線粒體通路在應(yīng)力介導(dǎo)的成肌細(xì)胞凋亡中的作用及其調(diào)控機(jī)制.pdf

1、摘要目的：面頜部肌肉組織尤其是翼外肌在牙、頜、面部畸形的發(fā)生、發(fā)展、治療和療效維持中發(fā)揮著重要作用。功能矯形前伸下頜后，翼外肌的功能和結(jié)構(gòu)可以發(fā)生適應(yīng)性改建，這正是矯形治療得以成功并保持穩(wěn)定的關(guān)鍵，成肌細(xì)胞是適應(yīng)性改建的主要體現(xiàn)者。翼外肌的改建主要包括細(xì)胞的增殖、分化和凋亡，以往研究多局限于細(xì)胞的增殖與分化，而凋亡方面的研究少見(jiàn)。本研究擬通過(guò)構(gòu)建成肌細(xì)胞體外培養(yǎng)一力學(xué)刺激模型，探討周期性張應(yīng)力對(duì)成肌細(xì)胞凋亡的影響，明確線粒體通路在應(yīng)力介

2、導(dǎo)的成肌細(xì)胞凋亡中的作用及作用機(jī)制，從而闡明機(jī)械應(yīng)力下面頜肌細(xì)胞適應(yīng)性改建可能存在的內(nèi)在分子調(diào)控機(jī)制，將為口腔臨床醫(yī)生應(yīng)用不同方法誘導(dǎo)或調(diào)控面頜肌肉改建提供理論指導(dǎo)。方法：以大鼠L6成肌細(xì)胞為研究對(duì)象，在成功構(gòu)建成肌細(xì)胞體外培養(yǎng)一力學(xué)刺激模型的基礎(chǔ)上，通過(guò)多通道細(xì)胞牽張應(yīng)力加載系統(tǒng)，分別對(duì)待測(cè)細(xì)胞持續(xù)施)JFllh、6h、12h、24h的周期性張應(yīng)力，加載力值為15％的細(xì)胞形變，頻率為10cycles／min，每一循環(huán)包括3sstret

3、ch／3srelaxation，不加力組為對(duì)照組；采用Hoechst33258熒光染料對(duì)凋亡細(xì)胞進(jìn)行形態(tài)學(xué)觀察；應(yīng)用流式細(xì)胞術(shù)分析成肌細(xì)胞的凋亡率；運(yùn)用RT—PCR和Westernblotting檢測(cè)周期性張應(yīng)力作用下各組之f司Caspase9和AIF的mRNA含量和蛋白含量；應(yīng)用Caspase9特異性抑制劑zLEHDFMK明確Caspase9在應(yīng)力介導(dǎo)的成肌細(xì)胞凋亡中的作用，進(jìn)一步明確線粒體途徑與應(yīng)力介導(dǎo)的成肌細(xì)胞凋亡的關(guān)系。采用統(tǒng)計(jì)

4、軟件SPSS170對(duì)所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果：1大鼠L6成肌細(xì)胞在受到15％，10cycles／min的周期性張應(yīng)力刺激后，可以保持良好的生長(zhǎng)狀態(tài)，生物學(xué)特性良好，長(zhǎng)時(shí)間的張力刺激后，細(xì)胞呈現(xiàn)順應(yīng)力場(chǎng)方向排列趨勢(shì)；Abstractobjective：Maxillofacialmuscleespeciallythelateralpterygoidmuscleplaysanimportantroleingenerating，develo

5、ping，treatingandretentionofdentofacialdeformityThestrU【ctureandfunctionoflateralpterygoidmusclecallbeadaptedafterfunctionalmandibularadvancementthatisthekeyofsuccessfulorthodontictreatmentMyoblastisthethemainvehicleforad

6、aptivereconstructionThereconstructionoflateralpterygoidmusclemainlyincludescellproliferationdifferentiationandapoptosisPreviousresearchislimitedtocellproliferationanddifferentiationbutapoptosisisrareTherefore，inthisstudy

7、，successfullyconstructsmyoblastinvitromodelofmechanicalstimulation，tostudyontheeffectsofdifferentmagnitudesofcyclicstretchonapoptosislevelsinmyoblastandtoinvestigatethepossiblemechanismsinvolvedandtheroleofmitochondrialp

8、athwayinthecyclicstretchinducedapoptosisofmyoblastsToclarifythepossibleinnermolecularregulationmechanismofmaxillofacialmuscleadaptivereconstructionwillprovidetheoreticalguidancetohelporalclinicaldoctorapplydifferentmetho

9、dstoinduceorcontroltheadaptivereconstructionofmaxillofacialmuscleMethods：Inthisresearch，ratsL6myoblastsweretheresearchobjectBasedonamyoblastsculturedinvitro—mechanicalstimulationmodel，itwoudthroughmulti—channelcellspulli

10、ngtensionstressloadingsystemtostretchthecellscontiuouslyfor16，12and24hoursrespectivelyLoadingforcevaluewas15％ofthecelldeformation，frequencyistencycles／min，eachcycleincluding3Sstretch／3S—relaxation，notstrengthgroupascontr

11、olgroupUsingHoechst33258fluorescentdyeonapoptosiscellsmorphologicalobservation；MyoblastscellsapoptosisrateWasanalyzedbyflowcytometry；Caspase9●andAIFmRNAcontentandproteincontentweredetectedbyRTPCRandWesternblotting；Caspas

12、e9specificinhibitorZLEHD——FMKWasappliedtocleartheroleofCaspase9instressmediatedmyoblastapoptosis，andtocleartherelationshipofraitochondrialpathwayandapoptoticcelldeathStatisticalanalysissoftware(SPSS17O)wasusedtoassessthe

13、differencebetweeneachgroupsResults：1TheRatL6myoblastscouldmaintainagoodstateofgrowthandthebiologicalcharacteristicsWasgoodafterimposedby15％，10cycles／mincyclicaltensilestressstimulationTheanangementofcellsfromdisorganized