【作者】 宋晓丹；

【导师】 张源淑；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 近年来,高血糖对机体的危害问题越来越引起人们的关注。高血糖发展到一定程度就称为糖尿病(dibaetesmellitus, DM).长期以来糖尿病一直被认为是一种严重危害健康的常见慢性终身疾病,而今已成为现代人类所面临的一个主要健康问题。乳中含有多种活性成分,有报道认为其有降血糖作用,但作用的有效成分并不明确,本文以SD大鼠为实验动物,研究主要的乳源活性肽β-酪啡肽7(β-casomorphin-7,β-CM7)对大鼠血糖水平的影响,探讨其可能的辅助降血糖作用及其机制。研究包括三个部分：试验一β-CM7辅助降血糖的作用及机制初探选用健康成年SD大鼠16只,随机分成对照组和试验组,分别灌喂生理盐水和β—CM7(7.5×10-6mol／L)。连续灌喂30天后,断颈处死,取血清、肝脏及肌肉等进行如下试验：1)氧化酶法测定血液中葡萄糖浓度；2)肝糖原、肌糖原含量的测定；3)血清中胰岛素(insulin)、胰高血糖素(glucagon)水平的RIA分析；4)小肠黏膜α-葡萄糖苷酶和Na+-K+-ATP酶活性分析；5)小肠黏膜组织中SGLT-1 mRNA表达分析。试验结果显示：连续灌喂p-CM7大鼠血糖浓度较灌喂生理盐水有降低,但二者之间差异不显著(p=0.35)；肝糖原、胰岛素、胰高血糖素、α-葡萄糖苷酶活力、Na+-K+-ATP酶活力二组之间均无明显差异；试验组肌糖原含量低于对照组(p=0.001)；SGLT-1mRNA表达显著低于对照组(p=0.02)。结论：β-CM7具有辅助降血糖作用,其机制可能是通过下调小肠黏膜上皮SGLT-1 mRNA的表达,减少肠道葡萄糖的转运,并增加肌糖原的消耗。试验二β-CM7对大鼠肠道葡萄糖吸收的抑制及其机制利用翻转的离体小肠模型研究了β-CM7对葡萄糖转运和小肠黏膜Na+-K+-ATP酶活力的影响,同时利用纳洛酮(NaL)阻断剂与β-CM7按2：1浓度混合,探讨了影响葡萄糖吸收的阿片机制。结果显示,在离体情况下β-CM7在7.5×10-7---7.5×10-5mol／L浓度范围内对葡萄糖的吸收均有一定的抑制作用,抑制作用与其浓度呈正相关(与0mol／L对照组相比,7.5×10-7 mol／Lβ-CM7组(L组)p=0.09；7.5×10-6 mol／Lβ-CM7组(M组)p=0.04；7.5×10-5mol／Lβ-CM7组(H组)p=0.01)；能够降低葡萄糖跨膜快速转运期的平均速率(L组p=0.09；M组p=0.04；H组p=0.007)。能够显著降低Na+-K+-ATP酶活力,与对照组相比,L组差异极显著p=0.004；SGLT-1和GLUT-2Real-timePCR结果发现：与对照组相比,L组、M组SGLT-1(p=0.02,0.05)和L组GLUT-2(p=0.03)mRNA表达水平显著下降。阿片特异性拮抗剂纳洛酮对β-CM7的抑制作用没有明显的逆转。结果认为提示β-CM 7可以通过非阿片样途径抑制小肠对葡萄糖的吸收,具有辅助降血糖作用,其机制可能是通过降低Na+-K+-ATP酶活力及下调SGLT-1、GLUT-2 mRNA的表达,从而减少了肠道葡萄糖的转运有关。试验三β-CM7降糖作用的细胞学验证试验一结果显示：β-CM7能够极显著的降低大鼠肌糖原含量,对肝糖原含量无影响。在这个试验中,我们将通过体外培养C2C12细胞和LO2细胞,对调控糖代谢的靶细胞进行筛选,同时观察不同培养条件下β-CM7对C2C12细胞和LO2细胞葡萄糖消耗和细胞活力的影响。结果发现β-CM7能明显的促进高糖状态下C2C12细胞对葡萄糖的消耗；在有胰岛素存在的情况下β-CM7可以大大提高C2C12细胞的葡萄糖消耗。而对LO2细胞作用不明显；低糖条件下,C2C12及LO2细胞对葡萄糖消耗均无影响。提示β-CM7作用的靶细胞是肌细胞,β-CM7本身具有促进C2C12细胞葡萄糖消耗的作用,也能协同胰岛素增加C2C12细胞葡萄糖消耗。更多还原

【Abstract】 Recently, the harm of hyperglycemia to the body has drawn more and more attention. Being hyperglycemia frequently can easily lead to dibaetesmellitus. It has always been thought that dibaetesmellitus has been a severe chronic disease for life. At present, it has become a main healthy problem faced by mankind.There are all kinds of active components in milk. It was reported that milk may have the effect of reducing blood glucose, but it has still not been cleared about its active components. Thus this paper was focused on the effect of P-casomorphin-7 which was the one of most important active peptides derived from milk on blood glucose in rats. We will explore it’s the effect of inducing blood glucose and mechanism. The research consistsof three parts:Experiment 1. Effect of P-casomorphin-7 on blood glucose in rats and preliminary study on its mechanismSixteen healthy adult SD rats were randomly derived into control group (administered with saline) and experiment group (P-casomorphin-7 group, administered with 7.5×10-6 mol/L of P-casomorphin-7). All rats of control group and experiment group were killed after they were oral administered continuously for 30 days, and immediately collected the blood, liver, muscles and small intestine mucosa of rats. we designed a few tests as follow:1) The concentration of blood glucose was tested by the method of oxidation enzyme; 2) The concentration of hepatic and muscle glycogen were detected; 3) the contents of insulin and glycagon in serum were analyze by RIA; 4) the activity ofα-glucosidase and Na+-K+-ATPase of small intestinal mucosa were observed; 5) the mRNA expression of sodium-glucose transporter (SGLT-1) in small intestinal mucosa were analyzed by the RT-PCR. The results showed the concentration of blood glucose decreased in experiment group, but there were no significant differences between two groups (p=0.35); the concentration of hepatic glycogen, the activity of Na+-K+-ATPase andα-glucosidase as well as the contents of insulin and