肌肉發育組化分析匯報人：ABBPart1Thesecells（progenitorcells）

delaminatefromthehypaxialedgeofthedorsalpartofthesomite,thedermomyotome,andmigrate

intothelimbbud,wheretheyproliferate,expressmyogenicdeterminationfactorsandsubsequentlydifferentiateintoskeletalmuscleTheepaxialdermomyotome,adjacenttotheneuraltubeandnotochord,givesrisetothedeepbackmuscleswhereastherestofthemusculatureofthebodyandthelimbsderivesfromthehypaxialextremityofthedermomyotomeThefirstmusclemasstoform,underthedermomyotome,isthemyotome,whichhasanepaxialandahypaxialcomponent,subsequentlyintegratedintothetrunkmusculatureOppositethelimbbuds,muscleprogenitorcellsdelaminatefromtheepitheliumofthehypaxialdermomyotomeandmigrateintothelimbfield,tothepositionswherethedorsalandventralmusclemasseswillforminitially.ItisthemesenchymalcellsofthelimbwhicharethoughttoprovidethepositionalcuesforthemuscleprogenitorcellscomingfromthesomiteBothdelaminationandmigrationdependonthepresenceofc-met,atyrosinekinasereceptorwhichinteractswithitsligandHGF,alsocalledscatterfactor,producedbynon-somiticmesodermalcellswhichthusdelineatethemigratoryrouteTranscriptionofthec-metgenedependsonPax3CellsthatmigratefromthesomitehavenotyetactivatedthemyogenicdeterminationgenesanditisonlywhentheyreachthelimbthattheybegintoexpressMyoDandMyf5ATP酶鈣鈷法腺苷三磷酸酶又稱ATP酶ATPADP+Pi+能量根據所用激活劑、抑制劑以及酶定位的不同，可分為：肌球蛋白腺苷三磷酸酶、膜性腺苷三磷酸酶、線粒體腺苷三磷酸酶

肌球蛋白腺苷三磷酸酶定位于骨骼肌最適PH為9.0~9.4被Ca++激活,而被Mg++抑制常用作區分二型肌纖維Ⅰ型(紅肌)----收縮慢,活動持久,其酶活性低,染色淡

Ⅱ型(白肌)----收縮快,活動不持久,其酶活性高,染色深ATPase

1.固定:將新鮮骨骼肌組織用雙面刀片切成約1mm厚的薄片,入固定液中固定10min,4℃.固定液配制:多聚甲醛4g;二甲砷酸鈉3.08g;氯化鈣0.75g;蔗糖11.5g;雙蒸水加至100ml,調節pH值為7.2.

2.OCT包埋,-20℃恒冷箱切片(8μm),室溫下自然干燥30min.

3.切片預處理15min.預處理液配制:0.1mol/L巴比妥鈉水溶液2ml,0.18mol/L氯化鈣水溶液2ml,蒸餾水6ml,用0.1mol/LNaOH調節至pH10.35.

4.入孵育液60min至75min.孵育液配制:0.1mol/L巴比妥鈉2ml;0.18mol/L氯化鈣1ml;蒸餾水7ml;ATP.Na230mg;調節pH為9.4.

5.1%氯化鈣洗3次,共10min.

6.入2%氯化鈷3min.

7.蒸餾水充分漂洗.

8.入2%硫化銨2min.

9.流水沖洗.

10.脫水、透明、封片.

結果:酶活性反應為不同色調黑褐色,Ⅰ型纖維色淡,Ⅱ型纖維色深.

ATP酶鈣鈷法·方法SDH孵育液37℃60分鐘；水洗；脫水透明封固。孵育液配制：琥珀酸鈉540mg+0.2mol/L磷酸緩沖液10ml+NBT10mg，調pH7.2至7.4.琥珀酸脫氫酶（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肌纖維的分類性狀I型

Ⅱa型

Ⅱb型顏色收縮特性代謝特性直徑線粒體ATPase活性脂質含量糖原含量紅慢、持久有氧氧化細多、大低高低紅快、易疲勞有氧、酵解中等中等高中等高白快、易疲勞無氧酵解粗少、小高低高肌纖維的分類方法Ⅱa或Ⅱx型Ⅰ型Ⅱb型（1）ATPase鈣鈷法在ATP酶活性處,出現黑色硫化鈷沉淀。主要用于顯示肌球蛋白ATPase，區分紅肌纖維和白肌纖維（2）琥珀酸脫氫酶（SDH）染色法Ⅱb型Ⅱa或Ⅱx型Ⅰ型肌肉系統的來源骨骼肌、平滑肌和心肌的肌肉系統均來自中胚層組織。體節形成除頭部肌肉以外的所有骨骼肌枕部直至骶部的體節成對排列于胚胎中央神經管的兩側;Limb-bud四肢的發生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