床意义。方法:纳入48例AD患者作为病例组(AD组);同期52名健康体检者作为对照组。采集受试者清晨空腹外周静脉血3 mL,离心,收集血清,采用酶联免疫吸附试验测定血清Ghrelin水平。比较两组血清Ghrelin水平及MMSE评分,不同特征AD患者及病情严重程度血清Ghrelin水平变化;采用ROC曲线分析Ghrelin对AD诊断价值;采用Pearson相关分析Ghrelin与不同临床资料相关性。结果:AD组Ghrelin水平和MMSE评分均低于对照组(P<0.05)。不同性别、年龄和体质量指数患者血清Ghrelin水平无统计学差异(P>0.05);病程>3年患者血清Ghrelin水平低于病程≤3年患者(P<0.05)。不同病情严重程度AD患者血清Ghrelin水平比较:重度组<中度组<轻度组(P<0.05)。ROC曲线分析显示,血清Ghrelin诊断AD敏感度、特异度分别为87.5%、91.7%。经Pearson分析,Ghrelin与病程负相关,与MMSE评分正相关(P<0.05)。结论:AD患者血清Ghrelin水平下降,且与患者病程>3年、病情重度及MMSE评分下降相关。

防风通圣散对肥胖症小鼠胆囊收缩素、胰高血糖素、促胰液素表达的影响

研究防风通圣散对于肥胖小鼠肠组织中胆囊收缩素、胰高血糖素、促胰液素的影响,探索防风通圣散治疗肥胖症的作用机理。方法以DOI小鼠为研究对象,高脂喂养8周建立单纯性肥胖小鼠模型,将小鼠随机分为空白组、模型组、中药组、西药组,每组6只,连续干预21天。观察并评价肥胖小鼠的体质量变化情况,油红染色观察脂肪组织病理学变化,ELISA法测定肥胖模型小鼠小肠组织中胆囊收缩素、胰高血糖素、促胰液素的表达水平,qPCR法检测于小鼠棕色脂肪中解耦连蛋白(UCP-1)mRNA变化。结果与模型组比较,中药组(防风通圣散)肥胖模型小鼠体质量减少,小鼠脂肪组织中染色减少,小肠组织胆囊收缩素显著升高,胰高血糖素显著升高,促胰液素显著升高。结论防风通圣散对肥胖小鼠的体质量具有抑制作用,可以减少脂肪组织中脂滴的数量与大小,其机制可能与升高肠道组织中的胆囊收缩素水平、胰高血糖素水平、促胰液素水平,提升棕色脂肪组织中的解耦连蛋白(UCP-1)mRNA水平有关。

Pancreatic function testing: Here to stay for the 21st century

The diagnosis of Chronic Pancreatitis （CP） is based on the detection of abnormal structure or function of the diseased pancreas. The pancreatic function tests more accurately determine the presence of CP than tests of structure, especially for early stage disease. The function tests can be divided into two categories： non- invasive and invasive. The invasive ＂tube＂ tests can reliably detect mild, early CP, but are only available at a few referral centers and tend to be poorly tolerated by patients. The non-invasive tests are easy to obtain, but tend to perform poorly in patients with early, mild disease. Therefore, no one test is useful in all clinical situations, and a detailed understanding of the rational, pathophysiologic basis, strengths, and limitations of various tests is needed. This review highlights the role of various pancreatic function tests in the diagnosis of CP including fecal fat analysis, fecal elastase, fecal chymotrypsin, serum trypsin, the secretin stimulation test, the cholecystokinin （CCK） stimulation test, the combined secretin-CCK stimulation test, the intraductal and endoscopic secretin stimulation tests, and the functional magnetic resonance imaging of the pancreas after secretin stimulation.

