茶多酚对铜蓝蛋白基因敲除小鼠糖代谢的影响及机制

发布时间：2018-05-03 22:04

  本文选题：铜蓝蛋白 + 铁过载　； 参考：《河北医科大学》2017年硕士论文

【摘要】：目的:糖尿病是一组以血浆葡萄糖水平增高为特征的慢性代谢性疾病,其病理特点是胰岛β细胞功能受损和/或胰岛素抵抗。近年来,越来越多的研究表明,铁过载可增加糖尿病的患病风险,体内铁过载是2型糖尿病的独立危险因素。铁是人体内含量最多的必需微量元素,铁稳态对于细胞信号转导和内环境的稳态至关重要。铜蓝蛋白(ceruloplasmin,Cp)是铁转运过程中的关键酶,主要在肝细胞合成,具有亚铁氧化酶活性,可将Fe2+氧化成Fe3+,促进铁与转铁蛋白结合。Cp基因敲除(Cp-/-)后可以导致组织铁过载,以及糖耐量异常,但其具体机制不详。茶多酚是一种天然的抗氧化剂,具有螯合铁的特性,目前茶多酚对Cp-/-小鼠糖代谢的影响机制尚未见详细报道。本课题组以Cp基因敲除(内源性铁过载)小鼠为研究对象,探讨铁过载导致糖代谢异常的具体作用机制;同时,应用茶多酚灌胃进行干预治疗,明确茶多酚对Cp基因敲除小鼠糖代谢的影响,以期为糖尿病患者提供新的治疗选择。方法:随机选取12只10月龄雌性BALB/c J×129Sv J品系铜蓝蛋白基因敲除(Cp-/-)小鼠作为实验组,另选取12只同性别、同月龄、同品系的野生型(Cp+/+)小鼠作为对照组,将实验组和对照组小鼠分别随机分为茶多酚干预组(TPG,n=6)和生理盐水处理组(NSG,n=6)进行干预,4周后,通过葡萄糖耐量实验和胰岛素耐量实验评估小鼠胰岛β细胞的储备功能和胰岛素敏感性。放射免疫法检测小鼠血清胰岛素水平;比色法检测肝脏非血红素铁含量;免疫组化法检测胰腺铁沉积情况;黄嘌呤氧化酶法测定超氧化物歧化酶(Superoxide dismutase,SOD)活性、硫代巴比妥酸法测定丙二醛(Malondialdehyde,MDA)含量以明确肝脏氧化应激水平;原位末端标记(terminal dexynucleotidyl transferase(Td T)-mediated d UTP nick end labeling,TUNEL)法检测肝实质细胞和胰岛β细胞的凋亡情况;Real-time PCR测定肝脏葡萄糖转运体2(glucose transporter,GLUT2)、胰岛素受体底物2(insulin receptor substrate 2,IRS2)mRNA表达水平;Western-blot法测定肝脏GLUT2、IRS2的蛋白表达水平。应用spss21.0统计软件进行统计分析,所有计量资料均进行正态性检验,正态分布数据以平均值±标准差(?x±sd)表示,非正态分布数据以(最小值,最大值)表示。正态分布且方差齐性数据,采用单因素方差分析,两两比较采用lsd检验。不符合正态分布的资料采用独立样本的kruskal-wallis秩和检验。以p0.05为差异有统计学意义。结果:1与cp+/+nsg小鼠相比,cp-/-nsg小鼠血糖显著升高(葡萄糖耐量实验各点血糖值:0min6.80±0.91mmol/l比4.98±0.71mmol/l,p0min=0.004;15min14.88±0.69mmol/l比12.70±0.67mmol/l,p15min=0.011;30min15.45±0.97mmol/l比8.85±0.79mmol/l,p30min0.001;60min10.30±1.61mmol/l比7.75±0.37mmol/l,p60min=0.004;120min8.00±0.70mmol/l比5.68±0.34mmol/l,p120min0.001),胰岛素敏感性显著下降(胰岛素耐量实验各点血糖值:0min7.15±1.20mmol/l比5.13±0.54mmol/l,p0min=0.001;15min5.58±0.87mmol/l比4.18±0.49mmol/l,p15min=0.014;30min3.55±0.60mmol/l比2.80±0.28mmol/l,p30min=0.013;60min2.30±0.22mmol/l比1.63±0.25mmol/l,p60min0.001)。cp+/+tpg与cp+/+nsg组间血糖无显著差异(葡萄糖耐量实验各点血糖值:0min4.35±0.68mmol/l比4.98±0.71mmol/l,p0min=0.245;15min12.70±1.44mmol/l比12.70±0.67mmol/l,p15min=1.000;30min8.23±0.75mmol/l比8.85±0.79mmol/l,p30min=0.450;60min7.68±0.54mmol/l比7.75±0.37mmol/l,p60min=0.919;120min4.90±0.78mmol/l比5.68±0.34mmol/l,p120min=0.091),胰岛素敏感性也无明显变化(胰岛素耐量实验各点血糖值:0min4.95±0.24mmol/l比5.13±0.54mmol/l,p0min=0.726;15min4.03±0.73mmol/l比4.18±0.49mmol/l,p15min=0.763;30min2.33±0.26mmol/l比2.80±0.28mmol/l,p30min=0.089;60min1.43±0.10mmol/l比1.63±0.25mmol/l,p60min=0.167)。与cp-/-nsg小鼠相比,cp-/-tpg小鼠血糖明显降低(葡萄糖耐量实验各点血糖值:0min5.65±0.53mmol/l比6.80±0.91mmol/l,p0min=0.044;15min13.23±1.08mmol/l比14.88±0.69mmol/l,p15min=0.042;30min12.70±1.74mmol/l比15.45±0.97mmol/l,p30min=0.005;60min7.23±1.08mmol/l比10.30±1.61mmol/l,p60min=0.001;120min5.45±0.47mmol/l比8.00±0.70mmol/l,p120min0.001),胰岛素敏感性显著升高(胰岛素耐量实验各点血糖值:0min5.10±0.35mmol/l比7.15±1.20mmol/l,p0min=0.001;15min4.13±0.61mmol/l比5.58±0.87mmol/l,p15min=0.011;30min2.53±0.15mmol/l比3.55±0.60mmol/l,p30min=0.002;60min1.48±0.17mmol/l比2.30±0.22mmol/l,p60min0.001)。2血清胰岛素水平:与cp+/+nsg相比,cp-/-nsg小鼠血清空腹胰岛素水平明显升高,但第一时相胰岛素分泌无统计学差异(0min37.93±3.36uiu/ml比33.91±2.38uiu/ml,p0min=0.036;79.31±7.84uiu/ml比68.81±6.89uiu/ml,p30min=0.068);cp+/+tpg和cp+/+nsg组间空腹血清胰岛素及第一时相胰岛素水平均无明显差异(31.58±2.09uiu/ml比33.91±2.38uiu/ml,p0min=0.198;69.11±8.38uiu/ml比68.81±6.89uiu/ml,p30min=0.956);茶多酚干预4周后,与cp-/-nsg组相比,cp-/-tpg组小鼠血清空腹胰岛素水平降低,但第一时相胰岛素分泌无统计学差异(33.88±1.44uiu/ml比37.93±3.36uiu/ml,p0min=0.035;71.35±6.37uiu/ml比79.31±7.84uiu/ml,p30min=0.154)。