cys蛋白酶-3对离体培养的骨骼肌蛋白降解的影响［摘要］目的：通过体外培养骨骼肌细胞，测定凋亡效应相关蛋口酶cys蛋白酶一3(caspase-3)及其抑制剂对骨骼肌细胞蛋白降解率的影响,探讨骨骼肌细胞凋亡的可能的调控因子。方法：0.5吒、1吒、2叱、4ug>8ug的肌动球蛋白复合体的反应体系中加入1单位的重组Caspase-3,通过WesternBlot电泳。体外培养骨骼肌细胞，并刺激其形成肌管，将培养的骨骼肌分为3组，分别为对照组、实验组A加入1单位Caspase-3,实验组B加入50nMCaspase-3的抑制剂Ac-DEVD-CH0,体系中均加入2mMATP(三磷酸腺昔)。用氨基酸全谱分析仪测定酪氨酸和3-甲基组氨酸的浓度。结果：0.5ug>1ug>2ug>4ug、8ug的肌动球蛋白复合体和1单位的重组Caspase-3,均可见到14kDa-actin条带,而肌动球蛋白复合体在0.5ug>lug剂量时条带消失，在2ug>4pg.8ng时条带逐渐增大。与对照组相比，给予caspase-3后骨骼肌细胞总蛋白降解率增加36.9%,肌纤维蛋白降解率增加66.4%,给TDEVD后总蛋白降解率下降17.7%,肌纤维蛋白降解率下降27.7%o结论：caspase3在体外可以降解肌动球蛋白复合体，而且还能改变骨骼肌蛋白降解率。caspase-3在骨骼肌蛋白降解中发挥了重要作用，参与了骨骼肌细胞的凋亡。［关键词］骨骼肌；蛋白代谢；细胞培养；凋亡［JQ591.2E文献标识码］A［］1008-6455(2014)07-0541-04TheinfluenceofcaspaseStotheculturedskeletalmusclecells'proteolysisWUYan-qiu,CHAT激a-ke,ZHANGHai-long,GENGYi-nan(BurnsInstitute,TheFirstAffiliatedHospitaltotheGeneralHospitalofPLA,Bei激ng100048,China)Abstract:ObjectiveWiththehelpofskeletalmusclecellculture,theinfluenceofapoptosiscaspase3anditsinhibitorontheskeletalmuscleproteolyticrateweredetected.Thepossibleregulatoryfactorsofapoptosisontheskeletalmusclewastingwasstudied・Methods1unitofcaspaseSwasaddedinto0.5ug,lug,2ug,4ugand8ugactomyosinrespectively.ThereactionproductsweredeterminedbyWesternBlot.TheskeletalmusclecelIswereculturedinvitroandwerestimulatedintoformingmyotube.Theculturedcellsweredevidedinto3groupsincludingthecontrolgroupandthetreatmentgroupAandB・1unitcaspaseSwasaddedintothetreatmentgroupA.50nMAc-DEVD-CIIO,oneofcaspase3inhibitors,wasaddedintothetreatmentgroupB.2mMATPwereaddedintoallofthethreegroups.Theconcentrationsoftyrosineand3~methy1histidineweredeterminedbyaminoacidanalyzer.ResultsldkDa-actinsmal1fragmentwereobservedinalloftheactomyosinandcaspase3reactionsystems.Whiletheactomyosinbandsweredisappearin0.5Mgandlugreactionsystemsandincreasedgraduallyin2ug,4ugand8ugreactionsystems.Comparedwiththecontrolgroup,withthehelpofcaspase3theskeletalmuscleproteolyticratewasincreasedby36.9%andthemusclefiberproteolyticratewasincreasedby66.4%.WhenDEVDwasgiven,theskeletalmuscleproteolyticratewasdecreasedby17.7%andthemusclefiberproteolyticratewasdecreasedby27.7%comparedwiththecontrolgroup.ConclusionCaspase3couldbreakdownactomyosincomplexandchangetheskeletalmuscleproteolyticrate.Caspase3playsanimportantroleinskeletalmuscleproteindegradationandtakespartintheapoptosisofskeletalcells.Keywords:skeletaimuscle;proteindegradation;cel1culture;apoptosis