缩胆囊素和促胰液素对豚鼠离体胃平滑肌运动的影响

用８个肌槽同时记录豚鼠胃不同部位肌条的收缩活动，以观察八肽缩胆囊素（ＣＣＫ－８）和促胰液素的影响。结果表明：ＣＣＫ－８能（１）增高各部位纵行肌和环行肌的张力；（２）加快胃体纵行肌，胃窦纵行肌、环行肌和幽门环行肌的收缩频率；（３）增大胃窦环行肌收缩波平均振幅和（４）增加幽门环行肌收缩波运动指数，但减少胃体和胃窦纵行肌收缩波平均振幅。上述作用均不能被阿托品和消炎病所阻断。而促胰液素对各部位肌条的收缩活动没有明显的影响。

乌药水提液对腹泻型肠易激综合征模型大鼠Ghrelin、MTL、SP、Sec水平的影响

目的探讨乌药水提液对腹泻型肠易激综合征(diarrhea-predominant irritable bowel syndrome,IBS-D)大鼠血清生长激素释放肽(Ghrelin)、胃动素(Motilin,MTL)、P物质(P substance,SP)、促胰液素(Secretin,Sec)水平的影响。方法将60只大鼠随机分成6组,采用单只孤养1周+番泻叶煎剂灌胃2周+慢性束缚应激1周造模法建立IBS-D大鼠模型,分别给予乌药水提液低、中、高剂量,匹维溴铵灌胃干预。采用酶联免疫吸附法(ELISA)检测各组大鼠血清Ghrelin、MTL、SP、Sec含量。结果与空白对照组比较,模型对照组SP、Ghrelin、MTL水平显著升高,Sec水平降低(P<0.01),提示造模成功;与模型对照组比较,乌药水提液低、中、高剂量组以及阳性对照组血清SP、MTL水平均明显降低,Sec水平均显著升高(P<0.01),乌药水提液低、中、高剂量组以及阳性对照组对Ghrelin影响无显著差异(P>0.05)。结论乌药水提液可能通过降低大鼠血清SP、MTL水平来改善IBS-D大鼠腹痛、腹泻症状。

ICP-AES法测定中国梨木虱分泌物中的元素

本文叙述了用电感耦合等离子体发射光谱法直接测定中国梨木虱分泌物中的钙、镁、锌、铜、铁、钡等多种元素。方法简便、可靠，具有良好的精密度和准确性，相对标准偏差为１．８％～５．８％，回收率为９４％～１０５％。

胰泌素增强磁共振胰胆管造影对健康成人胰腺外分泌功能的影响

目的:探讨胰泌素刺激磁共振胰胆管造影(S-magnetic resonance cholangiopancreatograp hy,S-MRCP)半定量和定量对健康成人胰腺外分泌功能评估的价值.方法:回顾性分析17例健康志愿者的3.0TMR影像学资料,其中女5例,男12例,平均年龄44.0岁±14.4岁(24-64岁),体质量指数(body mass index,BMI)平均为24.5±13.4(18.9-31.1).对受试者进行冠状位T2加权扫描和MRCP、S-MRCP、常规胰腺水模(measurement phantoms,MP)检查,绘制出时间-胰液分泌量曲线,并计算出十二指肠内的胰液的流率和胰液分泌的达峰时间.结果:水模试验证实了MP测得的水的体积和实际水的体积有很好的相关性.健康人粪便中的粪弹性蛋白酶(fecal elastase 1,FE-1)的均值是301.55-673.20g/g,均值是525.4g/g±94.4g/g.注射胰泌素后10min胰液流率是平均为8.17mL/min±1.11mL/min(6.2-11mL/min,分泌量达峰时间平均为5.2min±1.2min(3-7min).结论:S-MRCP半定量测量可以用来初步评估胰腺的外分泌功能,S-MRCP可以定量测量、准确评估胰腺的外分泌功能.S-MRCP为临床提供一种安全、无创、高效的评估胰腺的外分泌功能的检查方法.

郁金对十二肠液中HCO_3^-及血清胰泌素的影响

郁金煎剂经纤维胃镜注入十二指肠上段后,观察血清胰泌素水平及十二指肠液申HC0_3^-浓度的变化。观察对象为32例慢性浅表性胃炎患者。结果表明:注入郁金前十二指肠液HCO_3^-的浓度为7.04±5.6mmol/L,注入后为22.4±8.4mmol/L;注入前血清胰泌素的浓度为70.36±43.5pg/ml,注入后为93.4±49.7pg/ml,两组数据有统计学意义(P<0.05)。结论;郁金煎剂有刺激胰泌素分泌的作用,使血清胰泌素水平升高,使十二指肠液申HCO_3^-浓度增高。

