發(fā)布時間：2018-06-07 09:36

  本文選題：BAMBI + Wnt/β-catenin　； 參考：《西北農(nóng)林科技大學》2015年碩士論文

【摘要】：BAMBI是骨形成蛋白和激活素的跨膜抑制蛋白,其結構與轉化生長因子TGF-β家族I型受體的結構相似,但胞內(nèi)缺少激酶結構域。BAMBI主要通過增強Wnt/β-catenin或抑制TGF-β信號通路發(fā)揮作用,如調(diào)節(jié)細胞的增殖、遷移和分化等。Wnt/β-catenin通路又稱為經(jīng)典Wnt信號通路。當經(jīng)典Wnt配體存在時,細胞質(zhì)中的β-catenin降解復合體解體,β-catenin含量增加并轉移到細胞核中,與TCF/LEF共同調(diào)控下游靶基因的表達。BAMBI能與跨膜受體Fzd5、輔助受體LRP6以及胞內(nèi)蛋白Dvl2結合,增強復合物的穩(wěn)定性,進而促進β-catenin的核轉位和經(jīng)典Wnt信號通路的轉錄活性。經(jīng)典Wnt信號通路在胚胎肌肉發(fā)育、成年骨骼肌再生以及成肌細胞系體外增殖和分化過程中都具有重要作用。鑒于BAMBI與經(jīng)典Wnt信號通路的關系,我們推測BAMBI可能調(diào)控成肌分化,但目前還沒有相關報道。在本項研究中,我們首先檢測了BAMBI在C2C12成肌分化過程中的時序;然后,利用小干擾RNA抑制BAMBI表達,檢測成肌分化以及經(jīng)典Wnt信號通路活性的變化;最后,利用Li Cl激活經(jīng)典Wnt信號通路,驗證BAMBI是否通過經(jīng)典Wnt信號通路調(diào)控成肌分化。主要結果如下:1.BAMBI在成肌分化早期高表達。與誘導前(第0天)相比,BAMBI在誘導第1天的m RNA水平上調(diào)3倍,隨后逐漸下降,直至分化結束。2.干擾BAMBI表達抑制成肌分化。與對照組相比,小干擾RNA處理組中Myo D、Myo G和My HC的表達被抑制。同時,肌管數(shù)量、分化指數(shù)和融合指數(shù)也顯著下降。3.干擾BAMBI抑制經(jīng)典Wnt信號通路活性。與對照組相比,小干擾RNA處理組中Axin2 m RNA水平和β-catenin核轉位顯著下降。4.Li Cl恢復干擾BAMBI對經(jīng)典Wnt信號通路及成肌分化的抑制作用。當Li Cl存在時,干擾BAMBI不能有效降低Axin2 m RNA水平和β-catenin核轉位,且干擾BAMBI所導致的成肌關鍵基因表達下降、肌管數(shù)量、分化指數(shù)和融合指數(shù)下降等也得到恢復。綜上所述,在C2C12細胞系中,BAMBI能正調(diào)控經(jīng)典Wnt信號通路和成肌分化,并且BAMBI通過增強經(jīng)典Wnt信號通路促進成肌分化。
[Abstract]:BAMBI is a transmembrane inhibitor of bone morphogenetic protein and activin, and its structure is similar to that of type I receptor of TGF- 尾 family. However, the intracellular lack of kinase domain. BAMBI plays an important role by enhancing Wnt/ 尾 -catenin or inhibiting TGF- 尾 signaling pathway. The Wnt- 尾 -catenin pathway, which regulates cell proliferation, migration and differentiation, is also known as the classical Wnt signaling pathway. In the presence of classical Wnt ligands, the 尾 -catenin degradation complex in the cytoplasm disintegrates and the 尾 -catenin content is increased and transferred to the nucleus, and the expression of downstream target genes is regulated by TCF/LEF. BAMBI can bind to the transmembrane receptor Fzd5, the coreceptor LRP6 and the intracellular protein Dvl2. The stability of the complex was enhanced, which promoted the nuclear translocation of 尾 -catenin and the transcriptional activity of classical Wnt signaling pathway. Classical Wnt signaling pathway plays an important role in embryonic muscle development, adult skeletal muscle regeneration and proliferation and differentiation of myoblast in vitro. In view of the relationship between BAMBI and classical Wnt signaling pathway, we speculate that BAMBI may regulate myogenic differentiation, but there are no reports yet. In this study, we first examined the timing of BAMBI during myogenesis of C2C12; then, we used small interfering RNA to inhibit the expression of BAMBI, to detect the changes of myogenic differentiation and the activity of classical Wnt signaling pathway. The classical Wnt signaling pathway was activated by LiCl to verify whether BAMBI regulated myogenic differentiation through the classical Wnt signaling pathway. The main results were as follows: 1. High expression of BAMBI in early myogenic differentiation. Compared with pre-induction (day 0), the level of m RNA increased by 3 times on the first day of induction, and then decreased gradually until the differentiation ended. 2. Interfering with the expression of BAMBI inhibits myogenic differentiation. Compared with the control group, the expression of Myo D, Myo G and my HC in the small interfering RNA group was inhibited. At the same time, the number of myotubes, differentiation index and fusion index also decreased significantly. Interfering with BAMBI inhibits the activity of classical Wnt signaling pathway. Compared with the control group, the level of Axin2 m RNA and the nuclear translocation of 尾 -catenin in the small interfering RNA group decreased significantly. 4. LiCl restored the inhibitory effect of interfering BAMBI on the classical Wnt signaling pathway and myogenic differentiation. In the presence of LiCl, interfering BAMBI could not effectively decrease the level of Axin2 m RNA and 尾 -catenin nuclear translocation, and the decrease of myogenic key gene expression induced by interference with BAMBI could also restore the number of myotubes, differentiation index and fusion index. In conclusion, in C2C12 cell line, C2C12 can regulate the classical Wnt signaling pathway and myogenic differentiation, and BAMBI promotes myogenic differentiation by enhancing classical Wnt signaling pathway.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.2

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本文編號：1990719