本文選題：銅藍蛋白 + 鐵過載　； 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:糖尿病是一組以血漿葡萄糖水平增高為特征的慢性代謝性疾病,其病理特點是胰島β細胞功能受損和/或胰島素抵抗。近年來,越來越多的研究表明,鐵過載可增加糖尿病的患病風險,體內(nèi)鐵過載是2型糖尿病的獨立危險因素。鐵是人體內(nèi)含量最多的必需微量元素,鐵穩(wěn)態(tài)對于細胞信號轉導和內(nèi)環(huán)境的穩(wěn)態(tài)至關重要。銅藍蛋白(ceruloplasmin,Cp)是鐵轉運過程中的關鍵酶,主要在肝細胞合成,具有亞鐵氧化酶活性,可將Fe2+氧化成Fe3+,促進鐵與轉鐵蛋白結合。Cp基因敲除(Cp-/-)后可以導致組織鐵過載,以及糖耐量異常,但其具體機制不詳。茶多酚是一種天然的抗氧化劑,具有螯合鐵的特性,目前茶多酚對Cp-/-小鼠糖代謝的影響機制尚未見詳細報道。本課題組以Cp基因敲除(內(nèi)源性鐵過載)小鼠為研究對象,探討鐵過載導致糖代謝異常的具體作用機制;同時,應用茶多酚灌胃進行干預治療,明確茶多酚對Cp基因敲除小鼠糖代謝的影響,以期為糖尿病患者提供新的治療選擇。方法:隨機選取12只10月齡雌性BALB/c J×129Sv J品系銅藍蛋白基因敲除(Cp-/-)小鼠作為實驗組,另選取12只同性別、同月齡、同品系的野生型(Cp+/+)小鼠作為對照組,將實驗組和對照組小鼠分別隨機分為茶多酚干預組(TPG,n=6)和生理鹽水處理組(NSG,n=6)進行干預,4周后,通過葡萄糖耐量實驗和胰島素耐量實驗評估小鼠胰島β細胞的儲備功能和胰島素敏感性。放射免疫法檢測小鼠血清胰島素水平;比色法檢測肝臟非血紅素鐵含量;免疫組化法檢測胰腺鐵沉積情況;黃嘌呤氧化酶法測定超氧化物歧化酶(Superoxide dismutase,SOD)活性、硫代巴比妥酸法測定丙二醛(Malondialdehyde,MDA)含量以明確肝臟氧化應激水平;原位末端標記(terminal dexynucleotidyl transferase(Td T)-mediated d UTP nick end labeling,TUNEL)法檢測肝實質細胞和胰島β細胞的凋亡情況;Real-time PCR測定肝臟葡萄糖轉運體2(glucose transporter,GLUT2)、胰島素受體底物2(insulin receptor substrate 2,IRS2)mRNA表達水平;Western-blot法測定肝臟GLUT2、IRS2的蛋白表達水平。應用spss21.0統(tǒng)計軟件進行統(tǒng)計分析,所有計量資料均進行正態(tài)性檢驗,正態(tài)分布數(shù)據(jù)以平均值±標準差(?x±sd)表示,非正態(tài)分布數(shù)據(jù)以(最小值,最大值)表示。正態(tài)分布且方差齊性數(shù)據(jù),采用單因素方差分析,兩兩比較采用lsd檢驗。不符合正態(tài)分布的資料采用獨立樣本的kruskal-wallis秩和檢驗。以p0.05為差異有統(tǒng)計學意義。結果:1與cp+/+nsg小鼠相比,cp-/-nsg小鼠血糖顯著升高(葡萄糖耐量實驗各點血糖值: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),胰島素敏感性顯著下降(胰島素耐量實驗各點血糖值: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與cp+/+nsg組間血糖無顯著差異(葡萄糖耐量實驗各點血糖值: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),胰島素敏感性也無明顯變化(胰島素耐量實驗各點血糖值: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)。與cp-/-nsg小鼠相比,cp-/-tpg小鼠血糖明顯降低(葡萄糖耐量實驗各點血糖值: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),胰島素敏感性顯著升高(胰島素耐量實驗各點血糖值: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血清胰島素水平:與cp+/+nsg相比,cp-/-nsg小鼠血清空腹胰島素水平明顯升高,但第一時相胰島素分泌無統(tǒng)計學差異(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組間空腹血清胰島素及第一時相胰島素水平均無明顯差異(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);茶多酚干預4周后,與cp-/-nsg組相比,cp-/-tpg組小鼠血清空腹胰島素水平降低,但第一時相胰島素分泌無統(tǒng)計學差異(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肝臟組織鐵含量:與cp+/+nsg相比,cp-/-nsg小鼠肝臟發(fā)生了嚴重鐵沉積(21.47±3.53mg/gprot比3.04±0.61mg/gprot,p0.001),經(jīng)茶多酚干預4周后,與cp-/-nsg相比,cp-/-tpg小鼠肝臟鐵沉積得到了明顯改善(10.77±1.13mg/gprot比21.47±3.53mg/gprot,p0.001)。