图1 转基因小鼠模型的构建
纸质出版日期:2022-11-25,
网络出版日期:2022-03-30,
收稿日期:2021-12-19,
录用日期:2022-01-17
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为探讨miR-130a-3p调节有氧代谢对小鼠运动耐力的影响,选取3周龄生长性能相近的野生型(WT)、miR-130a-3p过表达(130OE)与敲除(130KO)型的FVB雄性小鼠各10只,在高脂饲料喂养条件下,每3 d记录1次小鼠采食量,每周记录1次体质量,并在第5周检测小鼠的肌肉力量、运动耐力,第6周测量小鼠有氧代谢情况。结果显示,与WT小鼠相比,130OE和130KO小鼠的采食量均无显著差异,但130OE小鼠的体增量显著降低(P<0.05),而130KO小鼠体质量和体增量均显著大于WT小鼠(P<0.05);相对于WT小鼠,130OE小鼠CO2产生量显著上升(P<0.01),130KO小鼠的耗氧量与CO2产生量均显著下降(P<0.01);而且130KO小鼠的产热量也显著低于WT小鼠(P<0.01)。在运动耐力方面,与WT小鼠相比,130OE小鼠的运动时间与距离均显著上升(P<0.05),而130KO小鼠均极显著下降(P<0.01)。结果表明,miR-130a-3p过表达能够提高小鼠的有氧代谢,降低小鼠体质量,增强小鼠运动耐力的作用。结果为深入研究miR-130a-3p对骨骼肌能量代谢及其运动机能的调节机制提供了前期基础。
The purpose of this study is to investigate the effect of miR-130a-3p in regulating aerobic metabolism in exercise endurance in mice. Ten of 3-week-old wild type (WT), miR-130a-3p overexpression (130OE) and knockout (130KO) FVB male mice with similar growth performance were selected respectively for each group. Under the condition of high-fat diet feeding, the food intake of the mice was recorded every 3 days, the body mass was recorded once a week, the muscle strength and exercise endurance were measured in the 5th week, and the aerobic metabolism was measured in the 6th week. The results showed that compared with WT mice, the food intake of 130OE and 130KO mice were not significantly different, but the body mass gain of 130OE mice was significantly reduced (P <0.05), while the body mass and body mass gain of 130KO mice were both higher than WT mice (P <0.05). Compared with that of WT mice, CO2 production in 130OE mice increased significantly (P < 0.01), while O2 consumption and CO2 production in 130KO mice decreased significantly (P < 0.01). 130KO mice also had lower heat production than that of WT mice (P<0.01). In terms of exercise endurance, the exercise endurance and distance of 130OE mice were increased (P<0.05) compared with WT mice, while those of 130KO mice were significantly decreased(P<0.01). The results showed that miR-130a-3p overexpression can improve aerobic metabolism, reduce body mass, and enhance the effect of exercise endurance in mice, and provided a preliminary basis for further research on the regulation mechanism of miR-130a-3p on skeletal muscle energy metabolism and motor function.
miRNA;
miR-130a-3p;
exercise endurance;
aerobic metabolism
MicroRNAs(miRNAs)是一类在转录后水平参与基因调控并发挥重要作用的非编码小RNA,主要通过抑制翻译和降解mRNA来调节其靶标[
近年来,有不少报道称miRNAs参与调节有氧代谢过程。研究表明,miR-181a/b-1在成骨过程中通过增加线粒体耗氧率和与ATP相关的呼吸代谢促进成骨分化[
骨骼肌作为动物机体参与运动、氧化代谢等活动的重要代谢器官,其自身的发育及其对体内能量物质的利用受到外部环境因素以及内部调节因子的共同作用,包括miRNAs。早在2006年,研究显示miR-1能促进成肌细胞分化,而miR-133刺激成肌细胞增殖[
