258 KB57 KB199420252002Jevšek, MarkoMarš, Tomažštevilka:4letnik:41str. 241-257ISSN:0025-8121COBISSID:15958745URN:URN:NBN:SI:doc-NWQ9R7DVslMedicinski razglediMedicinski razglediAcetilholinesterazaAcetylcholinesteraseAnatomy And HistologyCelične kulturecelične kutureCells, CulturedElectrophoresis, Polyacrylamide GelElektroforeza, poliakrilamid gelImmunohistochemistryImunohistokemijaInnervationMišica skeletnamolecular biologymolekularna biologijaMuscle, SkeletalNeuromuscular Junctionnevrobiologijasesalcisinapseživčno mišični stikŽivčnomišični stikIzvor acetilholinesteraze v živčno-mišičnem stiku sesalca| Origin of acetylcholinesterase in mammalian neuromuscular junction|The molecular mechanisms underlying synaptic formation are one of the central themes of modern neuroscience. The best-established role of acetylcholinesterase (AChE) is the termination of signal transmission in the neuromuscular junction (NMJ) and other cholinergic synapses by catalyzing hydrolysis of acetylcholine. Most of the AChE in the NMJ is bound to the specialized basal lamina located between the presynaptic membrane contributed by the nerve ending and the postsynaptic membrane belonging to skeletal musclefiber. Both cells can therefore be a source of synaptic AChE. Current evidence strongly supports a muscular origin for synaptic AChE; however, the possibility of neural origin has never been eliminated and is supported by another line of evidence. The objective of this work was to study the origin of synaptic AChE at the earlier stages of the formation of mammalian NMJ underin vitro conditions. At least part of the synaptic AChE found in the NMJ at the earlier stages of its formation is contributed by the motor neuron. In our study, an in vitro model was employed in which human muscle was co-cultured with the explants isolated from embryonic rat spinal cord. In these co-cultures the motor neurons originate from the rat spinal cord explants and form differentiated and long-lived NMJs with human muscle fibers.By using species-specific antibodies against human and rat AChE, respectively, we were able to distinguish between AChE of muscle (human) and neural (rat) origin. Species specificity of anti-AChE antibodies was tested byWestern blotting. Phase-contrast microscopy and fluorescent identification of acetylcholine receptors were used for visualization of the NMJs. (Abstract truncated at 2000 characters)Molekularni mehanizmi nastajanja sinaps so ena od osrednjih tem modernih nevrobioloških znanosti. Acetilholinesteraza (AChE) je encim, ki katalizira hidrolizo acetilholina in tako prekine holinergičen prenos signala v živčno-mišičnem stiku (ŽMS). Velika večina AChE je v tej sinapsi pričvrščena na bazalno lamino oz. membrano. Ta se nahaja med presinaptično membrano, ki jedel živčnega končiča, in postsinaptično membrano, ki je del mišičnega vlakna. Ker poteka sinteza AChE tako v motoričnem nevronu kot v mišičnem vlaknu, je teoretično možno, da je izvor AChE tako v enem kot v drugem tkivu. Dosedanje raziskave močno podpirajo mišični izvor, a dopuščajo tudi možnost živčnega izvora AChE v ŽMS. V tej nalogi smo preučevali izvor sinaptične AChE v zgodnejši fazi nastajanja sesalskega ŽMS v pogojih in vitro. V preučevani zgodnji fazi razvoja ŽMS prispeva motorični nevron vsaj del AChE, ki jo najdemo v sinaptični špranji. V poskusih smo uporabili in vitro sistem, v katerem smo človeške mišične cevčice gojili v kokulturi z eksplanti, izoliranimi iz podganje hrbtenjače. V teh kokulturah se med mišičnimi cevčicami in motoričnimi nevroni, ki izrastejo iz eksplantov, razvijejo visokodiferencirani ŽMS. Z uporabo vrstno specifičnih protiteles proti človeški in podganji AChE smo z imunofluorescenčno metodo ugotavljali, ali je sinaptična AChE človeškega (mišičnega) ali podganjega (živčnega) izvora. Vrstno specifičnost protiteles smo preverili z metodo Western blot. ŽMS smo identificirali s kombiniranjem faznokontrastne mikroskopije mišičnih in živčnih vlaken in flourescenčne mikroskopije skupkov acetilholinskih receptorjev. Slednje smo opazovali, prek vezave a-bungarotoksina, označenega zrodaminom. (Izvleček prekinjen pri 2000 znakih)TEXTznanstveno časopisjejournalsSlovenian National E-content AggregatorNational and University Library of SloveniaDruštvo Medicinski razgledi