但cp+/+tpg與cp+/+nsg組間未見顯著差異(2.68±0.55mg/gprot比3.04±0.61mg/gprot,p=0.827)。4胰島素免疫組化與鐵沉積普魯士藍染色結果顯示,鐵沉積主要發(fā)生在胰腺外分泌部的胰腺腺泡細胞,而非胰島β細胞。胰腺凋亡也主要發(fā)生在胰腺外分泌部的胰腺腺泡細胞,而非胰島β細胞。5氧化應激:與生理鹽水處理的野生組小鼠相比,cp-/-nsg小鼠sod水平顯著降低(1.70±0.27u/mgprot比2.11±0.09u/mgprot,p=0.005)、mda水平明顯升高(5.46±0.51nmol/mgprot比4.41±0.43nmol/mgprot,p=0.001);經(jīng)茶多酚干預4周后,cp-/-tpg小鼠sod水平顯著升高(2.46±0.21u/mgprot比1.70±0.27u/mgprot,p0.001)、mda水平明顯降低(4.75±0.48nmol/mgprot比5.46±0.51nmol/mgprot,p=0.019)。此外,與生理鹽水處理的野生組小鼠相比,茶多酚干預的野生組小鼠sod水平明顯升高(2.53±0.14u/mgprot比2.11±0.09u/mgprot,p=0.004)、mda水平顯著降低(3.76±0.25nmol/mgprot比4.41±0.43nmol/mgprot,p=0.031)。6cp-/-nsg小鼠肝細胞凋亡數(shù)顯著高于cp+/+nsg小鼠(67.00±4.58比13.67±2.08,p0.001),經(jīng)茶多酚灌胃干預后,cp-/-tpg小鼠肝細胞凋亡數(shù)較cp-/-nsg明顯降低(38.33±5.03比67.00±4.58,p0.001)。cp+/+tpg與cp+/+nsg兩組間肝細胞凋亡數(shù)目無顯著差異(12.67±2.52比13.67±2.08,p=0.754)。7與cp+/+nsg相比,cp-/-nsg小鼠肝臟irs2mrna和蛋白表達水平[(0.09,0.29)比(0.42,0.78),pmrna=0.011;(0.52±0.05比0.70±0.07),pprotein=0.024],以及肝臟glut2mrna和蛋白表達水平顯著降低[(0.03±0.02比0.97±0.13),pmrna0.001;(0.45±0.05比0.81±0.06),pprotein0.001];經(jīng)茶多酚為期4周的干預后,與cp-/-nsg組相比,cp-/-tpg組小鼠肝臟irs2mrna和蛋白表達水平[(0.46,0.88)比(0.09,0.29),pmrna=0.006;(0.69±0.05比0.52±0.05),pprotein=0.029],以及肝臟glut2mrna和蛋白表達水平[(0.96±0.23比0.03±0.02),pmrna0.001;(0.75±0.06比0.45±0.05),pprotein0.001]顯著升高。cp+/+nsg組和cp+/+tpg組小鼠肝臟irs2mrna和蛋白表達水平[(0.74,1.48)比(0.42,0.78),pmrna=0.077;(0.72±0.12比0.70±0.07),pprotein=0.759],以及肝臟glut2mrna和蛋白表達水平[(1.02±0.22比0.97±0.13),pmrna=0.755;(0.84±0.04比0.81±0.06),pprotein=0.478]無顯著變化。結論:1銅藍蛋白基因敲除可導致糖代謝異常,血糖升高的原因是胰島素抵抗而非胰島素分泌功能受損;2銅藍蛋白基因敲除可導致胰島素作用的靶器官(肝臟)發(fā)生鐵沉積,后者導致肝臟氧化應激紊亂,細胞凋亡增加,肝臟irs2、glut2表達降低,引起胰島素抵抗,并最終導致糖代謝異常。3茶多酚通過螯合肝臟組織鐵,減輕肝臟鐵沉積及機體的氧化應激水平,最終改善鐵過載引起的糖代謝異常。
[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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.1
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