3肝脏组织铁含量:与cp+/+nsg相比,cp-/-nsg小鼠肝脏发生了严重铁沉积(21.47±3.53mg/gprot比3.04±0.61mg/gprot,p0.001),经茶多酚干预4周后,与cp-/-nsg相比,cp-/-tpg小鼠肝脏铁沉积得到了明显改善(10.77±1.13mg/gprot比21.47±3.53mg/gprot,p0.001)。但cp+/+tpg与cp+/+nsg组间未见显著差异(2.68±0.55mg/gprot比3.04±0.61mg/gprot,p=0.827)。4胰岛素免疫组化与铁沉积普鲁士蓝染色结果显示,铁沉积主要发生在胰腺外分泌部的胰腺腺泡细胞,而非胰岛β细胞。胰腺凋亡也主要发生在胰腺外分泌部的胰腺腺泡细胞,而非胰岛β细胞。5氧化应激:与生理盐水处理的野生组小鼠相比,cp-/-nsg小鼠sod水平显著降低(1.70±0.27u/mgprot比2.11±0.09u/mgprot,p=0.005)、mda水平明显升高(5.46±0.51nmol/mgprot比4.41±0.43nmol/mgprot,p=0.001);经茶多酚干预4周后,cp-/-tpg小鼠sod水平显著升高(2.46±0.21u/mgprot比1.70±0.27u/mgprot,p0.001)、mda水平明显降低(4.75±0.48nmol/mgprot比5.46±0.51nmol/mgprot,p=0.019)。此外,与生理盐水处理的野生组小鼠相比,茶多酚干预的野生组小鼠sod水平明显升高(2.53±0.14u/mgprot比2.11±0.09u/mgprot,p=0.004)、mda水平显著降低(3.76±0.25nmol/mgprot比4.41±0.43nmol/mgprot,p=0.031)。6cp-/-nsg小鼠肝细胞凋亡数显著高于cp+/+nsg小鼠(67.00±4.58比13.67±2.08,p0.001),经茶多酚灌胃干预后,cp-/-tpg小鼠肝细胞凋亡数较cp-/-nsg明显降低(38.33±5.03比67.00±4.58,p0.001)。cp+/+tpg与cp+/+nsg两组间肝细胞凋亡数目无显著差异(12.67±2.52比13.67±2.08,p=0.754)。7与cp+/+nsg相比,cp-/-nsg小鼠肝脏irs2mrna和蛋白表达水平[(0.09,0.29)比(0.42,0.78),pmrna=0.011;(0.52±0.05比0.70±0.07),pprotein=0.024],以及肝脏glut2mrna和蛋白表达水平显著降低[(0.03±0.02比0.97±0.13),pmrna0.001;(0.45±0.05比0.81±0.06),pprotein0.001];经茶多酚为期4周的干预后,与cp-/-nsg组相比,cp-/-tpg组小鼠肝脏irs2mrna和蛋白表达水平[(0.46,0.88)比(0.09,0.29),pmrna=0.006;(0.69±0.05比0.52±0.05),pprotein=0.029],以及肝脏glut2mrna和蛋白表达水平[(0.96±0.23比0.03±0.02),pmrna0.001;(0.75±0.06比0.45±0.05),pprotein0.001]显著升高。cp+/+nsg组和cp+/+tpg组小鼠肝脏irs2mrna和蛋白表达水平[(0.74,1.48)比(0.42,0.78),pmrna=0.077;(0.72±0.12比0.70±0.07),pprotein=0.759],以及肝脏glut2mrna和蛋白表达水平[(1.02±0.22比0.97±0.13),pmrna=0.755;(0.84±0.04比0.81±0.06),pprotein=0.478]无显著变化。结论:1铜蓝蛋白基因敲除可导致糖代谢异常,血糖升高的原因是胰岛素抵抗而非胰岛素分泌功能受损;2铜蓝蛋白基因敲除可导致胰岛素作用的靶器官(肝脏)发生铁沉积,后者导致肝脏氧化应激紊乱,细胞凋亡增加,肝脏irs2、glut2表达降低,引起胰岛素抵抗,并最终导致糖代谢异常。3茶多酚通过螯合肝脏组织铁,减轻肝脏铁沉积及机体的氧化应激水平,最终改善铁过载引起的糖代谢异常。
[Abstract]:Objective: diabetes is a group of chronic metabolic diseases characterized by increased plasma glucose level. Its pathological characteristics are impaired pancreatic beta cell function and / or insulin resistance. In recent years, more and more studies have shown that iron overload can increase the risk of diabetes. Iron overload is an independent risk factor for type 2 diabetes. Iron is a human being. The most essential trace elements in the body are essential. Iron homeostasis is crucial to the homeostasis of cellular signal transduction and internal environment. Ceruloplasmin (Cp) is the key enzyme in the process of iron transport. It is mainly synthesized in liver cells, and has the activity of ferrous oxidase, which can oxidize Fe2+ into Fe3+ and promote the binding of iron to transferrin by.Cp gene knockout (Cp-/-) It can lead to iron overload and abnormal glucose tolerance, but the specific mechanism is unknown. Tea polyphenols are a natural antioxidant and have the characteristics of chelated iron. The mechanism of the influence of tea polyphenols