miR-130a-3p全身性敲除与过表达FVB小鼠由赛业(广州)生物科技有限公司通过CRISPR/CAS9基因编辑技术制备[
实时荧光定量PCR仪(Bio-Rad C1000 Touch,广州市昊洋贸易有限公司);小鼠跑步机(瑞沃德生物科技有限公司);抓握力测定仪器(BIO-GS3, Bioseb, France);代谢测量系统(Sable Systems International,美国);分光光度计(Nanodrop 2000,Thermo Fisher,美国)。
选用30只3周龄生长性能相近的FVB雄性小鼠,其中野生型(WT)、miR-130a-3p过表达(130OE)与miR-130a-3p敲除(130KO)小鼠各10只。在整个试验中,使用高脂饲料(HFD)喂养小鼠。所有小鼠均单笼饲养,每天光照和黑暗各 12 h,自由采食和饮水。环境相对湿度控制在 60%左右,温度控制在(25±1) ℃左右。每3 d记录1次采食量,每7 d记录1次体质量。
1.4.1 肌肉力量检测
在试验的第5周通过小鼠砝码抓握试验与抓握力测定试验检测肌肉力量。小鼠砝码抓握试验方法为:用手抓住小鼠尾巴把小鼠拎起来,迫使小鼠抓住一定质量的砝码,当小鼠能抓住砝码并坚持3 s以上时则认为小鼠可以承受该质量,进入下一质量的砝码检测。每一只小鼠均轮流进行检测,测试结束后统计每组小鼠的得分情况[
1.4.2 运动耐力测试
测试开始前将跑步机倾斜角度调为10°,每条跑道放入1只小鼠,先以11 m/min的速度运动,适应10 min后休息10 min。然后开始试验:给予小鼠一个11 m/min的初速度,每5 min加速4 m/min,直至小鼠静坐在电网上5 s,即判定为小鼠力竭,记录每只小鼠的运动时间与距离[
在饲喂高脂饲料的第6周,通过使用代谢测量系统得到小鼠的O2消耗量(VO2)、CO2产生量(VCO2)和产热量。
使用Trizol(Invitrogen)从组织中提取总RNA,使用分光光度计对RNA的浓度进行检测。使用 EZB 4 × EZscript Reverse Transcription Mix Ⅱ 逆转录试剂盒(海方生物,上海)将总量1 μg的RNA进行逆转录,其中miR-130a-3p使用特异性颈环引物逆转录,mRNA使用OligodT18逆转录。将得到的产物cDNA稀释5倍使用。
qRT-PCR通过 EZB 2× Color SYBR Green qPCR Master Mix(A0012) 在实时荧光定量PCR仪中进行检测,miRNA使用U6作为内参,将得到的结果通过2-ΔCt方法进行统计。
采用SPSS 24.0进行单因素方差分析和独立样本t检验分析。所有统计分析均用 Graghpad 7.0统计软件完成,结果用平均值±标准误(mean±S.E.M)表示,以P<0.05作为差异显著性判断标准。
为了探究miR-130a-3p是否参与调节骨骼肌发育以及机体的氧化代谢,我们建立了miR-130a-3p过表达(130OE)和miR-130a-3p基因敲除(130KO)小鼠模型。在琼脂糖凝胶电泳中,130OE小鼠中可以检测到插入的外源基因(
图1 转基因小鼠模型的构建
Fig.1 Construction of transgenic mouse model
“**”表示两组之间相比差异达P<0.01显著水平(t检验)。
在高脂饲喂条件下,每3 d测量1次小鼠采食量发现,与WT小鼠相比,130OE、130KO小鼠的采食量均无显著差异(
图2 过表达和miR-130a-3p敲除对小鼠采食量与体质量的影响
Fig.2 Effects of overexpression and miR-130a-3p knockout on feed intake and body mass in mice
“*” “**”分别表示两组之间相比差异达P<0.05、 P<0.01显著水平(t检验)。
随后我们在代谢笼中检测3种不同基因型小鼠的O2消耗量、CO2产生量以及产热量,由
图3 miR-130a-3p敲除或过表达对小鼠有氧代谢的影响
Fig.3 Effects of miR-130a-3p knockout or overexpression on aerobic metabolism in mice
“**”表示两组之间相比差异达P<0.01显著水平(t检验)。
由
图4 miR-130a-3p过表达和敲除对小鼠肌肉力量与运动耐力的影响
Fig.4 Effects of miR-130a-3p overexpression and knockout on muscle strength and exercise endurance in mice
“*” “**” “***”分别表示两组之间相比差异达P<0.05、P<0.01、P<0.001显著水平(t检验)。
运动与有氧氧化代谢是研究肌肉相关功能的重要课题。骨骼肌作为机体运动的重要器官,其收缩的能量主要由无氧代谢途径和有氧代谢途径提供,而有氧代谢途径所产生的能量远高于无氧代谢途径,是骨骼肌最有效的ATP来源[
对于肌纤维之间转换的研究一直是近几年来对于骨骼肌相关研究的热点。研究表明,运动与否与不同的运动形式都能使肌纤维代谢类型之间发生转变,长时间的耐力运动能增强有氧氧化代谢相关酶活、增加肌纤维的线粒体含量,促进肌纤维的有氧氧化代谢[
已有报道表明miR-130a参与了机体的发育与代谢调节,但其是否参与骨骼肌的发育还未见报道。碳水化合物作为骨骼肌在长时间(耐力型)运动中氧化的主要燃料,其来源在很大程度上取决了运动强度和持续时间,随着运动强度的增加,碳水化合物的贡献更大,通过摄入碳水化合物来提高长时间运动中碳水化合物的利用率一直是运动营养研究领域的主导[
本文以 miR-130a-3p全身性敲除与过表达小鼠为对象,通过分子生物学、在体生理指标测量等技术,初步探究了miR-130a-3p对小鼠有氧呼吸及其运动能力的作用。研究显示miR-130a-3p具有提高小鼠有氧代谢、降低体质量、增强运动耐力的作用,其结果为深入研究miR-130a-3p对骨骼肌能量代谢和运动机能的调节机制提供了前期基础。
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