本文選題：DEHP + 骨骼肌��； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：背景與目的塑化劑,又稱鄰苯二甲酸酯類化合物(Phthalate Esters,PAEs),是一種環(huán)境內(nèi)分泌干擾物,對生殖、肝腎功能、胚胎發(fā)育等多方面均有潛在的危害作用。目前,人們對塑化劑的研究主要集中在其對人體代謝以及生殖等方面,關(guān)于塑化劑對骨骼肌發(fā)育的影響鮮有報道。前期我們課題組研究發(fā)現(xiàn)塑化劑DEHP母體暴露后,仔鼠體重以及骨骼肌重量較對照組均明顯減少,肌肉生長抑制素(Myostatin,MSTN)在DEHP母體暴露的仔鼠肌肉中表達顯著上調(diào),提示MSTN可能在DEHP暴露引起的肌肉發(fā)育抑制中發(fā)揮作用。本研究利用Cre-lox P技術(shù)構(gòu)建骨骼肌中MSTN基因特異性敲除小鼠模型,進一步研究MSTN在DEHP母體暴露引起子代肌肉發(fā)育抑制中的作用及相關(guān)分子機制。方法1.利用Cre-loxP技術(shù)構(gòu)建骨骼肌中MSTN基因特異性敲除的小鼠模型,用PCR法進行基因型鑒定。2.在特定時間點(0、7、14、21天)分別測量野生型與MSTN基因敲除小鼠的體重,觀察兩組小鼠體重的變化;免疫熒光法檢測兩組小鼠腿部骨骼肌纖維橫截面積的大小;RT-qPCR法檢測兩組小鼠股四頭肌中Atrogin-1、MuRF-1等肌肉降解相關(guān)基因mRNA的表達。3.分別構(gòu)建野生型、MSTN基因敲除孕鼠模型,分成4組:野生型+玉米油(WT-con組),野生型+DEHP染毒(WT-DEHP組),MSTN基因敲除+玉米油(KO-con組)以及MSTN基因敲除+DEHP染毒(KO-DEHP組),DEHP與玉米油均采用隔天灌胃的方法,從觀察到陰栓時開始灌胃,灌胃時間至子代小鼠斷奶(第21天),在特定時間(0、7、14、21天)分別測量小鼠體重,觀察兩組小鼠體重變化,稱取骨骼肌重量,RT-qPCR法檢測骨骼肌合成與降解相關(guān)基因m RNA的表達。4.為了進一步探討DEHP影響骨骼肌發(fā)育的具體機制,采用CCK-8法檢測不同濃度(0、62.5、125、250、500、1000μM)MEHP(DEHP活性代謝產(chǎn)物)處理不同時間(48、72、96 h)對骨骼肌細胞C2C12增殖的影響;RT-qPCR法檢測MEHP對蛋白質(zhì)合成與降解相關(guān)基因m RNA表達的影響;熒光素酶標記法檢測MEHP對MSTN啟動子的影響;采用siRNA干擾技術(shù)干擾C2C12細胞中的轉(zhuǎn)錄因子C/EBPδ,觀察MEHP對其相關(guān)基因mRNA表達的影響。結(jié)果1.與對照組相比,MSTN基因敲除后仔鼠體重及骨骼肌重量均顯著增加,尤以第7和21天最為明顯(P0.05);免疫熒光結(jié)果顯示MSTN基因敲除組(KO組)仔鼠的肌纖維橫截面積較對照組(WT組)增大(P0.01);RT-q PCR結(jié)果提示KO組肌肉降解相關(guān)分子Atrogin-1和MuRF-1的mRNA表達水平較WT組下降(P0.01)。2.在特定時間點(0、7、14、21天)分別檢測4組仔鼠的體重及骨骼肌重量,結(jié)果顯示W(wǎng)T-DEHP組仔鼠的體重與骨骼肌重量較WT-Con組明顯降低,而KO-DEHP組與KO-Con組相比則降低不明顯。3.RT-q PCR檢測4組仔鼠相關(guān)基因表達情況,結(jié)果顯示W(wǎng)T-DEHP組仔鼠的肌肉降解因子Atrogin-1和MuRF-1 mRNA表達水平較WT-Con組升高,肌肉合成因子MyoD和Myogenin表達下降,而KO-DEHP組仔鼠較KO-Con組的Atrogin-1和MuRF-1并沒有明顯升高,MyoD和Myogenin也沒有明顯下調(diào);Western blot檢測仔鼠肌肉,WT-DEHP組仔鼠的MyoD和p-Akt蛋白水平低于WT-Con組,而KO-DEHP組仔鼠MyoD和p-Akt蛋白水平較KO-Con組沒有降低,這些結(jié)果均說明DEHP母體暴露抑制子代骨骼肌的發(fā)育通過了MSTN分子的介導(dǎo)。4.用MEHP(DEHP代謝產(chǎn)物)處理骨骼肌細胞C2C12,CCK-8實驗結(jié)果顯示,隨著MEHP濃度的增加以及暴露時間的延長,C2C12細胞的增殖活性顯著降低,說明MEHP對骨骼肌細胞C2C12的增殖有顯著的抑制作用,且呈現(xiàn)時間和劑量依賴效應(yīng);熒光素酶標記結(jié)果顯示MEHP組(125、500μM)的熒光相對表達量均較對照組高(P0.05),說明MEHP激活了MSTN的啟動子;同時,與動物實驗結(jié)果一致,RT-qPCR結(jié)果顯示C2C12細胞經(jīng)MEHP處理后,MSTN、Atrogin-1和MuRF-1的m RNA水平表達均升高(P0.05),而MyoD和Myogenin的表達均下調(diào)(P0.05);此外,MEHP組中轉(zhuǎn)錄因子C/EBPδ的m RNA表達水平較對照組升高。5.利用siRNA技術(shù)干擾C2C12細胞中C/EBPδ的表達,再經(jīng)MEHP處理,結(jié)果發(fā)現(xiàn)干擾C/EBPδ不僅明顯抑制了MEHP對MSTN以及Atrogin-1、Mu RF-1等肌肉降解因子表達的上調(diào),同時也抑制了MEHP對Myo D、Myogenin等肌肉合成因子表達的下調(diào),說明轉(zhuǎn)錄因子C/EBPδ在MEHP上調(diào)C2C12細胞中MSTN表達中發(fā)揮作用。結(jié)論1.利用Cre-LoxP技術(shù)成功構(gòu)建了骨骼肌中MSTN基因特異性敲除的小鼠模型。MSTN基因敲除導(dǎo)致仔鼠體重以及骨骼肌重量增加,肌纖維增大,其原因可能與肌肉降解因子Atrogin-1與MuRF-1等表達下調(diào)有關(guān)。2.MSTN在DEHP母體暴露引起子代肌肉發(fā)育抑制中發(fā)揮作用。MSTN敲除后,DEHP引起的子代肌肉發(fā)育抑制作用減弱,肌肉合成相關(guān)分子Myo D和Myogenin表達增加,肌肉降解相關(guān)分子Atrogin-1和Mu RF-1表達減少,Myo D和p-Akt蛋白水平增加。3.DEHP活性代謝產(chǎn)物MEHP通過影響轉(zhuǎn)錄因子C/EBPδ激活骨骼肌C2C12中MSTN的啟動子,從而增加MSTN表達水平,引起骨骼肌細胞的萎縮。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

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