肌肉發(fā)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(jù)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(qū)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A(yù)TPase

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(diào)節(jié)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(yù)處理15min.預(yù)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(diào)節(jié)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(diào)節(jié)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(jié)果:酶活性反應(yīng)為不同色調(diào)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(diào)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(lǐng)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(zhì)含量糖原含量紅慢、持久有氧氧化細(xì)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(xiàn)黑色硫化鈷沉淀。主要用于顯示肌球蛋白ATPase，區(qū)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(tǒng)的來源骨骼肌、平滑肌和心肌的肌肉系統(tǒng)均來自中胚層組織。體節(jié)形成除頭部肌肉以外的所有骨骼肌枕部直至骶部的體節(jié)成對排列于胚胎中央神經(jīng)管的兩側(cè);Limb-bud四肢的發(fā)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