【作者】 刘素芳；

【导师】 姚运纬；

【作者基本信息】 郑州大学 ， 生理学， 2003， 硕士

【摘要】 目的：胰高血糖素样肽-1（glucagon-like peptide-1,GLP-1）是一种组织特异性的胃肠道激素，主要由回肠末端L细胞、胰腺内分泌部A细胞以及神经细胞合成和分泌。GLP-1和胰高血糖素来源于共同的前体前胰高血糖素原，其33-61位氨基酸编码胰高血糖素，72-108位氨基酸则编码GLP-1。GLP-1尽管和胰高血糖素有48％的同源性，它们的作用却不同。GLP-1酶解去掉6肽，末端酰胺化后，即生成有活性的GLP-1（7-36）NH₂。GLP-1（7-36）NH₂可以促进胰岛素分泌，但是关于其促胰岛素分泌的机制，目前国内研究较少。本实验中利用GLP-1（7-36）NH₂作用于离体培养的大鼠胰岛细胞，观察GLP-1（7-36）NH₂的促胰岛素分泌作用，并进一步探讨GLP-1（％36）NH₂促进胰岛素分泌的机制。 方法：胰岛的分离参照王玲等的方法，每次实验取新生3-5天SD大鼠，无菌条件下剖腹取出胰腺，剪切为＜0.5mm³的组织块，V型胶原酶消化30min后，离心洗涤，悬浮于完全培养基，接种入50ml培养瓶，于5％CO₂、95％空气条件下培养20h，转板纯化，接种于96孔培养板培养24h，按实验要求进行实验。实验分四项：①GLP-1（7-36）NH₂对胰岛素分泌的影响。对照组培养液中不加GLP-1（7-36）NH₂，实验组分别以浓度为2.5nmol／L、5.0nmol／L、10.0nmol／L、20.0nmol／L、40.0nmol／L的GLP-1（7-36）NH₂作用于胰岛细胞，培养24h后，进行放射免疫分析（γ计数），测定胰岛素分泌量。②8-bromo-cyclic 3′，5′-adenosine monophosphate（8-Br-cAMP）对胰岛素分泌的影响。实验分两组：对照组培养液中不含8-Br-cAMP，实验组培养液中含有0.1mmol／L的8-Br-cAMP，体外培养胰岛规H大学医学院硕」刀防生毕业论文6U卜1（7-36卜氏蒯眼岛素分泌作用和胞内动州P浓度改变与胰岛素m砂叭表达24h后，测定胰岛素分泌量。③G廿一1（7一36）NH:对胞内cAMP的影响。实验分两组:对照组培养液中不含G廿一1口一36）NHZ，实验组培养液中含有浓度为20nmof/L GLP一1（7一36）NH2，培养24h后，进行放射免疫分析（p计数），测定胞内cAMP含量。④GLP一1（7一36）NH:对胰岛素mRNA表达的影响。对照组培养液中不含G廿一1（7一36）NHZ，实验组培养液中含有20nmol/L GLP一1（7一36）NHZ，培养24h后，用0.25%胰蛋白酶消化胰岛分散细胞，涂片后利用针对胰岛素mRNA的寡核甘酸探针进行细胞原位杂交，DAB显色，高清晰度病理图文分析系统（highpathologieal image analysis system，HP认s）对细胞着色的平均光密度（m ean即tiealdensity， MoD）量化分析，观察实验组和对照组胰岛素mRNA的表达情况。 实验结果均用X土SD表示，根据实验要求分别采用多个样本均数比较的方差分析方法和两独立样本均数比较的t检验，以Q二0.05作为检验水准。结果:LG廿一1（7一36）NHZ对胰岛素分泌的影响 以2 .snmol/L、5.0刊rnoUL、10.onmol/L、20.0nm01/L、40.加mol/L的G廿一1（7一36卿玩作用于离体胰岛团24h后，胰岛素分泌量分别为68.76土n.71、72.30士13.13、104.16士15.57、110.98土7.29、111.58士10.65mIU瓜与正常对照组 （55.53土5.63mnJ凡）相比，差异有显著性（尸<0.05）;2.snmoljl刀L卫一1（7一36）NHZ组与5.Olllno呱GLP一1（7一36）NH:组之间胰岛素分泌量差异无显著性（P>0.05），10.Onmol/L GLP一1（7一36）NH:组、20.0刊口01/L石廿一1（7·36）NH2组和40.onmol/LG廿一1（7一36）NH:组三组间胰岛素分泌量差异无显著性伊>0.05），但是2.snm。比、5.0nm01几GLP一1（7一36）NH:组分别与10.onmoUL、20刀刊mol/L、40.onmol/LG廿一1（7一36）NH:组之间胰岛素分泌量差异有显著性.（尸<0.05）。将G廿一1（7一36）NH:对胰岛素分泌的影响用曲线表示，可见随着G廿一1口一36)NH:作用浓度的增加，胰岛素分泌逐渐增加。2.8一B卜cAMP对胰岛素分泌的影响 对照组24h胰岛素分泌量为52.59士19.77 m1U/L，实验组为117.03士50.04mIU/L，两组间24h胰岛素分泌量差异有显著性伊<0.05)。3.GLP一1（7一36）NH:对胞内cAMp的影响 对照组胰岛细胞内cAMP含量28.n土1.41nmo讥，实验组为173.88士郑州大学医学写测)切仁歹赵胜毕日州仑文G口‘1口拐娇旧2的促胰岛素分泌作用和胞内。划”P湘夏改变与胰岛素mRNA表达3.62nmol/L，两组相比差异有显著性伊<0.05)。4.GLP一1（7一36）NH:对胰岛素mRNA表达的影响 光学显微镜下观察，对照组细胞胞浆着色多为黄色或浅黄，实验组细胞胞浆着色多为深黄或棕黄，经HP认S系统对细胞着色的平均光密度（mean oPticaldensities，MoD）定量分析，对照组276个胰岛B细胞着色的MoD为0.07436士0.01794，其中最大值为0.1943，最小值为0.03798，细胞着色的平均光密度值集中在0.05一0.1，只有少数细胞着色的平均光密度值超过0.1;GLP一l（7一36）NH:实验组胰岛B细胞着色的MOD为0.2404士0.03368，其中最大值为0.3428，最小值为0.1911，细胞着色的平均光密度值集中在0.2一0.3，只有少数细胞着色的平均光密度值低于0.2。两组细胞MOD值相比差异有显著性伊<0 .05)。结论: GLP一l(7一3句NH:可以促进胰岛素分泌，在一定范围内呈剂量依赖性，?更多还原