5-羟色胺对大鼠膈下迷走神经传入放电的影响及机制

目的探讨大鼠外周静脉注射5-羟色胺以及促胰液素(secretin,SEC)对膈下迷走神经传入冲动的影响,以及5-羟色胺对膈下迷走神经传入冲动影响的机制。方法采用电生理学方法记录膈下迷走神经传入自发放电为观察指标,观察外周静脉注射不同剂量的5-HT(3、10、30μg/kg)、5-HT3受体的拮抗剂格拉司琼(1mg/kg)以及5-HT+SEC对其的影响。结果5-HT对膈下迷走神经传入自发放电具有兴奋作用,给予5-HT3受体拮抗剂后神经放电的效应被抑制,大剂量5-羟色胺+促胰液素可增强由单独的大剂量5-HT所致的迷走神经传入放电的兴奋作用。结论5-HT可能是作用于与迷走传入相关的5-HT3受体兴奋了膈下迷走传入神经自发放电;促胰液素可以增强5-HT对迷走神经传入自发放电的兴奋作用。

促胰液素通过大鼠Cajal间质细胞促进胃平滑肌舒张

观察Cajal间质细胞 (interstitialcellsofCajal,ICC)在胃平滑肌收缩的起搏作用 ,以及促胰液素对ICC促进胃平滑肌细胞舒张的影响。采用大鼠胃体上 1/ 3起搏区及胃窦环行肌肌条以Kreb′s液恒温灌流 ,通过张力换能器输入生理记录仪记录胃肌条的机械运动。用美蓝加光照选择性损伤ICC ,观察促胰液素对胃平滑肌舒张的作用。结果显示 :(1)带有ICC的胃体起搏区和胃窦肌条记录到稳定的收缩活动 ,胃窦收缩频率和振幅较胃体起搏区高。 (2 )损伤胃环肌层ICC后 ,导致胃体起搏区和胃窦平滑肌收缩频率与振幅明显下降 ,运动几乎消失。 (3)促胰液素 0 0 6～0 5mg/L明显减少胃体起搏区和胃窦区的收缩频率和振幅 ,呈剂量依赖性减少。损伤ICC后几乎完全取消促胰液素促进胃肌的舒张作用。

Morphological and functional evaluation of chronic pancreatitis with magnetic resonance imaging

Magnetic resonance imaging(MRI)techniques for assessment of morphology and function of the pancreas have been improved dramatically the recent years and MRI is very often used in diagnosing and follow-up of chronic pancreatitis(CP)patients.Standard MRI including fat-suppressed T1-weighted and T2-weighted imaging techniques reveal decreased signal and glandular atrophy of the pancreas in CP.In contrast-enhanced MRI of the pancreas in CP the pancreatic signal is usually reduced and delayed due to decreased perfusion as a result of chronic inflammation and fibrosis.Thus,morphological changes of the ductal system can be assessed by magnetic resonance cholangiopancreatography(MRCP).Furthermore,secretin-stimulated MRCP is a valuable technique to evaluate side branch pathology and the exocrine function of the pancreas and diffusion weighted imaging can be used to quantify both parenchymal fibrotic changes and the exocrine function of the pancreas.These standard and advanced MRI techniques are supplementary techniques to reveal morphological and functional changes of the pancreas in CP.Recently,spectroscopy has been used for assessment of metabolite concentrations in-vivo in different tissues and may have the potential to offer better tissue characterization of the pancreas.Hence,the purpose of the present review is to provide an update on standard and advanced MRI techniques of the pancreas in CP.

脾虚大鼠胃肠道SEC和GIP mRNA表达差异及其对胰腺外分泌的影响

以利血平大鼠脾虚模型为研究对象,采用RT-PCR技术,研究脾虚大鼠胃肠道不同组织中促胰液素和抑胃肽mR-NA表达变化及其对胰腺外分泌的影响。结果显示,与各自对照组相比,脾虚大鼠SECmRNA表达在十二指肠和回肠都显著降低(P<0.05);GIP mRNA表达在胃窦显著降低(P<0.05)。表明,SEC和GIP mRNA下调是引起脾虚的重要原因。

Is irritable bowel syndrome an organic disorder?

