【作者】 张瑞鹏；

【导师】 李杰；

【作者基本信息】 吉林大学 ， 内科学， 2023， 硕士

【摘要】 肌少症是一种与年龄相关的骨骼肌力量、质量降低和(或)伴有相关躯体功能丧失的临床综合征,严重影响着老年人的生活质量,发病率也呈现出逐年上升的趋势,且目前的治疗选择非常有限[1-4]。近年来有研究发现肌萎缩症与铁死亡(Ferroptosis)之间存在着密切关联,而铁死亡是以铁依赖性细胞内脂质活性氧(L-ROS)积累为主要特征的细胞死亡方式[5],主要表现为细胞的精细结构中出现较小的线粒体,线粒体膜收缩,线粒体嵴减少或消失,外膜断裂,但细胞核的形态变化不明显。铁死亡与体内脂质代谢、氨基酸代谢、铁代谢和基因调控密切相关[6,7]。在之前的研究中发现,lncRNAGPRC5D-AS1能够调控骨骼肌肌肉萎缩,其下游靶基因包含铁死亡的抑制基因SLC7A11和调控该基因的ATF4、ARID1A,由此可以推断lncRNAGPRC5D-AS1通过调控SLC7A11来参与骨骼肌的铁死亡这一过程。谷胱甘肽过氧化酶4(GPX4)是一种谷胱甘肽依赖性的关键调节因子,在铁死亡调控通路中也起到关键作用,通过清除多余的自由基,水解脂质过氧化物(Lipidhydroperoxide,LOP),降低脂质过氧化物的积累,进而起到保护细胞的作用。GPX4的缺失或者抑制GPX4蛋白的表达,可以促进铁死亡发生,换言之,GPX4蛋白的表达与铁死亡的发生存在密切关联[5-8]。但目前铁死亡与骨骼肌萎缩方面的文章比较少。因此,本课题通过地塞米松诱导的小鼠C2C12细胞肌萎缩模型,探究lncRNAGPRC5D-AS1对肌萎缩及铁死亡相关蛋白的影响。首先本课题使用地塞米松诱导C2C12细胞肌萎缩模型建立。通过MTT对地塞米松最佳量效及时效进行筛选。成功诱导C2C12细胞后,细胞实验验证lncRNAGPRC5D-AS1对C2C12细胞肌萎缩的影响。通过qPCR检测lncRNAGPRC5D-AS1在各分组中的表达水平。构建lncRNAGPRC5D-AS1过表达载体,通过qPCR检测过表达效率。ELISA检测过表达lncRNAGPRC5D-AS1后各分组中GPX4酶、Fe2+、丙二醛(Malondialdehyde,MDA)含量。流式细胞术检测各分组中ROS含量。透射电镜观察各组细胞内线粒体数量以及形态结构。WesternBlot检测过表达 lncRNAGPRC5D-AS1 后,ACSL4、SLC7A11、GPX4和TFRC铁死亡相关蛋白的表达水平。免疫荧光检测分组中MyoD的表达水平,证实lncRNAGPRC5D-AS1能够通过铁死亡信号通路,调节地塞米松诱导的小鼠C2C12细胞肌萎缩进展。在本研究中,应用MTT法筛选出地塞米松诱导C2C12的浓度和时间为100ug/ml浓度下诱导48小时效果最佳。通过qPCR检测各分组中lncRNAGPRC5D-AS1的表达水平及干扰率,得知正常C2C12细胞组lncRNAGPRC5D-AS 1的表达水平显著高于地塞米松诱导C2C12细胞组(p<0.001),且过表达lncRNAGPRC5D-AS1后,肌萎缩症明有明显的减轻趋势,进一步验证了 lncRNAGPRC5D-AS1在肌肉萎缩症状中起到了 一定的作用。过表达lncRNAGPRC5D-AS 1后,ELISA检测各组中GPX4、Fe2+、丙二醛(Malondialdehyde,MDA)含量,与正常C2C12细胞组相比,地塞米松诱导C2C12细胞组中,Fe2+、MDA含量显著升高(p<0.05),同时GPX4含量显著下降(p<0.05)。而当过表达lncRNAGRPC5D-AS1后,与地塞米松诱导C2C12细胞组相比Fe2+、MDA含量显著降低(p<0.05),同时GPX4含量显著升高(p<0.05)。通过流式细胞术检测ROS含量,地塞米松诱导C2C12组较正常C2C12细胞组,ROS含量明显提高。地塞米松诱导C2C12细胞+lncRNAGPRC5D-AS1-OE组的ROS含量较地塞米松诱导C2C12细胞+lncRNAGPRC5D-AS1-NC组含量明显下降。地塞米松诱导C2C12细胞后,地塞米松诱导C2C12细胞组SLC7A11、GPX4蛋白的表达水平明显低于正常C2C12细胞组,而ACSL4、TFRC蛋白的表达水平明显高于正常C2C12细胞组。地塞米松诱导C2C12细胞+lncRNAGPRC-AS1-OE组的SLC7A11、GPX4蛋白的表达水平明显高于地塞米松诱导C2C12细胞组,而ACSL4、TFRC蛋白的表达水平明显低于地塞米松诱导C2C12细胞组。通过免疫荧光检测地塞米松诱导C2C12组较正常C2C12细胞组,MyoD表达明显下降,地塞米松诱导C2C12细胞+lncRNAGPRC5D-AS1-OE 组较地塞米松诱导 C2C12 细胞+lncRNAGPRC5D-AS 1-NC 组,MyoD 表达明显升高。本次研究表明:(1)通过地塞米松诱导小鼠C2C12细胞建立本课题所需的实验模型,采用MTT对地塞米松诱导的小鼠C2C12细胞进行存活率测试。证实可以通过地塞米松诱导小鼠C2C12细胞建立肌萎缩症模型。(2)肌萎缩细胞中lncRNAGPRC5D-AS1的表达明显下降,促进铁死亡相关蛋白的表达明显增加。(3)lncRNAGPRC5D-AS1的过表达抑制了肌萎缩的发展,抑制促进铁死亡发生的相关蛋白的表达。lncRNA GPRC5D-AS1可能通过介导SLC7A11/GPX4/ACSL4信号轴对地塞米松诱发的成肌细胞铁死亡起到保护作用,促进细胞修复再生。更多还原

【Abstract】 Sarcopenia is an age-related clinical syndrome of reduced skeletal muscle strength and mass and/or associated loss of physical function that seriously affects quality of life in older adults.The incidence of sarcopenia is also on the increase with limited treatment options[1-4].In recent years,it has been found that muscular dystrophy is closely related to Ferroptosis,a pattern of cell death primarily characterized by the build-up of lipid activated oxygen species(L-ROS)in iron-dependent