糖尿病大鼠心室肌细胞钙库操纵钙内流及相关蛋白的变化

发布时间：2018-05-04 17:26

本文选题：糖尿病心肌病 + 钙库操纵钙内流　；参考：《皖南医学院》2015年硕士论文

【摘要】：目的:建立糖尿病大鼠模型,明确钙库操纵钙内流(store-operated calcium entry,SOCE)及其相关蛋白在糖尿病大鼠心室肌细胞中是否改变,探讨大鼠心室肌细胞SOCE在糖尿病心肌病的发生、发展中所起到的作用。方法:选择健康雄性Sprauge Dawley(SD)大鼠,随机分为实验组和对照组(n=20)。实验组大鼠予以链脲佐菌素(streptozocin,STZ)60mg·kg-1一次性腹腔注射,对照组予以等剂量柠檬酸钠溶液腹腔注射,72h后测尾静脉血糖≥16.0mmol·L-1,且出现多饮、多尿、多食等症状,确定糖尿病大鼠造模成功,后予以饲养8周,期间每天予以足量食水,勤换垫料,每周定期测尾静脉血糖维持在≥16.5mmol·L-1用于实验。8周后,实验组及对照组禁食、禁水8h后,进行称重及测血糖,予以10%水合氯醛以3 ml/kg进行腹腔麻醉,待大鼠肌张力减弱后固定于手术台上,采用BL-420E多道生理信号采集处理系统,正确插入电极,测定心电图变化,记录完毕后处死,取出心脏后进行称重,取心尖组织行HE染色,确定糖尿病大鼠心室肌是否发生肥大及纤维化等改变;取同一部位心肌组织进行培养、孵育及染色,测钙细胞内浓度确定SOCE是否异常开放;取心室肌组织,运用蛋白免疫印迹法(Western Blot)技术,对Ca2+释放激活Ca2+通道蛋白质(calcium release-activated calcium channel protein,Orai)和基质相互作用分子(stromal interaction molecule,STIM)蛋白含量进行测定。结果:1.饲养8周后:①生存状态改变:实验组动物腹腔注射STZ 24h后逐渐出现多饮、多尿、多食及精神萎靡等症状,在生理方面发生显著的变化,正常组饮食、饮水及大小便排泄正常;②体重改变:实验组与对照组显著降低,差异有统计学意义(P0.001);③末次血糖:实验组与对照组相比显著升高,差异有统计学意义(P0.001);④心脏重量:实验组与对照组相比有统计学意义(P0.001);⑤心脏重量/体重:实验组与对照组相比增加,差异有统计学意义(P0.001)。2.心电图检查:①心电图波形改变:对照组心电图心律齐、波形一致,T波正常,实验组出现不同程度的心律失常,如:T波低平及倒置、室性早搏等;②心率改变:实验组与对照组之间差异无统计学意义(P0.05)。3.HE染色结果:对照组心肌纤维排列致密,细胞形态正常,实验组心肌纤维排列疏松、紊乱,细胞肿胀变性。4.钙浓度测定:①加入毒胡萝卜素(thapsigargin,TG)后钙库释放能力:实验组与对照组相比明显下降,差异有统计学意义(P0.05);②Ca2+内流比较:实验组与对照组相比明显升高,差异有统计学意义(P0.05)。5.Western blot:①Orai1蛋白含量比较:通过半定量分析显示,实验组比对照组表达增强,差异有统计学意义(P0.05);②Orai2蛋白含量比较:实验组比对照组表达增强,差异有统计学意义(P0.01);③Orai3蛋白含量比较:实验组比对照组表达增强,差异无统计学意义(P0.05);④STIM1:蛋白含量比较:实验组比对照组表达增强,差异有统计学意义(P0.01);⑤STIM2:蛋白含量比较:实验组比对照组表达减弱,差异有统计学意义(P0.001)。结论:1.糖尿病大鼠心肌细胞中SOCE较正常组异常激活,其相关蛋白Orai1、Orai2和STIM1表达增加,STIM2表达减少;2.SOCE是糖尿病心肌病的发生、发展中的重要因素。
[Abstract]:Objective: to establish a diabetic rat model to determine whether the store-operated calcium entry (SOCE) and its related proteins change in the ventricular myocytes of diabetic rats, and explore the role of SOCE in the development of diabetic cardiomyopathy in rats. Methods: selecting healthy male Sprauge Dawley (SD). Rats were randomly divided into experimental group and control group (n=20). Rats in the experimental group were injected with streptozotocin (streptozocin, STZ) 60mg. Kg-1 at one time. The control group was given the same dose of sodium citrate in the abdominal cavity. After 72h, the blood glucose of the tail vein was more than 16.0mmol. L-1, and the symptoms of polyuria, polyuria and polydipsia were found, and the model of diabetic rats was established. After 8 weeks, the work was given for 8 weeks. During the period every day, the amount of water was fed and changed frequently. The blood sugar of the tail vein was maintained at more than 16.5mmol. L-1 a week. After the experiment.8 weeks, the experimental group and the control group were fasting and the water was forbidden 8h to weigh and measure the blood sugar. The 10% chloral chloral was given to the abdominal anesthesia with 3 ml/ kg. After the muscle tension was weakened, the rats were immobilized to the hand after the muscle tension was weakened. The rats were fixed to the hands after the muscle tension was weakened. On the table, the BL-420E multichannel physiological signal collection and processing system was used, the electrode was inserted correctly, the changes of electrocardiogram were measured. After the record was completed, the heart was weighed after taking out the heart and the apex tissue was stained with HE to determine whether the ventricular myocytes of the diabetic rats were hypertrophy and fibrosis, and the same part of the myocardium was cultured and incubated. The intracellular concentration of calcium was