MSTN在DEHP母体暴露引起子代肌肉发育抑制中的作用及相关机制研究

发布时间：2018-06-05 17:37

本文选题：DEHP + 骨骼肌　；参考：《第三军医大学》2017年硕士论文

【摘要】：背景与目的塑化剂,又称邻苯二甲酸酯类化合物(Phthalate Esters,PAEs),是一种环境内分泌干扰物,对生殖、肝肾功能、胚胎发育等多方面均有潜在的危害作用。目前,人们对塑化剂的研究主要集中在其对人体代谢以及生殖等方面,关于塑化剂对骨骼肌发育的影响鲜有报道。前期我们课题组研究发现塑化剂DEHP母体暴露后,仔鼠体重以及骨骼肌重量较对照组均明显减少,肌肉生长抑制素(Myostatin,MSTN)在DEHP母体暴露的仔鼠肌肉中表达显著上调,提示MSTN可能在DEHP暴露引起的肌肉发育抑制中发挥作用。本研究利用Cre-lox P技术构建骨骼肌中MSTN基因特异性敲除小鼠模型,进一步研究MSTN在DEHP母体暴露引起子代肌肉发育抑制中的作用及相关分子机制。方法1.利用Cre-loxP技术构建骨骼肌中MSTN基因特异性敲除的小鼠模型,用PCR法进行基因型鉴定。2.在特定时间点(0、7、14、21天)分别测量野生型与MSTN基因敲除小鼠的体重,观察两组小鼠体重的变化;免疫荧光法检测两组小鼠腿部骨骼肌纤维横截面积的大小;RT-qPCR法检测两组小鼠股四头肌中Atrogin-1、MuRF-1等肌肉降解相关基因mRNA的表达。3.分别构建野生型、MSTN基因敲除孕鼠模型,分成4组:野生型+玉米油(WT-con组),野生型+DEHP染毒(WT-DEHP组),MSTN基因敲除+玉米油(KO-con组)以及MSTN基因敲除+DEHP染毒(KO-DEHP组),DEHP与玉米油均采用隔天灌胃的方法,从观察到阴栓时开始灌胃,灌胃时间至子代小鼠断奶(第21天),在特定时间(0、7、14、21天)分别测量小鼠体重,观察两组小鼠体重变化,称取骨骼肌重量,RT-qPCR法检测骨骼肌合成与降解相关基因m RNA的表达。4.为了进一步探讨DEHP影响骨骼肌发育的具体机制,采用CCK-8法检测不同浓度(0、62.5、125、250、500、1000μM)MEHP(DEHP活性代谢产物)处理不同时间(48、72、96 h)对骨骼肌细胞C2C12增殖的影响;RT-qPCR法检测MEHP对蛋白质合成与降解相关基因m RNA表达的影响;荧光素酶标记法检测MEHP对MSTN启动子的影响;采用siRNA干扰技术干扰C2C12细胞中的转录因子C/EBPδ,观察MEHP对其相关基因mRNA表达的影响。结果1.与对照组相比,MSTN基因敲除后仔鼠体重及骨骼肌重量均显著增加,尤以第7和21天最为明显(P0.05);免疫荧光结果显示MSTN基因敲除组(KO组)仔鼠的肌纤维横截面积较对照组(WT组)增大(P0.01);RT-q PCR结果提示KO组肌肉降解相关分子Atrogin-1和MuRF-1的mRNA表达水平较WT组下降(P0.01)。2.在特定时间点(0、7、14、21天)分别检测4组仔鼠的体重及骨骼肌重量,结果显示WT-DEHP组仔鼠的体重与骨骼肌重量较WT-Con组明显降低,而KO-DEHP组与KO-Con组相比则降低不明显。3.RT-q PCR检测4组仔鼠相关基因表达情况,结果显示WT-DEHP组仔鼠的肌肉降解因子Atrogin-1和MuRF-1 mRNA表达水平较WT-Con组升高,肌肉合成因子MyoD和Myogenin表达下降,而KO-DEHP组仔鼠较KO-Con组的Atrogin-1和MuRF-1并没有明显升高,MyoD和Myogenin也没有明显下调;Western blot检测仔鼠肌肉,WT-DEHP组仔鼠的MyoD和p-Akt蛋白水平低于WT-Con组,而KO-DEHP组仔鼠MyoD和p-Akt蛋白水平较KO-Con组没有降低,这些结果均说明DEHP母体暴露抑制子代骨骼肌的发育通过了MSTN分子的介导。4.用MEHP(DEHP代谢产物)处理骨骼肌细胞C2C12,CCK-8实验结果显示,随着MEHP浓度的增加以及暴露时间的延长,C2C12细胞的增殖活性显著降低,说明MEHP对骨骼肌细胞C2C12的增殖有显著的抑制作用,且呈现时间和剂量依赖效应;荧光素酶标记结果显示MEHP组(125、500μM)的荧光相对表达量均较对照组高(P0.05),说明MEHP激活了MSTN的启动子;同时,与动物实验结果一致,RT-qPCR结果显示C2C12细胞经MEHP处理后,MSTN、Atrogin-1和MuRF-1的m RNA水平表达均升高(P0.05),而MyoD和Myogenin的表达均下调(P0.05);此外,MEHP组中转录因子C/EBPδ的m RNA表达水平较对照组升高。5.利用siRNA技术干扰C2C12细胞中C/EBPδ的表达,再经MEHP处理,结果发现干扰C/EBPδ不仅明显抑制了MEHP对MSTN以及Atrogin-1、Mu RF-1等肌肉降解因子表达的上调,同时也抑制了MEHP对Myo D、Myogenin等肌肉合成因子表达的下调,说明转录因子C/EBPδ在MEHP上调C2C12细胞中MSTN表达中发挥作用。结论1.利用Cre-LoxP技术成功构建了骨骼肌中MSTN基因特异性敲除的小鼠模型。MSTN基因敲除导致仔鼠体重以及骨骼肌重量增加,肌纤维增大,其原因可能与肌肉降解因子Atrogin-1与MuRF-1等表达下调有关。2.MSTN在DEHP母体暴露引起子代肌肉发育抑制中发挥作用。MSTN敲除后,DEHP引起的子代肌肉发育抑制作用减弱,肌肉合成相关分子Myo D和Myogenin表达增加,肌肉降解相关分子Atrogin-1和Mu RF-1表达减少,Myo D和p-Akt蛋白水平增加。3.DEHP活性代谢产物MEHP通过影响转录因子C/EBPδ激活骨骼肌C2C12中MSTN的启动子,从而增加MSTN表达水平,引起骨骼肌细胞的萎缩。
[Abstract]:Background and purpose plasticizer, also known as Phthalate Esters (PAEs), is an environmental endocrine disruptor, which has potential harm to many aspects, such as reproduction, liver and kidney function, embryo development and so on. At present, the main collection of plasticizers on plasticizers in human metabolism and reproduction, and other aspects of plasticizers The effect on skeletal muscle development was rarely reported. In our previous study, we found that after the exposure of the plasticizer DEHP mother, the weight of the offspring and the weight of skeletal muscle were significantly