to the glucose metabolism of Cp-/- mice is not yet reported. The research group is based on Cp gene knockout (endogenous iron overload) mice. At the same time, the effect of tea polyphenols on gastric perfusion was used to determine the effect of tea polyphenols on the glucose metabolism in Cp knockout mice, so as to provide new treatment options for diabetic patients. Methods: 12 10 month old female BALB/ C J x 129Sv J strains were randomly selected as the gene knockout (Cp-/-). As the experimental group, 12 mice of the same sex, the same month age and the same strain of the wild type (Cp+/+) mice were selected as the control group. The experimental group and the control group were randomly divided into the tea polyphenols intervention group (TPG, n=6) and the physiological saline treatment group (NSG, n=6). After 4 weeks, the glucose tolerance test and insulin tolerance test were used to evaluate the mice pancreas. The reserve function of islet beta cells and insulin sensitivity. Radioimmunoassay was used to detect serum insulin levels in mice; colorimetric assay was used to detect non heme iron content in the liver; immunohistochemical method was used to detect iron deposition in the pancreas; the activity of Superoxide dismutase (SOD) was measured by xanthine oxidase method and thiobarbituric acid method was used for the determination of C two The levels of aldehyde (Malondialdehyde, MDA) were determined to determine the level of liver oxidative stress; in situ terminal terminal labeling (terminal dexynucleotidyl transferase (Td T) -mediated D UTP nick end) was used to detect the apoptosis of liver parenchymal cells and islet beta cells. The expression level of the island hormone receptor substrate 2 (insulin receptor substrate 2, IRS2) and the protein expression level of the liver GLUT2 and IRS2 were measured by Western-blot method. The statistical analysis was carried out by the spss21.0 statistical software. All the measured data were tested in normality, and the normal distribution data were expressed with the mean standard deviation (? X +. SD), and the non normal distribution data were obtained. The minimum, maximum value) expressed. Normal distribution and variance homogeneity data, using single factor analysis of variance, 22 comparison using LSD test. The data that did not conform to normal distribution used Kruskal-Wallis rank sum test of independent sample. The difference of P0.05 was statistically significant. Results: 1 compared with cp+ /+nsg mice, the blood sugar was significantly higher (Portuguese blood glucose). The blood sugar values of the glucose tolerance test were: 0min6.80 + 0.91mmol/l ratio 4.98 + 0.71mmol/l, p0min=0.004, 15min14.88 + 0.69mmol/l ratio 12.70 + 0.67mmol/l, p15min=0.011, 30min15.45 + 0.97mmol/l ratio 8.85 + 0.79mmol/l, p30min0.001, 7.75 + 5.68 + N0.001), insulin sensitivity decreased significantly (0min7.15 + 1.20mmol/l ratio 5.13 + 0.54mmol/l, p0min=0.001; 15min5.58 + 0.87mmol/l ratio 4.18 + 0.49mmol/l, p15min=0.014; 30min3.55 + 0.60mmol/l 2.80 + 0.28mmol/l) There