| Treatment of vocal cord paralysis caused by recurrent laryngeal nerve injury has beena difficult and hot issues in Laryngology.Although denerved laryngeal muscle can beregenerated by nerve reinnervation, but still laryngeal muscle hardly returned to normal.One of the important reasons is the dysfunctional throat muscle satellite cells (myoblasts).Muscle satellite cells (myoblasts) as well as skeletal myoblasts stem cells, migrating to thedamaged parts of the skeletal muscle is the key to the regeneration process. Signalingmolecules released from damaged muscle fibers impelled myoblast stem cells migrating tothe site of injury, which through generate new myogenic fibers ordirect integrate withdamaged muscle fibers, this involves complex chemical chemotacticrecruit, cytoskeletalreorganization and other procedural molecular and cellular events. Studies have shown thattime and space difference expression of different P2purinergic receptors is closely relatedto functional regulation of muscle stem cells, our group’s research shows that P2Y6wereupregulated and specifically polarity distribution, strongly suggesting that P2Y6may beinvolved in migrating of muscle precursor cells and myotubes, but the exact mechanism isunclear. Therefore, this paper proposed by genetic modification, specific drug interventiontechniques, to clarify the role and signal transduction mechanisms of P2Y6signalingpathways regulating muscle stem cells migration. This study aims to reveal the exactmechanism of P2Y6regulating myoblast stem cell to promote denervated laryngeal muscleregeneration and recovery, providing new treatment ideas and theoretical basis. The studyis divided into three parts.The first part The construction of P2Y6RNAi lentivirus myoblast stable strainObjective:Using shRNA interference technology to silencing P2Y6receptor expressionand constructing the shP2Y6myoblasts stable strain. Methods:According to the differentgene locusto building a series of shP2Y6C2C12cells and a blank virus shCtrl C2C12cellsby the shRNA interference technology, and verifying transfection efficiency by qPCR andWB, and finally picking out the two best interference efficiency cells.Results:There were ablank virus strains C2C12UETP and eight lentiviral stable strains C2C12shB202-C2C12shB209, the C2C12shB203and C2C12shB206were the best stable strains,andinterference efficiency were (78.1%±6.4%) and (71.8%±2.9%),respectively. Conclusion:We had successfully constructed P2Y6RNAi lentiviral myoblast stable strains, and C2C12 shB203was the highest interference efficiency.The second part The effect of P2Y6receptor regulating the myoblasts proliferation,differentiation and migrationObjective: To investigate the P2Y6receptor regulation of skeletal muscle stem cellproliferation, differentiation, migration. Methods:The genes interference group wasdivided into an empty plasmid C2C12shCtrl group and lentivirus C2C12shP2Y6-1,shP2Y6-2group;The drug stimulation group was divided into:10μM,50μM,100μM UDPtreated group and1μM,5μM,10μM MRS2578treated group and a control group;To detectthe cells proliferation changes in the two groups by MTS and CCK-8assay; To detect thedifferences of myoblast differentiation markers Myogenin, MyoD in mRNA levels betweengene interference groups byqPCR;To detect myoblast migrtation ability by scratch andTranswell migration method in the two groups.Results:Compared with the controlgroup,the decreased myoblasts proliferation ability had no statistically significant in thedifferent treated groups (p>0.05); Compared with the control group, MyoD, MyogeninmRNA changes had nostatistically significant at different time points (0d,1d,2d,3d,5d,6d) in gene interference in groups(p>0.05);The migration ability had increasedsignificantly in gene interference group and10μM MRS2578treated group compared withthe control group (p <0.05).Conclusion:P2Y6receptor is involved in regulating theproliferation and differentiation of myoblast in the part of the movement of cell migrationThe third part The mechanism study of P2Y6receptor in regulation of myoblastmigrationObjective:To detect the β-catenin, N-cadherin and MMP2protein changes by WB ingene interference group;To analyze the P2Y6receptor signaling pathways associated withregulation of a muscle cell migration using gene chips.Methods: A comparison of mRNAmicroarray analysis of shCtr and shP2Y6silencing C2C12cells and identify genes whichchanged more than2-fold, and p-value<0.05, and the migration related signal pathwayswere predicted by IPA analysis of the identified genes.Results:Compared with the controlgroup, β-catenin protein expression had significantly increasedin the interference group (p<0.01). A total of Related124increased and21decreased expression of genes, of whichtotal42was the most significant, the changes of21genes expression in30migrationrelated molecule indicated an increase in migration, the analysis28kinds of related signaling pathways, as well as HGF and NF-κB signaling pathways network werepredicted by mRNAmicroarray analysis. Conclusions:Cell migration-related moleculesexpression was significantly increased in P2Y6RNAi silencing C2C12by microarrayanalysis, which indicated the increased migration ability, which may was related tocytoskeletal reconstruction caused by β-catenin.The HGF and NF-κB signaling pathwaywere predicted associated to migration ability by IPA analysis, the exact pathway was to befurther confirmed.Conclusion:The specific expression patterns of P2Y6receptor suggestted that it may be involvedin regulating the development and regeneration of skeletal muscle myoblasts in mouseskeletal muscle development and injuryed regeneration process. Function experiments hadconfirmed the P2Y6receptor is involved in myoblasts migration and cytoskeletonregulation of the proliferation and differentiation process. Microarray had confirmed thatP2Y6receptor regulated downstream target genes were associated primarily with theregulation of cell motility and skeleton, and the classic HGF and NF-κB pathway maynetwork were the main signal transduction pathways associated with P2Y6receptorsregulated myoblast migration. |
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