P2Y6受体调控成肌细胞迁移对失神经喉肌再生的作用

发布时间：2018-04-12 23:37

本文选题：骨骼肌成肌细胞 + 嘌呤能P2受体　；参考：《第二军医大学》2014年博士论文

【摘要】：喉返神经损伤导致的声带麻痹的治疗一直是喉科领域的难点和热点问题，尽管神经修复可使失神经喉肌获得再生，但喉肌功能仍难以恢复正常。其重要原因之一是喉肌肌卫星细胞（成肌细胞）功能受损。肌卫星细胞是骨骼肌成肌细胞，其向受损部位迁移是骨骼肌再生过程的关键环节。受损肌纤维释放的信号分子诱导成肌细胞迁移至受损部位，其通过直接融合生成新生肌纤维或与受损肌纤维融合而完成修复，此过程涉及复杂的化学趋向诱导、细胞骨架重组等程序化的分子细胞事件。研究表明嘌呤能受体P2家族不同成员的时空差异性表达与成肌细胞的功能调控密切相关，我们课题组已研究表明在骨骼肌损伤后成肌细胞P2Y6表达上调并呈特异性的极性分布，提示P2Y6受体可能参与前体细胞和肌管的迁移，但确切机制尚不清楚。本课题拟通过基因修饰、特异性药物干预等技术，阐明P2Y6受体信号通路调控成肌细胞迁移的作用及信号转导机制。本研究旨在揭示P2Y6受体调控成肌细胞对失神经喉肌再生的作用和确切机制，为促进失神经喉肌等骨骼肌的再生和功能恢复，提供新的治疗思路及理论依据。本研究共分为三个部分。第一部分P2Y6RNAi慢病毒成肌细胞稳定株的构建目的：采用shRNA干扰技术沉默P2Y6受体表达并构建shP2Y6成肌细胞。方法：利用shRNA干扰技术针对不同的基因位点构建一系列的shP2Y6C2C12细胞及空白病毒shCtrl C2C12细胞，并通过qPCR和WB验证转染效率，并挑选出干扰效率最好的两株细胞。结果：共构建空白病毒对照株C2C12UETP和8个慢病毒稳定株C2C12shB202-C2C12shB209，其中C2C12shB203和C2C12shB206干扰效率最好，分别为（78.1%±6.4%）和（71.8%±2.9%）。结论：成功构建P2Y6RNAi慢病毒成肌细胞稳定株，且shB203的干扰效率最好。第二部分P2Y6受体调控成肌细胞增殖、分化、迁移的作用目的：探讨P2Y6受体调控骨骼肌成肌细胞增殖、分化和迁移的确切作用。方法：基因干预组分为空质粒C2C12shCtrl组和慢病毒C2C12shP2Y6-1、shP2Y6-2组。药物刺激组分为：10μM、50μM、100μMUDP处理组、1μM、5μM、10μMMRS2578处理组和空白对照组。MTS和CCK-8法检测不同处理组细胞增殖能力的差异；qPCR方法检测基因干扰组成肌细胞分化指标Myogenin、MyoD的mRNA水平的差异；细胞划痕和Transwell迁移方法检测不同处理组成肌细胞迁移能力的差异，结果：与对照组相比，不同处理组间成肌细胞的增殖活性降低无统计学意义（p0.05）；与对照组相比，基因干扰组成肌细胞在分化不同时间点（0d、1d、2d、3d、5d、6d）的MyoD、Myogenin转录水平的变化差异无统计学意义（p0.05）。而基因干扰组和10μMMRS2578处理组的成肌细胞的迁移能力显著增加，与对照组比较，差异有统计学意义（p0.05）。结论：P2Y6受体参与调控成肌细胞的增殖和分化环节中细胞迁移运动。第三部分P2Y6受体调控成肌细胞迁移作用的机制研究目的：用WB检测基因干扰组β-catenin、N-cadherin、MMP2的蛋白表达；利用基因芯片分析P2Y6受体调控成肌细胞迁移相关的信号通路。方法：利用慢病毒C2C12shP2Y6干扰组和空质粒C2C12shCtrl组，通过基因芯片分析比较并找出两组间的基因表达水平差异超过2倍（p0.05）的基因，将这些基因提交IPA分析，，预测出迁移相关信号通路。结果：与对照组相比，基因干预组β-catenin的蛋白表达明显升高，差异具有统计学意义（p0.01）。基因芯片分析得出124个表达升高和21个表达降低的基因，其中42个差异表达最显著，30个迁移相关分子中有21个的表达变化预示迁移能力的增加，分析得出28种相关信号通路，并预测P2Y6受体调控成肌细胞迁移相关的经典HGF和NF-κB信号通路网络。结论：基因芯片分析显示P2Y6沉默后大量迁移分子的激活预示着细胞迁移的功能被激活，迁移运动与细胞骨架运动蛋白β-catenin的调控相关，IPA分析预测其调控成肌细胞迁移的信号通路可能是经典HGF和NF-κB通路，确切作用途径有待进一步证实。全文结论：小鼠骨骼肌发育和损伤再生过程中，P2Y6受体的特异性时空表达模式提示其可能通过调控成肌细胞功能参与骨骼肌发育和再生。功能学实验证实了P2Y6受体为参与调控成肌细胞的增殖和分化环节中成肌细胞的迁移能力及细胞骨架调控直接相关。基因芯片证实了P2Y6受体调控的下游靶基因主要是与细胞运动及骨架调控相关的基因，而经典HGF和NF-κB通路网络可能是P2Y6受体调控成肌细胞迁移的主要信号转导途径。
[Abstract]:Treatment of recurrent laryngeal nerve injury caused by vocal cord paralysis is always a difficult and hot problem in the field of throat, although nerve repair can make denervated laryngeal muscle regeneration, but still difficult to restore the normal laryngeal muscle function. One of the important reasons is the laryngeal muscle satellite cells (myoblasts) function of muscle satellite cells are damaged. Skeletal muscle cells, the migration is a key link to the damaged parts of the skeletal muscle regeneration process. The damaged muscle fibers induced release of signal molecules into muscle cells migrate to the site of injury, by direct fusion of angiogenesis and muscle fiber or damaged muscle fiber fusion and complete repair, this process involves complex chemotaxis induced. Molecular and cellular events cytoskeletal reorganization and other procedures. The results indicate that the expression of purinergic regulation of temporal and spatial differences of different members of P2 family receptors and muscle cell function is closely related to our research group The study showed that in the skeletal muscle myoblasts and P2Y6 expression was polar distribution specificity, suggesting that migration of P2Y6 receptors may be involved in precursor cells and myotubes, but the exact mechanism is not clear. This paper through genetic modification of specific drug intervention techniques, clarify the regulation of P2Y6 receptor signaling pathway into effect and the signal transduction mechanism of muscle cell migration. The purpose of this study is to reveal the P2Y6 receptor expression in myoblasts on denervated laryngeal muscle regeneration and the exact mechanism, in order to promote the recovery of denervated skeletal muscle and laryngeal muscle function, and provide a new treatment ideas and theoretical basis. This research is divided into three parts.
Construction of a stable strain of P2Y6RNAi lentivirus myoblast
Objective: the expression of shRNA silencing P2Y6 receptor and construct shP2Y6 myoblasts. Methods: using shRNA interference technology in different locus to construct a series of shP2Y6C2C12 cells and blank virus shCtrl C2C12 cells, and the transfection efficiency of qPCR and WB verification, and pick out the interference efficiency of two cell lines. The best results: Construction Control line C2C12UETP and 8 lentiviral stable strains C2C12shB202-C2C12shB209, C2C12shB203 and C2C12shB206 interference efficiency best, respectively (78.1% + 6.4%) and (71.8% + 2.9%). Conclusion: the successful construction of lentivirus P2Y6RNAi myoblasts stable strains, and the best jamming efficiency of shB203.