was no significant difference between the blood sugar and the cp+/+nsg group (the glucose tolerance test points: 0min4.35 + 0.68mmol/l ratio 4.98 + 0.71mmol/l, p0min=0.245; 15min12.70 + 1.44mmol/l ratio 12.70 + 0.67mmol/l, p15min=1.000; 30min8.23 + 0.75mmol/l 8.85 + 0.79mmol/l. .90 + 0.78mmol/l ratio 5.68 + 0.34mmol/l, p120min=0.091), and no significant changes in insulin sensitivity (blood glucose value of insulin tolerance test points: 0min4.95 + 0.24mmol/l ratio 5.13 + 0.54mmol/l, p0min=0.726, 15min4.03 + 0.73mmol/l ratio 4.18 + 0.49mmol/l, p15min=0.763, 2.80 + 0.10 Mmol/l was 1.63 + 0.25mmol/l, p60min=0.167). Compared with the cp-/-nsg mice, the blood sugar of cp-/-tpg mice was significantly reduced (the blood sugar value of each point in the glucose tolerance test: 0min5.65 + 0.53mmol/l was 6.80 + 0.91mmol/l, p0min=0.044; 15min13.23 + 1.08mmol/l 14.88 + 0.69mmol/l, 0.042, 15.45 +, 6. 0min7.23 + 1.08mmol/l ratio 10.30 + 1.61mmol/l, p60min=0.001, 120min5.45 + 0.47mmol/l ratio 8 + 0.70mmol/l, p120min0.001), insulin sensitivity increased significantly (the blood glucose value of each point of insulin tolerance test: 0min5.10 + 0.35mmol/l ratio 7.15 + 1.20mmol/l, p0min=0.001, 5.58 + 0.15 Mmol/l was 3.55 + 0.60mmol/l, p30min=0.002, 60min1.48 + 0.17mmol/l ratio 2.30 + 0.22mmol/l, p60min0.001).2 serum insulin level: compared with cp+/+nsg, the serum insulin level of cp-/-nsg mice increased significantly, but there was no significant difference between the first phase insulin secretion (0min37.93 + 3.36uiu/ml ratio 33.91 +, 79.31 + 7) .84uiu/ml was 68.81 + 6.89uiu/ml, p30min=0.068); there was no significant difference in serum insulin and first phase insulin levels between cp+/+tpg and cp+/+nsg groups (31.58 + 2.09uiu/ml than 33.91 + 2.38uiu/ml, p0min=0.198; 69.11 + 8.38uiu/ml ratio 68.81 + 6.89uiu/ml, p30min=0.956). After the intervention of tea polyphenols for 4 weeks, the group was smaller than the cp-/-nsg group. The level of fasting insulin in the rat serum was reduced, but there was no significant difference in the first phase insulin secretion (33.88 + 1.44uiu/ml, 37.93 + 3.36uiu/ml, p0min=0.035; 71.35 + 6.37uiu/ml ratio 79.31 + 7.84uiu/ml, p30min=0.154).3 liver tissue iron content: severe iron deposition in the liver of cp-/-nsg mice (21.47 + 3.53mg/gprot ratio 3) compared with cp+/+nsg 4 + 0.61mg/gprot, p0.001), after 4 weeks of tea polyphenols intervention, the liver iron deposition in cp-/-tpg mice was significantly improved (10.77 + 1.13mg/gprot ratio 21.47 + 3.53mg/gprot, p0.001), but there was no significant difference between cp+/+tpg and cp+/+nsg group (2.68 + 0.55mg/gprot ratio 3.04 + 0.61mg/gprot, p=0.827) immunization of insulin and iron deposition Prussian blue staining showed that the iron deposition mainly occurred in the pancreatic acinar cells in the exocrine part of the pancreas, not the islet beta cells. The apoptosis of the pancreas was also mainly in the pancreatic acinar cells in the exocrine pancreas, and not the.5 oxidative stress in the islet beta cells: the SOD level of the cp-/-nsg