Irritable bowel syndrome(IBS) is a common gastrointestinal disorder that is generally considered to be functional because there appears to be no associated anatomical defect. Stress and psychological factors are thought to play an important role in IBS. The gut neuroendocrine system(NES),which regulates all functions of the gastrointestinal tract,consists of endocrine cells that are scattered among the epithelial cells of the mucosa,and the enteric nervous system. Although it is capable of operating independently from the central nervous system(CNS),the gut NES is connected to and modulated by the CNS. This review presents evidence for the presence of an anatomical defect in IBS patients,namely in the gastrointestinal endocrine cells. These cells have specialized microvilli that project into the lumen and function as sensors for the luminal content and respond to luminal stimuli by releasing hormones into the lamina propria,which starts a chain reaction that progresses throughout the entire NES. The changes in the gastrointestinal endocrine cells observed in IBS patients are highly consistent with the other abnormalities reported in IBS patients,such as visceral hypersensitivity,dysmotility,and abnormal secretion.

促胰液素对血管灌流大鼠离体胃运动的抑制作用

本工作采用血管灌流大鼠离体胃制备,探讨促胰液素对胃运动的影响。结果表明:(1)促胰液素能明显抑制胃窦自发和五肽胃泌素兴奋的胃运动;(2)抗促胰液素血清可完全取消促胰液素的抑制胃窦运动作用;(3)抗生长抑素血清和消炎痛都能阻断促胰液素的抑制胃窦运动作用。上述结果提示,促胰液素的抑制作用除通过直接作用于促胰液素受体外,还可能部分通过胃窦局部生长抑素和前列腺素介导来抑制胃的运动。

促胰液素抑制胃扩张引起膈下迷走神经传入放电的电生理研究

目的 :探讨促胰液素对扩胃 -迷走传入放电反应的影响。方法 :实验在Wistar大鼠上进行 ,记录扩张胃引起的膈下迷走传入放电的变化 ,观察静脉注射不同剂量促胰液素、十二指肠灌注HCl和静脉注射兔促胰液素抗血清后再灌注HCl对迷走传入放电的影响。结果 :不同剂量的促胰液素 (2 .5 ,5 .0和 10 pmol/kg/h)静脉注射对扩胃 -迷走传入放电反应的影响不同 ,2 .5 pmol/kg/h(n =6 )没有明显作用 ,5 .0pmol/kg/h(n =7)对扩胃 -迷走传入放电反应有明显抑制 (P <0 .0 1) ,10 pmol/kg/h(n =7)出现抑制作用快而持久。但 10min后 5 .0与 10pmol/kg/h之间无统计学意义。十二指肠灌注HCl可以引起内源性促胰液素释放由此也引起胃扩张 -迷走传入反应的抑制 (n =8,P <0 .0 1)以 2 0min后作用更明显。在观察的 35min期间一直保持抑制效应。用兔促胰液素抗血清 (titer=1:1,0 0 0 ,0 0 0 ) 0 .1mliv .2 0～ 4 0min后上述HCl的抑制效应消失 (n =8,P <0 .0 1)。结论 :迷走神经传入冲动在促胰液素对胃扩张的抑制中发挥了重要作用。

胞内cAMP在促胰液素和生长抑素引起胃平滑肌细胞舒张中的作用

用大鼠游离平滑肌细胞，观察促胰液素和生长抑素对胃平滑肌细胞的舒张效应及胞内ｃＡＭＰ的作用。结果表明：（１）促胰液素对胃体和胃窦平滑肌细胞的作用均为舒张反应。这一作用只引起环肌舒张，而对纵肌细胞无直接作用。促胰液素抗血清可阻断促胰液素对胃环肌细胞的舒张反应。Ｆｏｒｓｋｏｌｉｎ可加强促胰液素对胃环肌细胞的舒张作用，而ｃＡＭＰ抑制剂则减弱其舒张作用。促胰液素作用于胃环肌细胞后胞内ｃＡＭＰ含量显著升高。（２）生长抑素对胃体和胃窦平滑肌细胞无直接的舒张作用，但可以抑制由五肽胃泌素引起的胃平滑肌细胞的收缩作用，这一作用可被生长抑素抗血清所阻断。生长抑素作用下的胃平滑肌细胞内ｃＡＭＰ无显著变化。上述结果提示：促胰液素通过特异受体引起胃平滑肌细胞舒张，其收缩作用通过胞内ｃＡＭＰ介导。生长抑素对五肽胃泌素所致胃平滑肌细胞收缩的抑制作用不通过ｃＡＭＰ途径。