瘦素对成年大鼠心肌细胞收缩功能作用及机制研究

发布时间：2018-03-24 02:18

本文选题：瘦素　切入点：钙瞬变　出处：《广州医科大学》2014年硕士论文

【摘要】：背景和目的瘦素（Leptin）是肥胖基因（OB基因）的表达产物，肥胖者血浆Leptin明显升高。肥胖是心力衰竭等心血管疾病的重要危险因素，与心力衰竭的发病密切相关。本研究意义在于明确Leptin对成年大鼠心肌细胞收缩功能作用及与钙敏感性、氧化应激和自噬的作用机制。方法利用成年大鼠心肌细胞进行实验，新鲜分离的心肌细胞用2μmol/L Fura-2/AM避光孵育30min（37oC），分别以1、10、100nM Leptin灌流，采用IonOptix光谱荧光检测系统记录30min内心肌细胞钙瞬变和收缩性的各项指标；钙敏感性实验以不同浓度（1.8、2.4、3.6、5.4mM）外钙灌流已孵育Leptin的心肌细胞，，记录灌流外钙后心肌细胞钙瞬变荧光强度值和收缩性的各项指标；用Leptin灌流已经孵育Apocynin100μM、Tempol100μM和Rapamycin100nM的心肌细胞，观察心肌细胞钙瞬变、收缩性和钙敏感性的各项指标；Western blotting检测心肌细胞p70S6K及其磷酸化蛋白、PKCε和自噬相关蛋白LC3B、Beclin1的表达情况。结果（1）心肌细胞钙瞬变结果显示，Lep1nM在30min内钙瞬变值只有peak值有明显下降的趋势（P0.05），bl、TB50%和TP50%值没有明显变化（P0.05）；Lep10nM在30min内bl、peak、TB50%和TP50%钙瞬变值均有明显下降的趋势（P0.05）；Lep100nM对bl和peak钙瞬变值有明显下降的趋势（P0.05）。（2）心肌细胞收缩结果显示，Lep1nM在30min内使TPS50%、TR50%收缩值明显增加（P0.05），PS、±dL/dtmax值没有明显变化（P0.05）；Lep10nM使PS和+dL/dtmax的收缩值明显下降（P0.05）；Lep100nM使收缩值除TPS50%没有明显变化外，PS、TR50%和±dL/dtmax值均有明显差异（P0.05）。（3）钙敏感性实验结果显示，Leptin能降低由不同浓度（1.8、2.4、3.6、5.4mM）外钙灌流心肌细胞的敏感性水平，PS和±dL/dtmax值均明显减弱，TR50%值时间显著延长。（4）Apocynin和Tempol作用的心肌细胞，与CTL组相比，其钙瞬变和收缩性指标与钙敏感性结果均无明显变化，两者分别与Leptin同时作用于心肌细胞，均能阻断Leptin减少细胞内游离钙离子浓度、减弱细胞收缩力的作用和降低钙敏感性水平。Rapamycin作用的心肌细胞，与CTL组相比，其钙瞬变和收缩值无明显变化，但与Leptin同时作用于心肌细胞，Rapamycin能阻断Leptin减少细胞内游离钙离子浓度和减弱细胞收缩功能的作用。（5）Western blotting结果显示，与CTL组相比，Lep组自噬相关蛋白LC3B和Beclin1的表达均下降（P0.05），PKCε蛋白水平升高（P0.05），但都能分别被Apocynin和Tempol阻断。同样地，Rapamycin与Leptin的实验结果中，与CTL组相比，Lep组自噬相关蛋白LC3B和Beclin1的表达均下降（P0.05），p-p70S6K蛋白水平升高（P0.05），但能被Rapamycin阻断。结论 Leptin减少细胞内游离钙离子浓度，减弱心肌的收缩力，并降低钙敏感性水平，Apocynin、Tempol和Rapamycin均能阻断Leptin对心肌细胞的作用，其机制与氧化应激和自噬相关。
[Abstract]:Background and purpose. Leptin (Leptin) is the expression product of obesity gene (OB gene), and plasma Leptin is significantly increased in obese patients. Obesity is an important risk factor for cardiovascular diseases such as heart failure. The purpose of this study is to clarify the effects of Leptin on the contractile function of cardiac myocytes in adult rats and its mechanism of calcium sensitivity, oxidative stress and autophagy. Method. The freshly isolated cardiomyocytes were incubated with 2 渭 mol/L Fura-2/AM for 30 mins at 37oC, then perfused with 110100nM Leptin respectively. The indexes of calcium transient and contractility of myocardial cells in 30min were recorded by IonOptix fluorescence detection system. The calcium sensitivity test was conducted with different concentrations of 1.8U 2.4C 3.6mM) to incubate cardiomyocytes with Leptin, and to record the indexes of calcium transient fluorescence intensity and contractility of cardiomyocytes after perfusion, and to incubate the cardiomyocytes of Apocynin100 渭 Mil Tempol100 渭 M and Rapamycin100nM with Leptin. The changes of calcium transient, contractility and calcium sensitivity in cardiomyocytes were observed. Western blotting was used to detect the expression of p70S6K and its phosphorylated protein, PKC 蔚, and autophagy associated protein LC3B, Beclin1. Results. (1) the results of