發(fā)布時間：2018-05-04 17:26

  本文選題：糖尿病心肌病 + 鈣庫操縱鈣內(nèi)流��； 參考：《皖南醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的:建立糖尿病大鼠模型,明確鈣庫操縱鈣內(nèi)流(store-operated calcium entry,SOCE)及其相關(guān)蛋白在糖尿病大鼠心室肌細(xì)胞中是否改變,探討大鼠心室肌細(xì)胞SOCE在糖尿病心肌病的發(fā)生、發(fā)展中所起到的作用。方法:選擇健康雄性Sprauge Dawley(SD)大鼠,隨機分為實驗組和對照組(n=20)。實驗組大鼠予以鏈脲佐菌素(streptozocin,STZ)60mg·kg-1一次性腹腔注射,對照組予以等劑量檸檬酸鈉溶液腹腔注射,72h后測尾靜脈血糖≥16.0mmol·L-1,且出現(xiàn)多飲、多尿、多食等癥狀,確定糖尿病大鼠造模成功,后予以飼養(yǎng)8周,期間每天予以足量食水,勤換墊料,每周定期測尾靜脈血糖維持在≥16.5mmol·L-1用于實驗。8周后,實驗組及對照組禁食、禁水8h后,進(jìn)行稱重及測血糖,予以10%水合氯醛以3 ml/kg進(jìn)行腹腔麻醉,待大鼠肌張力減弱后固定于手術(shù)臺上,采用BL-420E多道生理信號采集處理系統(tǒng),正確插入電極,測定心電圖變化,記錄完畢后處死,取出心臟后進(jìn)行稱重,取心尖組織行HE染色,確定糖尿病大鼠心室肌是否發(fā)生肥大及纖維化等改變;取同一部位心肌組織進(jìn)行培養(yǎng)、孵育及染色,測鈣細(xì)胞內(nèi)濃度確定SOCE是否異常開放;取心室肌組織,運用蛋白免疫印跡法(Western Blot)技術(shù),對Ca2+釋放激活Ca2+通道蛋白質(zhì)(calcium release-activated calcium channel protein,Orai)和基質(zhì)相互作用分子(stromal interaction molecule,STIM)蛋白含量進(jìn)行測定。結(jié)果:1.飼養(yǎng)8周后:①生存狀態(tài)改變:實驗組動物腹腔注射STZ 24h后逐漸出現(xiàn)多飲、多尿、多食及精神萎靡等癥狀,在生理方面發(fā)生顯著的變化,正常組飲食、飲水及大小便排泄正常;②體重改變:實驗組與對照組顯著降低,差異有統(tǒng)計學(xué)意義(P0.001);③末次血糖:實驗組與對照組相比顯著升高,差異有統(tǒng)計學(xué)意義(P0.001);④心臟重量:實驗組與對照組相比有統(tǒng)計學(xué)意義(P0.001);⑤心臟重量/體重:實驗組與對照組相比增加,差異有統(tǒng)計學(xué)意義(P0.001)。2.心電圖檢查:①心電圖波形改變:對照組心電圖心律齊、波形一致,T波正常,實驗組出現(xiàn)不同程度的心律失常,如:T波低平及倒置、室性早搏等;②心率改變:實驗組與對照組之間差異無統(tǒng)計學(xué)意義(P0.05)。3.HE染色結(jié)果:對照組心肌纖維排列致密,細(xì)胞形態(tài)正常,實驗組心肌纖維排列疏松、紊亂,細(xì)胞腫脹變性。4.鈣濃度測定:①加入毒胡蘿卜素(thapsigargin,TG)后鈣庫釋放能力:實驗組與對照組相比明顯下降,差異有統(tǒng)計學(xué)意義(P0.05);②Ca2+內(nèi)流比較:實驗組與對照組相比明顯升高,差異有統(tǒng)計學(xué)意義(P0.05)。5.Western blot:①Orai1蛋白含量比較:通過半定量分析顯示,實驗組比對照組表達(dá)增強,差異有統(tǒng)計學(xué)意義(P0.05);②Orai2蛋白含量比較:實驗組比對照組表達(dá)增強,差異有統(tǒng)計學(xué)意義(P0.01);③Orai3蛋白含量比較:實驗組比對照組表達(dá)增強,差異無統(tǒng)計學(xué)意義(P0.05);④STIM1:蛋白含量比較:實驗組比對照組表達(dá)增強,差異有統(tǒng)計學(xué)意義(P0.01);⑤STIM2:蛋白含量比較:實驗組比對照組表達(dá)減弱,差異有統(tǒng)計學(xué)意義(P0.001)。結(jié)論:1.糖尿病大鼠心肌細(xì)胞中SOCE較正常組異常激活,其相關(guān)蛋白Orai1、Orai2和STIM1表達(dá)增加,STIM2表達(dá)減少;2.SOCE是糖尿病心肌病的發(fā)生、發(fā)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.

【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.1

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