低温联合硫氢化钠对PC12细胞凋亡及GSK-3β和Bcl-2表达的影响

发布时间：2018-05-10 07:09

  本文选题：PC12细胞 + H_2O_2　； 参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]用H202氧化应激损伤PC12细胞,复制神经细胞损伤模型,初步探索低温、硫化氢(hydrogen sulfide,H2S)联合干预后,对氧化应激损伤的PC12细胞的保护作用,以及低温联合H2S在对抗神经细胞的氧化应激损伤方面的可能机制。[方法]按照常规方法培养PC12细胞,选取贴壁80%-90%、生长状态良好的细胞,调整H202浓度为10Oμmol/L,过氧化损伤PC12细胞12h,得到神经细胞氧化应激模型,采用RT-QPCR方法分别检测氧化应激损伤前后PC12细胞IRAK1和TNF-α两个指标mRNA的表达,获得本次实验的目标细胞;分组(n=3),空白对照(37℃,5%的C02,培养24h);低温干预组:将目标细胞分别置于37℃、32℃、27℃,置于5%的C02条件下培养24h;低温联合H2S干预组:以NaHS为H2S供体,向培养基中加入NaHS,调整终浓度至100μmol/L,分别置于37℃、32℃、27℃,5%的C02培养箱条件下培养24h;NaHS对照组:调整NaHS的浓度到100μmol/L,37℃,5%的C02培养条件下培养24h。CCK-8检细胞增殖;Hoechst33258染色观察核形态变化;FITC、PI双染,FCM检测细胞凋亡;通过电泳、蛋白质印迹(Western Blot)技术和RT-QPCR技术检测Bcl-2和GSK-3β在不同干预组的蛋白和mRNA含量变化情况,用SPSS17.0对结果进行统计学分析。[结果]1.细胞在27℃-37℃内生长良好;2.PC12细胞H202氧化应激模型,有效可行;3.单纯低温干预,能减弱H202对Bcl-2的蛋白、mRNA表达量的下调作用,H2S联合低温可进一步加强这种作用;4.亚低温能减弱H202对GSK-3β的蛋白表达的上调作用,在深低温组和亚低温组差异不明显,H2S联合低温可进一步减轻H202对GSK-3β蛋白表达的上调作用;低温能减轻H202对GSK-3β mRNA的上调作用,H2S联合低温能进一步减轻这种作用。[结论](1)体外培养大鼠嗜铬细胞瘤(PC12)细胞模型操作简便,重复性好,能模拟氧化应激损伤后低温治疗过程,是一种较好的体外神经元低温模型。(2)PC12细胞在氧化应激损伤过后,Bcl-2的蛋白和mRNA的表达量下调,GSK-3β蛋白和mRNA表达上调。(3)在27℃-37℃范围内,随着温度的降低Bcl-2蛋白和mRNA表达量逐渐升高,表明低温能促进Bcl-2蛋白转录、表达,减轻细胞凋亡,H2S可加强这种作用。(4)在27℃-37℃范围内,随着温度的降低GSK-3β蛋白和mRNA表达量逐渐减少,H2S可加强这种作用。(5).低温联合H2S对GSK-3 β和Bcl-2蛋白质和mRNA表达量的影响可能是低温脑保护的作用机制之一。
[Abstract]:[objective] to study the protective effects of hypothermia and hydrogen sulfide (H2S) on PC12 cells injured by oxidative stress (H202), and to establish a model of nerve cell injury, and to explore the protective effect of H202 on PC12 cells injured by oxidative stress. And the possible mechanism of hypothermia combined with H _ 2S in antagonizing oxidative stress injury of nerve cells. [methods] PC12 cells were cultured according to the conventional method. The cells with good growth state were selected from 80 to 90 cells attached to the wall. The oxidative stress model of nerve cells was obtained by adjusting the concentration of H202 to 10 O 渭 mol / L for 12 h after peroxidation injury of PC12 cells. The expression of IRAK1 and TNF- 伪 in PC12 cells before and after oxidative stress injury was detected by RT-QPCR method, and the target cells were obtained. They were divided into three groups, the control group was incubated with C02 at 37 鈩，

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