calcium transient of myocardial cells showed that the transient values of Lep1nM in 30min only showed a significant decrease in peak value. There was no significant change in TB50% and TP50% values of P0.05 P0.05 + Lep10nM in 30min. There was a significant decrease in TB50% and TB50% calcium transient values of Lep1nM in 30min and the transient changes of calcium in both Bl and peak were observed in Lep100nM. There is a significant downward trend in the value of P0. 05%. 2) the contractile results of cardiac myocytes showed that Lep1nM significantly increased the contraction value of TPS50 / TPS50 / TPS50 / TPS50 / TPS50, and the 卤dL/dtmax value did not significantly change P0.05Lep10nM decreased the contractile values of PS and dL/dtmax significantly (P0.05Lep100nM). There were significant differences in the contractile values of PS50% and 卤dL/dtmax (P0.05nM) except TPS50%. The results of calcium sensitivity test showed that leptin could decrease the sensitivity level of cardiac myocytes perfused with calcium from different concentrations of 1.8U 2.4U 3.6mM, PS value and 卤dL/dtmax value were significantly weakened and the time of TR50% was significantly prolonged. Compared with CTL group, the calcium transient and contractility indexes and calcium sensitivity of cardiac myocytes treated with Apocynin and Tempol had no significant changes. Both of them treated with Leptin at the same time could block the reduction of intracellular free calcium concentration by Leptin. Compared with CTL group, there was no significant change in calcium transient and contractile values in cardiac myocytes with decreased cell contractility and calcium sensitivity. But at the same time as Leptin, Rapamycin could block the effect of Leptin on reducing intracellular free calcium concentration and decreasing cell contraction function. The results of Western blotting showed that the expression of LC3B and Beclin1 of autophagy associated protein in CTL group was significantly lower than that in CTL group, but both of them could be blocked by Apocynin and Tempol, respectively. In the same way, in the experimental results of Rapamycin and Leptin, the expression of LC3B and Beclin1 in Lep group was decreased, and the level of PKC-蔚 protein in P0.05 was increased, but both could be blocked by Apocynin and Tempol, respectively. Compared with CTL group, the expression of autophagy associated protein (LC3B) and Beclin1 in Lep group decreased significantly, and the level of p-p70S6K protein increased, but could be blocked by Rapamycin. Conclusion. Leptin reduced intracellular free calcium concentration, decreased myocardial contractility, and decreased calcium sensitivity. Both Apocyninine Tempol and Rapamycin could block the effect of Leptin on cardiomyocytes, and its mechanism was related to oxidative stress and autophagy.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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