抗增殖蛋白Prohibitin1在C2C12小鼠成肌细胞中对线粒体功能及F0F1-ATP合成酶的影响

发布时间：2018-08-16 08:04

【摘要】：目的:调控C2C12细胞内PHB1的含量,确认PHB1对F0F1-ATP酶含量及其合成活性的影响;观察PHB1对细胞能量代谢、氧化应激(ROS)、ATP含量的影响。为下一步研究PHB1调控细胞内F0F1-ATP酶的作用机制及对运动能力的影响,揭示运动时线粒体自身调节的分子机制,以及探寻PHB1是否能够作为调控能量代谢分子靶点提供科学依据。方法:构建PHB1高表达和RNA干扰腺病毒载体,建立完整的C2C12细胞系培养体系。将PHB1高表达和RNA干扰腺病毒载体转染进小鼠成肌细胞C2C12内,最佳转染条件的摸索及确定PHB1的转染效果。利用荧光倒置显微镜观察细胞内荧光分布情况,采用流式细胞仪确定C2C12细胞中PHB1转染效率高低;并采用Western blot检测细胞中PHB1表达水平的变化,确认两种载体在细胞中对PHB1表达升高和抑制的效果。采用实时荧光定量PCR方法检测升高或降低细胞中PHB1的表达后,细胞中F0F1-ATP酶mRNA水平变化;采用Western blot观察升高和降低C2C12细胞中PHB1的表达后,F0F1-ATP酶蛋白表达水平的变化。升高和降低C2C12细胞中PHB1的表达后,采用线粒体呼吸链复合物V活性试剂盒检测细胞中F0F1-ATP酶合成活性的变化;采用活性氧(ROS)测试盒检测细胞内ROS变化;采用ATP含量试剂盒检测C2C12细胞内ATP含量的变化;采用XF细胞线粒体应激检测试剂盒检测C2C12细胞耗氧率(OCR)变化。结果:1.腺病毒载体(Ad-GFP)转染C2C12细胞,最佳转染条件为MOI=150,48h2.腺病毒高表达载体Ad-phb1转染C2C12细胞,PHB1的表达水平明显升高;RNA干扰载体Ad-phb1 shRNA转染C2C12细胞,PHB1的表达水平明显降低。3.与对照组相比,外源性升高PHB1表达,C2C12细胞中FOF1-ATP酶mRNA水平和F0F1-ATP酶蛋白表达水平均明显升高;与对照组相比,外源性降低PHB1表达,C2C12细胞中FOF1-ATP酶mRNA水平和F0F1-ATP酶蛋白表达水平均明显降低。4.与对照组相比,外源性升高PHB1的表达,C2C12细胞中F0F1-ATP合成酶活性显著升高;与对照组相比,外源性降低PHB1的表达,C2C12细胞中F0F1-ATP合成酶性显著降低。5.与对照组相比,外源性升高PHB1的表达,C2C12细胞中ATP含量和细胞耗氧率(OCR)显著增高,细胞呼吸功能显著增强,细胞能量代谢增强;与对照相组比,外源性降低C2C12细胞内PHB1的表达,ATP含量和细胞耗氧率(OCR)显著降低,细胞呼吸功能显著降低,细胞能量代谢减弱。6.与对照组相比,外源性升高PHB1的表达,C2C12细胞中ROS生成显著降低;与对照组相比,外源性降低PHB1的表达,C2C12细胞中ROS生成显著升高。结论:1.通过调控PHB1的表达,发现PHB1可以使F0F1-ATP合酶的表达及活性升高,从而促进细胞能量代谢的增强。2.通过调控PHB1的表达,使得细胞中ROS的产生减少,表明PHB1可能与稳定线粒体结构有关。
[Abstract]:Aim: to regulate the content of PHB1 in C2C12 cells, to confirm the effect of PHB1 on the content and synthesis of F0F1-ATP, and to observe the effect of PHB1 on cell energy metabolism and (ROS) content under oxidative stress. In order to further study the mechanism of PHB1 regulating intracellular F0F1-ATP enzyme and its effect on motor ability, to reveal the molecular mechanism of mitochondrial self-regulation during exercise, and to explore whether PHB1 can be used as a molecular target to regulate energy metabolism. Methods: the high expression of PHB1 and RNA interfering adenovirus vector were constructed and the complete culture system of C2C12 cell line was established. The high expression of PHB1 and RNA interference adenovirus vector were transfected into mouse myoblast C2C12. The optimal transfection conditions were explored and the transfection effect of PHB1 was determined. The fluorescence distribution in cells was observed by fluorescence inverted microscope, the transfection efficiency of PHB1 in C2C12 cells was determined by flow cytometry, and the changes of PHB1 expression in C2C12 cells were detected by Western blot. To confirm the effect of the two vectors on the increase and inhibition of PHB1 expression in cells. Real time fluorescence quantitative PCR was used to detect the changes of mRNA level of F0F1-ATP enzyme after increasing or decreasing the expression of PHB1 in C2C12 cells, and Western blot was used to observe the changes of F0F1-ATPase protein expression in C2C12 cells after increasing and decreasing the expression of PHB1. After increasing and decreasing the expression of PHB1 in C2C12 cells, mitochondrial respiratory chain complex V activity kit was used to detect the activity of F0F1-ATP synthesis, and reactive oxygen species (ROS) kit was used to detect the changes of ROS in cells. The changes of ATP content in C2C12 cells were detected by ATP assay kit, and the changes of C2C12 cell oxygen consumption rate (OCR) were detected by XF cell mitochondrial stress assay kit. The result is 1: 1. Adenovirus vector (Ad-GFP) was used to transfect C2C12 cells. The expression level of PHB1 in C2C12 cells transfected with adenovirus overexpression vector Ad-phb1 was significantly increased. The expression level of PHB1 in C2C12 cells transfected with Ad-phb1 shRNA was significantly decreased. Compared with the control group, exogenous PHB1 increased the expression of FOF1-ATP enzyme mRNA and F0F1-ATP protein in C2C12 cells, and compared with the control group, the expression of FOF1-ATP enzyme in C2C12 cells was significantly higher than that in control group. Exogenous reduction of PHB1 expression in C2C12 cells significantly decreased the mRNA level of FOF1-ATP and the expression of F0F1-ATP protein in C2C12 cells. Compared with the control group, exogenous increased the expression of PHB1 in C2C12 cells and significantly increased the activity of F0F1-ATP synthase in C2C12 cells, while exogenous decreased the expression of PHB1 in C2C12 cells. Compared with the control group, exogenous PHB1 increased the ATP content and cell oxygen consumption rate (OCR) in C2C12 cells, the respiratory function and energy metabolism of C2C12 cells were significantly increased, compared with those in the radiographic group. Exogenous reduction of PHB1 expression in C2C12 cells and cell oxygen consumption rate (OCR) decreased significantly, cell respiratory function decreased significantly, cell energy metabolism weakened .6. Compared with the control group, exogenous increased PHB1 expression and significantly decreased ROS production in C2C12 cells, while exogenous decreased PHB1 expression significantly increased ROS production in C2C12 cells. Conclusion 1. By regulating the expression of PHB1, it was found that PHB1 could increase the expression and activity of F0F1-ATP synthase, thus promoting the enhancement of cell energy metabolism. By regulating the expression of PHB1, the production of ROS was reduced, which suggested that PHB1 might be related to the stabilization of mitochondrial structure.
【学位授予单位】：天津体育学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：G804.7

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