【Abstract】 Purposes: Enhancement of insulin secretion from the islet B cells is a principal goal for treatment of patients with type 2 diabetes. Some gastrointestinal hormones are associated with insulin secretion. The glucagon-like peptide-l(GLP-l) is a kind of gastrointestinal hormone formed by alternative tissue-specific cleavages in the L cells of the intestine, the A cells of the endocrine pancreas and neurons in the brain. GLP-1 is based on the proglucagon gene sequence. It is secreted mainly as "truncated" GLP-l(7-36 )amide with more activities. Though GLP-1(7-36)NH2 can increase insulin secretion, but how does GLP-1(7-36)NH2 stimulate insulin secretion? The answer to this question is fundamental for the assessment of the therapeutic potential of the gut hormone. In this experiment, radio-immunoassay and hybridization in situ were applied to observe the insulinotropic activities of GLP-1 (7-36)NH2 and reveal the mechanisms underlying this process.Methods: Rat pancreases were removed from 3-5 day-old Sprague-Dawley rats and dissected into 0.5mm3 segments and islets were isolated by the collagenase digestion method of Wangling et al. Thoroughly washed islets and suspended in modified RPMI-1640 medium supplemented with 10% fetal bovine serum, and added to 50ml cell culture flasks. The islets were incubated for 20 hours at 37癈 with 95% air and 5% CO2. After 24 hours of incubation, the medium containing the suspended islets were carefully removed and washed with serum-free midium, then resuspended in modified RPMI-1640 medium. The islets were added into 96-well tissue cultureplates(containing 20? islets per well) for 24 hours. The effects of GLP-1(7-36)NH2 with different concentrations on insulin secretion were measured by radioimmuno-assay(RIA). Effects of 8-bromo-cyclic 3’,5’-adenosine monophosphate (8-Br-cAMP) on insulin secretion and effects of GLP-1(7-36)NH2 on intracellular cAMP were determined by RIA. Influences of GLP-1(7-36)NH2 (20nmol/L) on insulin mRNA expression were determined by hybridizaiton in situ. The mean optical densities (MOD) of B cells stained by DAB were analyzed by high pathological image analysis system (HPIAS).The data were showed as Means盨D and analyzed using the Student’s t-test and one-way ANOVA. P<0.05 is considered as significant. Results:1. The effects of GLP-1(7-36)NH2 on insulin secretionGLP-1(7-36)NH2 with the concentration of 2.5nmol/L, 5..0nmol/L, l0.0nmol/L, 20.0nmol/L, 40.0nmol/L respectively were added to the medium as different experimental groups , 24 hours later, insulin amount are 68.76?1.71 72.30?3.13 104.16?5.57 110.98?.29 111.58?0.65mIU/L respectively, and the insulin account is 55.53?.63mIU/Lin the control group. There was no significant difference between the groups with 2.5nmol/Land 5.0nmol/L GLP-1(7-36)NH2 respectively; and there was not significant difference among the groups with lO.Onmol/L, 20.0nmol/L and 40.0nmol/L GLP-1(7-36)NH2 respectively. But the difference is significant between experimental groups and control group(P<0.05).The data show that with the rising concentration of GLP-1(7-36)NH2, there is an increasing amount of insulin.2. The actions of 8-Br-cAMP on insulin secretionInsulin concentration of control group is 52.59 ?9.77mIU/L, and that of the experiment group is 117.03?0.04mIU/L, there is significant difference between them(P<0.05).3. The effects of GLP-1(7-36)NH2 on intracellular cAMPThe amount of cAMP of control group is 28.11 ?.41nmol/L, and that of the experiment group is 173.88?.62nmol/L, there is significant difference between them (P<0.05).4. The effects of GLP-1(7-36)NH2 on insulin mRNA expressionThe B cell cytoplasma color of the experiment group is brown stained by DAB, but that of control group is weak. The mean optical densities(MOD) of the control group are 0.07436+0.01794, the maximum is 0.1943 and the minimum is 0.03798. The MOD of the experiment group are 0.2404+0.03368, the maximum is 0.3428 and the minimum is 0.1911. The difference is significant between two groups (F更多还原