【作者】 赵聪颖；

【导师】 俞惠民； 江克文； 李晓明；

【作者基本信息】 浙江大学 ， 儿科学， 2014， 博士

【摘要】 目的：β-catenin胞浆蛋白是Wnt经典信号通路的一个重要下游分子,它在各系统组织中作用广泛β-catenin已经被证实在神经肌肉接头(neuromuscular junction, NMJ)形成中起着重要作用。而骨骼肌而并非突触前神经元的B-catenin在NMJ中作用尤为重要。我们之前的研究已经表明：特异性地敲除小鼠骨骼肌细胞中的B-catenin (HSA-β-cat-/-)会引起出生后小鼠的早期死亡。目前在关于骨骼肌B-catenin缺失的研究中,主要认为这与骨骼肌B-catenin影响NMJ发育相关,为了更好地理解其中蕴含的机制,我们获取HSA-β-cat-/-敲除小鼠,检测其和同窝对照小鼠骨骼肌细胞的电生理差异,并结合体外用靶向β-catenin的小片段干扰RNAs (β-catenin siRNAs)转染培养的小鼠成肌纤维细胞(C2C12)的方法进一步证实这种差异,从而来探讨骨骼肌β-catenin缺失后小鼠致死的可能原因和引起RMP去极化的可能机制,并进一步为β-catenin在NMJ发育中的作用研究提供补充证据和新的思路。方法：1.实验分组：1)HSA-B-cat-/-敲除PO小鼠组和同窝β-catloxp/loxpP0小鼠对照组,各4-6只；2)分别转染了β-catenin siRNA259s β-catenin siRNA577、β-catenin siRNA1312的三组C2C12细胞作为实验组和转染了scrambled siRNA的C2C12细胞作为对照组；2.实验方法：杂交小鼠获取敲除组和实验组小鼠,分别对各组小鼠膈肌、腓肠肌、趾长伸肌进行细胞内记录；转染C2C12细胞,对各组进行全细胞记录；Western blot法检测各组转染后C2C12细胞和各组小鼠肌肉中钠/钾ATP酶(Na,K-ATPase)a2亚单位(a2NKA)、Na,K-ATPase的a1亚单位(alNKA)、Kv3.4蛋白的表达；实时荧光定量逆转录-PCR检测两组小鼠肌肉中a2NKAmRNA的水平。结果：1.我们发现敲除组小鼠和对照组小鼠相比,β-catenin的缺失会使膈肌,腓肠肌,趾长伸肌的骨骼肌细胞静息膜电位(RMP)发生去极化；2.被转染了β-catenin siRNA的C2C12细胞与对照组细胞相比,RMP同样发生了去极化；3.无论是体内敲除还是体外敲低骨骼肌细胞的β-catenin都会使钠a2NKA的表达水平减少,而相应细胞中a1NKA和Kv3.4蛋白的表达都没有减少；4.接下来,我们进一步验证了小鼠骨骼肌β-catenin的缺失引起了转录水平上a2NKA mRNA的减少。结论：1.骨骼肌β-catenin对NMJ的突触后骨骼肌细胞RMP的维持起重要作用；2.骨骼肌细胞β-catenin的缺失引起a2NKA表达减少；3.揭示了β-catenin通过a2NKA调节RMP从而影响骨骼肌正常功能,这一可能机制。更多还原

【Abstract】 Objective:β-catenin cytoplasmic protein is a key downstream component of the canonical (β-catenin-dependent) Wnt signaling pathway, and its function is very extensive in all tissues and systems. Muscle β-catenin has been shown to play a role in the formation of the neuromuscular junction (NMJ). Our previous studies showed that muscle-specific conditional knockout of B-catenin (HSA-β-cat-/-) results in early postnatal death in mice. Recently, the previous studies considered that early postnatal death of skeletal muscle-specific B-catenin-deficient mice mainly involved in development of NMJ. To understand the underlying mechanisms, we investigated the electrophysiological properties of muscle cells from HSA-B-cat-/-and the control mice. Furthermore, to confirm the difference above, we combined the method in vitro by transfecting a primary line of mouse myoblasts (C2C12) with small-interfering RNAs targeting B-catenin. We explored a possible cause underlying the early death and a possible mechanism underlying the depolarized RMP in the absence of muscle B-catenin, and provided additional evidence supporting a role for B-catenin in the development of the NMJs.Methods: 1. Groups:1) HSA-B-caf-/-P0mice group and6-catloxp/loxp P0mice control group, n=3-6mice per group.2) C2C12cells transfected with β-catenin siRNA259, β-catenin siRNA577, B-catenin siRNA1312and scrambled siRNA (control);2. Research methods: We generated the β-catenin mutant mice and control mice, and investigated the electrophysiological properties of muscle cells from the diaphragm, gastrocnemius, and extensor digitorum longus muscles by intracellular recording; We transfected C2C12cells and detected the RMP by whole-cell clamp recording; We detected the expression levels of the a2subunit of sodium/potassium adenosine triphosphatase (a2NKA), the a1subunit of sodium/potassium adenosine triphosphatase (a1NKA), and Kv3.4by Western blotting; We investigated the a2NKA mRNA level of muscle cells from the two group mice by real-time quantitative reverse-transcription PCR.Results:1. We found that in the absence of muscle B-catenin, the resting membrane potential (RMP) depolarized in muscle cells from the diaphragm, gastrocnemius, and extensor digitorum longus muscles;2. Furthermore, C2C12cells transfected with small-interfering RNAs targeting B-catenin, the RMP depolarized as well;3. We found that the expression levels of the a2NKA were reduced by B-catenin knockdown in vitro or deletion in vivo;4. We further confirmed that a deletion of B-catenin affects the transcriptional activity of a2NKA (mRNA).Conclusion:1. Skeletal muscle B-catenin is crucial for the maintenance of the RMP of postsynaptic skeletal muscle cells of the NMJ;2. Skeletal muscle cells lacking B-catenin induces a decreased expression of a2NKA; 3. We suggested a likely mechanism by which β-catenin regulates the RMP and affects the normal function of skeletal muscle.更多还原