determined to determine whether SOCE was unusually open, and the content of Ca2+ release activation of the Ca2+ channel protein (calcium release-activated calcium channel protein, Orai) and the protein content of the matrix were determined by using the technique of Western Blot. Results: 1. after 8 weeks of feeding: (1) changes in the state of survival: after intraperitoneal injection of STZ 24h in the experimental group, the symptoms of polypodities, polyuria, polydipsia and mental malaise were gradually changed, the normal diet, drinking water and feces excretion were normal, and the body weight was changed: the experimental group and the control group were significantly reduced, the difference was statistically significant Significance (P0.001); third time blood glucose: the experimental group was significantly higher than the control group (P0.001); the heart weight: the experimental group was statistically significant compared with the control group (P0.001); fifth, the heart weight / weight: the experimental group was compared with the control group, the difference was statistically significant (P0.001).2. electrocardiogram examination: (1) electrocardiogram wave Shape change: in the control group, the electrocardiogram was homogeneous, the waveform was the same, the T wave was normal, the experimental group had different degrees of arrhythmia, such as T wave low level and inversion, ventricular premature beat and so on; the difference of heart rate between the experimental group and the control group was not statistically significant (P0.05).3.HE staining fruit: the control group was dense, the cell morphology was normal, the experimental group was normal. Myocardial fibers arranged loosely, disorder, and cell swelling and denaturation.4. calcium concentration: (1) the release capacity of calcium library after the addition of thapsigargin (TG): the experimental group was significantly lower than the control group, and the difference was statistically significant (P0.05); (2) compared with the control group, the experimental group was significantly higher than the control group, and the difference was statistically significant (P0.05).5.W Estern blot: (1) Orai1 protein content comparison: through semi quantitative analysis, the experimental group was more expressed than the control group, the difference was statistically significant (P0.05), and the content of Orai2 protein in the experimental group was compared with the control group, the difference was statistically significant (P0.01); (3) the content of Orai3 protein was compared: the expression of the experimental group was stronger than the control group, and there was no difference in the expression of the experimental group. Study significance (P0.05); (4) comparison of STIM1: protein content: the expression of the experimental group was stronger than the control group, the difference was statistically significant (P0.01), and the content of STIM2: protein was compared: the expression of the experimental group was weaker than the control group, and the difference was statistically significant (P0.001). Conclusion: 1. the SOCE in the myocardial cells of the diabetic rats was more abnormal than that in the normal group, and its related protein Orai1, O The expression of rai2 and STIM1 increased, and the expression of STIM2 decreased. 2.SOCE is an important factor in the occurrence and development of diabetic cardiomyopathy.

【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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