reduced, and the expression of Myostatin (MSTN) in the muscles of the DEHP mother exposed rats was significantly up-regulated, suggesting that MSTN may be in the muscle caused by DEHP exposure. This study uses Cre-lox P technology to construct MSTN gene specific knockout mouse model in skeletal muscle, and further studies the role and molecular mechanism of MSTN in the inhibition of offspring muscle development induced by DEHP maternal exposure. Method 1. using Cre-loxP technology to construct MSTN gene specific knockout mice in skeletal muscles The PCR method was used to identify the weight of the wild type and MSTN gene knockout mice at a specific time point (0,7,14,21 days), and the body weight of the two groups of mice was observed. The size of the cross section of the skeletal muscle fibers in the legs of the two groups of mice was detected by immunofluorescence, and the RT-qPCR method was used to detect Atrogin-1, MuRF-1 in the four muscles of the two groups of mice. The expression of.3. gene mRNA was divided into 4 groups: wild type and corn oil (group WT-con), wild type +DEHP (group WT-DEHP), MSTN gene knockout + corn oil (KO-con group) and MSTN gene knockout +DEHP (KO-DEHP group), DEHP and corn oil were gavage every other day. The weight of mice was measured at a specific time (0,7,14,21 days), the weight of the mice was measured at a specific time (0,7,14,21 days), the weight of the two groups was observed, the weight of the skeletal muscle was weighed, and the expression of M RNA in the skeletal muscle synthesis and degradation gene was detected by the RT-qPCR method to further explore the effect of DEHP on the skeletal muscle hair. The effect of different concentration (0,62.51252505001000 M) MEHP (DEHP active metabolite) on the proliferation of C2C12 in skeletal muscle cells at different time (48,72,96 h) was detected by CCK-8 method, and the effect of MEHP on the m RNA table of protein synthesis and degradation related genes was detected by RT-qPCR method, and the luciferase labeling method was used to detect the initiation of C2C12. SiRNA interference technique interfered with the transcription factor C/EBP Delta in C2C12 cells to observe the effect of MEHP on its related gene mRNA expression. Results 1. compared with the control group, the weight and skeletal muscle weight of the offspring increased significantly after the MSTN knockout, especially at the seventh and twenty-first day (P0.05); the immunofluorescence results showed MSTN knockout. The cross section of muscle fibers in the group (group KO) was larger than that in the control group (group WT) (P0.01), and RT-q PCR results suggested that the mRNA expression level of Atrogin-1 and MuRF-1 in the KO group was lower than that of the WT group (P0.01).2. at a specific time point, respectively, to detect the weight of the 4 groups of offspring and the weight of the skeletal muscles respectively. Weight and skeletal muscle weight were significantly lower in group WT-Con than in group WT-Con, while in group KO-DEHP compared with group KO-Con, the expression of related genes was