骨骼肌发育过程

肌肉发育组化分析汇报人：ABBPart1Thesecells（progenitorcells）

delaminatefromthehypaxialedgeofthedorsalpartofthesomite,thedermomyotome,andmigrate

intothelimbbud,wheretheyproliferate,expressmyogenicdeterminationfactorsandsubsequentlydifferentiateintoskeletalmuscleTheepaxialdermomyotome,adjacenttotheneuraltubeandnotochord,givesrisetothedeepbackmuscleswhereastherestofthemusculatureofthebodyandthelimbsderivesfromthehypaxialextremityofthedermomyotomeThefirstmusclemasstoform,underthedermomyotome,isthemyotome,whichhasanepaxialandahypaxialcomponent,subsequentlyintegratedintothetrunkmusculatureOppositethelimbbuds,muscleprogenitorcellsdelaminatefromtheepitheliumofthehypaxialdermomyotomeandmigrateintothelimbfield,tothepositionswherethedorsalandventralmusclemasseswillforminitially.ItisthemesenchymalcellsofthelimbwhicharethoughttoprovidethepositionalcuesforthemuscleprogenitorcellscomingfromthesomiteBothdelaminationandmigrationdependonthepresenceofc-met,atyrosinekinasereceptorwhichinteractswithitsligandHGF,alsocalledscatterfactor,producedbynon-somiticmesodermalcellswhichthusdelineatethemigratoryrouteTranscriptionofthec-metgenedependsonPax3CellsthatmigratefromthesomitehavenotyetactivatedthemyogenicdeterminationgenesanditisonlywhentheyreachthelimbthattheybegintoexpressMyoDandMyf5ATP酶钙钴法腺苷三磷酸酶又称ATP酶ATPADP+Pi+能量根据所用激活剂、抑制剂以及酶定位的不同，可分为：肌球蛋白腺苷三磷酸酶、膜性腺苷三磷酸酶、线粒体腺苷三磷酸酶

肌球蛋白腺苷三磷酸酶定位于骨骼肌最适PH为9.0~9.4被Ca++激活,而被Mg++抑制常用作区分二型肌纤维Ⅰ型(红肌)----收缩慢,活动持久,其酶活性低,染色淡

Ⅱ型(白肌)----收缩快,活动不持久,其酶活性高,染色深ATPase

1.固定:将新鲜骨骼肌组织用双面刀片切成约1mm厚的薄片,入固定液中固定10min,4℃.固定液配制:多聚甲醛4g;二甲砷酸钠3.08g;氯化钙0.75g;蔗糖11.5g;双蒸水加至100ml,调节pH值为7.2.

2.OCT包埋,-20℃恒冷箱切片(8μm),室温下自然干燥30min.

3.切片预处理15min.预处理液配制:0.1mol/L巴比妥钠水溶液2ml,0.18mol/L氯化钙水溶液2ml,蒸馏水6ml,用0.1mol/LNaOH调节至pH10.35.

4.入孵育液60min至75min.孵育液配制:0.1mol/L巴比妥钠2ml;0.18mol/L氯化钙1ml;蒸馏水7ml;ATP.Na230mg;调节pH为9.4.

5.1%氯化钙洗3次,共10min.

6.入2%氯化钴3min.

7.蒸馏水充分漂洗.

8.入2%硫化铵2min.

9.流水冲洗.

10.脱水、透明、封片.

结果:酶活性反应为不同色调黑褐色,Ⅰ型纤维色淡,Ⅱ型纤维色深.

ATP酶钙钴法·方法SDH孵育液37℃60分钟；水洗；脱水透明封固。孵育液配制：琥珀酸钠540mg+0.2mol/L磷酸缓冲液10ml+NBT10mg，调pH7.2至7.4.琥珀酸脱氢酶（SDH）染色·方法Confocalimagesoffluorescenttylabeledembryosandthree-dimensionalimagesoffixedtissuerevealaball-and-socketshapetotheseparationofsomitesOfromthesegmentalplate(sp).Fig.1.Time-lapseseriesofDil-labeledcellswithinthesegmentaiplate.(AtoF)Cellsnearthenodefrequentlyexchangeneighborsanddisperse.(A)Smallnumbersofcellsarelabeledatlocationsnearthecaudalendoftheembryo.(B)SubgroupsoftheDil-labeledcellsin(A)arecircledbydifferentcolorsforclarity(r,rostral;c,caudal).[(C)and(D)¡After4hours,celldispersalandtissuemovementsspreadthecellswithinthesegmentalplateshortlyafterreleasefromthenode.[(E)and(F)]After16hours,cellshavespreadextensivelywithinthesegmentalplate(sp).(GtoJ)Withintherangeofsegmentation,cellsundergominimalmovementsandmaintainanteroposteriorspacing.[(C)and(H)]SmallnumbersofcellsareDillabeledatthreedifferentlocationsalongthesegmentalplateononesideoftheneuraltube(n)inschematic(C)andrawdata(H).Thelatestformedsomiteboundaryislabeled(arrow),[(i)and(J)]Over6hours,theDil-labeledcellsdispersebutremainwithinonesomitelengthfromtheinitialinjectionsites.Somiteformationproceedsatthenormalrate(-1.5hourspersomitepair)andoccursintheostralto-caudaldirection.bodipy-ceramide-labeledembryosPart2肌纤维的分类性状I型

Ⅱa型

Ⅱb型颜色收缩特性代谢特性直径线粒体ATPase活性脂质含量糖原含量红慢、持久有氧氧化细多、大低高低红快、易疲劳有氧、酵解中等中等高中等高白快、易疲劳无氧酵解粗少、小高低高肌纤维的分类方法Ⅱa或Ⅱx型Ⅰ型Ⅱb型（1）ATPase钙钴法在ATP酶活性处,出现黑色硫化钴沉淀。主要用于显示肌球蛋白ATPase，区分红肌纤维和白肌纤维（2）琥珀酸脱氢酶（SDH）染色法Ⅱb型Ⅱa或Ⅱx型Ⅰ型肌肉系统的来源骨骼肌、平滑肌和心肌的肌肉系统均来自中胚层组织。体节形成除头部肌肉以外的所有骨骼肌枕部直至骶部的体节成对排列于胚胎中央神经管的两侧;Limb-bud四肢的发生SomiteformationMyotomeformationMyoblastfusionFig.1.LabellednuclearprofilesinE20EDL(A$)andanadultsoleus(C).Thesectionsweredoublestainedwithanti-BrdUlinkedtoEODIPY(green)andanti-myosin(A&)oranti-collagenIV(C)antibodieslinkedtoTexasRed.TheBODIPYandTexasRedfluorescencesweresequentiallyexcitedwiththe488and568nmlineofaKrypton-Argonlaserofaconfocalmicroscope.Theredandgreenimageswerethenelectronicallycombined.A:InjectionofBrdUonE15.Allthelabelled-myonuclearprofileswerelocatedwithinprimarymyotubes.Theintensityoflabellednuclearprofilescanbedividedintoverystrong(triplearrows),strong(doublearrows),andweak(singlearrow).Onlyverystronglyandstronglylabellednucleiwerecountedinthisstudy.B:InjectionofBrdUonE18.Bothprimarymyotubes(arrow)andsecondarymyotubes(arrowhead)containErdU-positivenuclei.Primaryandsecondaryrnyotubeswereidentifiedonthe