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目的评价LYRM1基因过表达对成熟骨骼肌细胞线粒体功能的影响。方法体外培养大鼠骨骼肌细胞株(L6),分别将pcDNA3.1及LYRM1-pcDNA3.1转染L6,通过G418筛选稳定表达株,应用Western blot技术验证其转染情况。诱导L6分化成熟后电镜观察线粒体形态;经Mitotracker red染色,应用流式细胞仪检测线粒体膜电位变化。结果 Western blot技术证实LYRM1表达质粒转染成功;LYRM1-pcDNA3.1组细胞线粒体凝集、形态扭曲变形及线粒体空泡化、线粒体嵴排列不规则,甚至断裂、溶解、消失;流式细胞仪检测结果显示LYRM1-pcDNA3.1组荧光值为3.7667±0.1007,pcDNA3.1组荧光值为8.7633±0.2286,二组比较差异有统计学意义(P<0.05)。结论 LYRM1基因在骨骼肌细胞中过表达能明显改变线粒体形态,降低骨骼肌细胞线粒体的膜电位,提示LYRM1基因在骨骼肌细胞中过表达影响了线粒体的功能。
Objective To evaluate the effect of LYRM1 gene overexpression on the mitochondrial function of adult skeletal muscle cells. Methods The rat skeletal muscle cell line (L6) was cultured in vitro. The recombinant plasmid pcDNA3.1 and LYRM1-pcDNA3.1 were transfected into L6 cells respectively. The stable expression strain was screened by G418. The transfection efficiency was verified by Western blot. Mitochondrial morphology was induced by L6 differentiation and electron microscopy. Mitotracker red staining was used to detect mitochondrial membrane potential changes by flow cytometry. Results Western blot showed that LYRM1 expression plasmid was successfully transfected. The mitochondria of LYRM1-pcDNA3.1 group were aggregated, deformed, and vacuolized. The mitochondrial cristae were irregularly arranged and even ruptured, dissolved and disappeared. The results of flow cytometry The fluorescence of LYRM1-pcDNA3.1 group was 3.7667 ± 0.1007, and the fluorescence value of pcDNA3.1 group was 8.7633 ± 0.2286. The difference between the two groups was statistically significant (P <0.05). Conclusion Overexpression of LYRM1 gene in skeletal muscle cells can significantly change mitochondrial morphology and mitochondrial membrane potential in skeletal muscle cells, suggesting that over-expression of LYRM1 gene in skeletal muscle cells may affect mitochondrial function.