cells[5],mainly represented by small mitochondria in the fine structure of cells and mitochondrial membrane contraction.mitochondrial cristae reduced or disappeared,and the external membrane was disrupted,but the morphological alteration of the core was not obvious.Iron death is closely related to lipid metabolism,amino acid metabolism,iron metabolism and gene regulation in vivo[6,7].In a prior study,lncRNAGPRC5D-AS1 can regulate skeletal muscle atrophy,and its downstream target genes include the iron death suppressor SLC7A11 and the regulation genes ATF4 and ARID 1 A.It can be inferred that lncRNAGPRC5D-AS1 is involved in iron death in skeletal muscle by regulating SLC7A11.Glutathione peroxidase 4(GPX4)is a key regulator of glutathione dependence.It also plays a key role in the iron death regulation pathway.By cleaning up excess free radicals and hydrolyzing lipidhydrooxides(LOP),it decreases the build-up of lipid peroxides,thereby protecting cells.The absence of GPX4 or the inhibition of GPX4 protein expression can promote the occurrence of iron death,in other words,the expression of GPX4 protein is closely related to the occurrence of iron death[5-8].However,there are few articles on iron death and skeletal muscle atrophy.Therefore,in this study,the effects of lncRNAGPRC5D-AS1 on muscle atrophy and ferroptosis related proteins were explored through dexamethasone-induced muscle atrophy model of mouse C2C12 cells.Firstly,the model of muscle atrophy induced by dexamethasone in C2C12 cells was established.The optimal dosification and aging of dexamethasone were screened by MTT,and C2C12 cells were successfully induced.The effect of lncRNAGPRC5D-AS1 on muscle atrophy in C2C12 cells was verified by cell experiments.The exposure level of lncRNAGPRC5D-AS1 in all groups was examined by qPCR.lncRNAGPRC5D-AS1 overexpression vector was constructed and the interference efficiency was detected by qPCR.The contents of GPX4 enzyme,Fe2+and Malondialdehyde(MDA)in lncRNAGPRC5D-AS 1 groups were determined by ELISA.ROS content in each group was determined by flow cytometry.The number and morphology of mitochondria in each group were observed by transmission electron microscopy.After lncRNAGPRC5D-AS1 expression was detected by WesternBlot,ferroptosis related proteins of ACSL4,SLC7A11,GPX4 and TFR4 were detected.Immunofluorescence detection of MyoD expression level in the group confirmed that lncRNAGPRC5D-AS 1 could regulate dexamethasone-induced muscle atrophy progression in mouse C2C12 cells through the ferroptosis signaling pathway.In this study,MTT method was used to screen out the best effect of dexamethasone induction of C2C12 at 100ug/ml concentration and time for 48 hours.The expression level and interference rate of lncRNAGPRC5D-AS1 in each group were detected by qPCR.The exposure level of lncRNAGPRC5D-AS1 