decreased by.3.RT-q PCR. The results showed that the mRNA expression level of muscle degradation factor Atrogin-1 and MuRF-1 in WT-DEHP group was higher than that of WT-Con group, and MyoD and Myogenin expression of muscle synthetic factors were decreased. The Atrogin-1 and MuRF-1 of the KO-DEHP group were not significantly higher than that of the KO-Con group, and the MyoD and Myogenin did not decrease obviously. The Western blot detected the muscle of the offspring, and the MyoD and p-Akt protein levels in the WT-DEHP group were lower than those in the WT-Con group. The development of the skeletal muscle of the body exposure inhibition progeny was mediated by the MSTN molecule,.4. used MEHP (DEHP metabolite) to treat the C2C12 of skeletal muscle cells. The results of CCK-8 experiment showed that the proliferation activity of C2C12 cells decreased significantly with the increase of MEHP concentration and the prolongation of exposure time. It said that MEHP had a significant inhibition on the proliferation of C2C12 in skeletal muscle cells. The results showed that the fluorescence relative expression of MEHP group (125500 mu M) was higher than that of the control group (P0.05), indicating that MEHP activates the promoter of MSTN; at the same time, it is consistent with the animal experimental results, and RT-qPCR results show that C2C12 cells are treated with MEHP, MSTN, Atrogin-1 and MuRF-1 M levels. The expression of MyoD and Myogenin decreased (P0.05), and the m RNA expression level of C/EBP Delta in MEHP group was higher than that of control group, and the expression of C/EBP Delta in C2C12 cells was disturbed by siRNA technology. The up regulation of the expression of the muscle degrading factor also inhibited the down regulation of MEHP on the expression of Myo D, Myogenin and other muscle synthetic factors, indicating that the transcription factor C/EBP delta played a role in the MEHP up regulation of MSTN expression in C2C12 cells. Conclusion 1. a mouse model.MSTN gene knockout in skeletal muscle by MSTN gene specific knockout was constructed by Cre-LoxP technique. In addition to the weight of the offspring and the increase of skeletal muscle weight and the increase of muscle fiber, the reason may be related to the downregulation of the expression of muscle degradation factor Atrogin-1 and MuRF-1 related to the function of.2.MSTN to play a role in the inhibition of the muscle development of the progeny of the DEHP mother body. The inhibition of the muscle development of the progenies caused by DEHP is weakened and the muscle synthesis is related. The expression of Myo D and Myogenin increased, and the expression of muscle degradation related molecules Atrogin-1 and Mu RF-1 decreased. Myo D and p-Akt protein levels increased.3.DEHP active metabolites. The expression level of skeletal muscle cells was increased by affecting the transcription factor C/EBP delta activated the promoter of skeletal muscle, causing the atrophy of skeletal muscle cells.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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