glycagon had no apparently differences between two groups; the content of muscle glycogen from experiment group was apparently lesser than that of control group (p=0.001); The expression of SGLT-1 mRNA from small intestinal mucosa of experiment group decreased, and had a significant variance compared with control group (p=0.02). The results believed that oral administering ofβ-casomorphin-7 had a little effect on decreasing blood sugar of rats. It is very possible thatβ-casomorphin-7 reduced blood sugar by inhibiting the mRNA expression level of SGLT-1 in small intestine and increasing the consumption of muscle glycogen.Experiment 2. Inhibition ofβ-casomorphin-7 on the absorption of glucose in vitro and its mechanismFormer studys have showed thatβ-casomorphin-7 may induce the decrease of blood sugar and the expression of SGLT-1. In this experiment, we will investigate the influence ofβ-CM-7 on the absorption of glucose and its absorption mechanism, In this study used reverted sacs of adult rats in vitro which were dipped into culture sample including P-casomorphin-7 and glucose, we explored the transport of glucose and the activity of Na+-K+-ATPase in small intestinal mucosa were analyzed. We also researched its opioid mechanism by choosing opioid special inhibitor called naloxone, which was mixed withβ-casomorphin-7 in the proportion of 2:1. The results showed that Luminally appliedβ-casomorphin-7 of 7.5×10-7-7.5×10-5 mol/L significantly induced glucose concentration;β-casomorphin-7 dose-dependently inhibited the transmitting of glucose in vitro (compared with control group, the low dose group p=0.09;. the middle dose group p=0.04; the high dose group p=0.01); and slowed the transmitted velocity of glucose on average in intestinal sacs at the first transmitted-period; the activity of Na+-K+-ATPase was obviously reduced(p=0.004,0.73,0.54); the mRNA expressions of SGLT-1 and GLUT-2 were also decreased. Compared with control group, the mRNA expressions of SGLT-1 of low-dose group and middle-dose group were significantly different(p=0.02 and P=0.05); The inhibition ofβ-casomorphin-7 cannot be blocked by the naloxone. Conclusion: The present data demonstrated thatβ-casomorphin-7 could inhibit the absorption of glucose of the intestinal sac in vitro without depending on opioid receptor. It is possible that P-casomorphin-7 reduce the absorption of glucose by inhibiting the activity of Na+-K+-ATPase and the mRNA expression level of SGLT-1, GLUT-2 in small intestine of rats.Experiment 3. Cytology evidence of the glucose-lowering effect ofβ-casomorphin-7Previous studies showed that P-casomorphin-7 may significantly induce the concentration of muscle glycogen and had no effect on the concentration of hepatic glycogen. In this study we will sieve the target cell of glycometabolic regulate by observing the effect of P-casomorphin-7 of the glucose consumption and cell activities of C2C12 and LO2 cells under of the different culture condition. The result showed that In the hyperglycaemia culture, P-casomorphin-7 may not only increase the glucose consumption of C2C12 cell, but also significantly increase the glucose consumption of C2C12 together with insulin. And P-casomorphin-7 had no any effect to the glucose consumption of LO2 cell. Under the culture of low carbohydrates, P-casomorphin-7 have no effct on the glucose consumption of C2C12 and LO2 cells. The result indicated that the target cell of P-casomorphin-7 was muscle cell; P-casomorphin-7 may increase the glucose consumption of C2C12, also may improve the glucose consumption of C2C12 in coordination with insulin. We think that one of the mechanism of lowering blood glucose ofβ-casomorphin-7 is to improve the glucose consumption of muscle cell.更多还原