was markedly higher in natural C2C12 cells than in the dexamethasone induced C2C12 cells(p<0.001),and the overexpression of lncRNAGPRC5D-AS1 showed a significant remission trend of muscular dystrophy,further confirming the role of lncRNAGPRC5D-AS1 in the symptoms of muscular dystrophy.GPX4,Fe2+,Malondialdehyde(MDA)contents were determined by ELISA after lncRNAGPRC5D-AS1 overexpression.Compared with normal C2C12 cells,dexamethasone-induced concentrations of Fe2+and Malondialdehyde(MDA)were significantly increased(p<0.05)and GPX4 content was significantly decreased(p<0.05).When lncRNAGRPC5D-AS1 was overexpressed,Fe2+and MDA contents were significantly decreased compared with dexamethasone-induced C2C12 cell group(p<0.05)and GPX4 content was significantly increased(p<0.05).Flow cytometry revealed that the ROS content of dexamethasone-induced C2C12 cells was obviously greater than that of regular C2C12 cells.The ROS amounts in the dexamethasone-induced C2C12 cells+lncRNAGPRC5D-AS1-OE group were distinctly lower than those in the dexamethasone-induced C2C12 cells+lncRNAGPRC5D-AS1-NC group.The expression levels of SLC7A11 and GPX4 in dexamethasone-induced C2C12 cells were significantly lower than those in normal C2C12 cells,and the expression levels of ACSL4 and TFRC were notably greater than in commercial C2C12 cells.The expression levels of SLC7A11 and GPX4 in dexamethasone-induced C2C12 cells+lncRNAGPRC-AS1-OE group were significantly higher than those in dexamethasone-induced C2C12 cells,and the expression levels of ACSL4 and TFRC were obviously inferior to those in the dexamethasone-induced C2C12 cells.MyoD expression was apparently diminished in dexamethasone-induced C2C12 cells in comparison with normal C2C12 cells.The expression of Myod in dexamethasone-induced C2C12 cells+lncRNAGPRC5D-AS 1-OE group was significantly decreased compared with dexamethasone-induced C2C12 cells+lncRNAGPRC5D-AS1-NC group.The expression of MyoD was significantly increased.This study shows that:(1)The experimental model required for this subject was established by dexamethasone-induced mouse C2C12 cells,and the survival rate of dexamethasone-induced mouse C2C12 cells was tested by MTT.It was confirmed that dexamethasone could induce mouse C2C12 cells to establish a muscular dystrophy model.(2)The expression of lncRNAGPRC5D-AS1 in muscular atrophy cells was significantly decreased,and the expression of iron death related proteins was significantly increased.(3)Overexpression of lncRNAGPRC5D-AS1 inhibited the development of muscular atrophy and inhibited the expression of related proteins that contribute to the occurrence of iron death.lncRNA GPRC5D-AS1 may play a protective role in dexamethasone-induced iron death of myoblasts by mediating SLC7A11/GPX4/ACSL4 signaling axis and promote cell repair and regeneration.更多还原