The effect of the second part of P2Y6 receptor on the proliferation, differentiation and migration of myocytes
Objective: To investigate the P2Y6 receptor expression in skeletal myoblast proliferation, differentiation and migration of the exact role. Methods: the gene intervention group were divided into C2C12shCtrl group and empty plasmid lentivirus C2C12shP2Y6-1, shP2Y6-2 group. Drug stimulation group was divided into: 10 M, 50 M, 100 MUDP group, 1 M, 5 M, 10 MMRS2578 treatment group and blank control was detected the proliferation ability of.MTS group and CCK-8 method difference; qPCR method for detection of gene interference index of differentiation of muscle cells Myogenin, the difference of MyoD mRNA level; differences in detection of cell scratch assay and Transwell migration method of different treatment composition of muscle cell migration results: Compared with the control group, among different treatment groups, the proliferation of skeletal muscle cells decreased activity was not statistically significant (P0.05); compared with the control group, the muscle cells in the differentiation of gene interference at different time points (0d, 1D, 2D, 3D, 5D, 6D, MyoD, Myogenin) There were no significant differences in the transcriptional level (P0.05). The gene interference group and 10 MMRS2578 group of myoblast migration increased significantly, compared with the control group, the difference was statistically significant (P0.05). Conclusion: the P2Y6 receptor is involved in the regulation of cell migration into the link of the proliferation and differentiation of muscle cells in motion.
The mechanism of the third part of P2Y6 receptor to regulate the migration of myoblast
Objective: to group beta -catenin, WB was used to detect N-cadherin gene interference, the expression of MMP2 protein by gene chip analysis; P2Y6 receptor regulates myoblast migration related signaling pathways. Methods: using lentiviral C2C12shP2Y6 interference group and empty plasmid group C2C12shCtrl by gene chip analysis was found between the two groups of gene expression over 2 times (P0.05) genes, these genes will be submitted to IPA analysis, predict the migration related signaling pathways. Results: compared with control group, intervention group -catenin beta gene protein expression was significantly increased, the difference was statistically significant (P0.01). The gene chip analysis of 124 and 21 increased expression of decreased expression of genes. One of the 42 most significant differential expression, increased expression of 21 indicates the migration of 30 migration related molecules, analyzed 28 kinds of signaling pathways, and predicted by P2Y6 Body control myoblast migration related to classical HGF and NF- kappa B signaling network. Conclusion: the gene chip analysis showed that P2Y6 gene silencing molecules indicates that activation of the great migration of cell migration function is activated, the regulation of migration and cytoskeleton movement protein beta -catenin, IPA signal pathway analysis prediction of myoblast migration the regulation may be the classic HGF and NF- B pathway, the exact mechanism needs to be further confirmed.
The full text conclusion:
Mouse skeletal muscle development and injury regeneration process, expression pattern suggests that it may be through the regulation of myoblast functions involved in the development and regeneration of skeletal muscle specific temporal P2Y6 receptor. Functional experiments confirmed that the P2Y6 receptor is involved in the regulation of proliferation and differentiation of muscle cells to link the directly related to migration and cytoskeletal regulation of myoblast the gene chip confirmed the downstream target gene of P2Y6 receptor regulation is mainly associated with cell motility and skeleton regulated genes HGF and NF-, and the classical B pathway network may be P2Y6 receptor signal transduction regulation into muscle cell migration pathway.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R767.4

【参考文献】