mice was significantly lower than that in the wild group treated with saline. The level of MDA was significantly higher (1.70 + 0.27u/mgprot than 2.11 + 0.09u/mgprot, p=0.005), and the level of MDA increased significantly (5.46 + 0.51nmol/mgprot than 4.41 + 0.43nmol/mgprot, p=0.001). After the intervention of tea polyphenols for 4 weeks, the SOD level of cp-/-tpg mice increased significantly (2.46 + 0.21u/mgprot than 1.70 + 0.27u/mgprot, p0.001), and the level was significantly lower (4.75 + than 5.46 +. L/mgprot, p=0.019). In addition, compared with the wild group treated with saline, the level of SOD in the wild group mice increased significantly (2.53 + 0.14u/mgprot ratio 2.11 + 0.09u/mgprot, p=0.004), and the MDA level was significantly decreased (3.76 + 0.25nmol/mgprot ratio 4.41 + 0.43nmol/ mgprot, p=0.031). +/+nsg mice (67 + 4.58 versus 13.67 + 2.08, p0.001), the apoptosis number of hepatocytes in cp-/-tpg mice was significantly lower than that of cp-/-nsg (38.33 + 5.03, 67 + 4.58, p0.001) and cp+/+nsg two (12.67 + 2.52 compared to 13.67 + 2.08, p=0.754).7 and cp+/+nsg, cp-/-nsg mice Liver irs2mrna and protein expression level [(0.09,0.29) ratio (0.42,0.78), pmrna=0.011; (0.52 + 0.05 to 0.70 + 0.07), pprotein=0.024], and the level of glut2mrna and protein expression in the liver significantly decreased [(0.03 + 0.02 to 0.97 + 0.13), pmrna0.001; (0.45 + 0.05 / 0.81 + 0.06), pprotein0.001]; the prognosis of the tea polyphenols for 4 weeks, and cp-/-nsg group The expression level of irs2mrna and protein in the liver of cp-/-tpg Group [(0.46,0.88) ratio (0.09,0.29), pmrna=0.006, (0.69 + 0.05 to 0.52 + 0.05), pprotein=0.029], and the expression level of liver glut2mrna and protein [(0.96 + 0.23 to 0.03 + 0.02), pmrna0.001, (0.75 + 0.06 / 0.45 + 0.05), pprotein0.001] significantly increased.Cp+/+nsg and cp+/+tpg groups The expression level of irs2mrna and protein in the liver of mice [(0.74,1.48) ratio (0.42,0.78), pmrna=0.077; (0.72 + 0.12 to 0.70 + 0.07), pprotein=0.759], and the level of glut2mrna and protein expression in the liver [(1.02 + 0.22 to 0.97 + 0.13), pmrna=0.755; (0.84 + 0.04 / 0.81 + 0.06), pprotein=0.478] without significant change. Conclusion: 1 knockout of 1 ceruloprotein gene can lead to The cause of glucose metabolism is abnormal. The cause of the increase of blood sugar is insulin resistance and the damage of non insulin secretion. 2 the knockout of the gene can cause iron deposition in the target organ of insulin, which leads to the oxidative stress disorder of the liver, the increase of cell apoptosis, the decrease of the expression of IRS2 and GLUT2 in the liver, the insulin resistance, and ultimately lead to the insulin resistance. Abnormal glucose metabolism.3 tea polyphenols by chelating liver tissue iron, alleviating liver iron deposition and the level of oxidative stress in the body, and eventually improving the abnormal glucose metabolism caused by iron overload.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
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本文编号：1840266