www.medenosrce.net/arhimed   arhimed(a-t>medenosrce.net   [23/04/2026 12:22:31] http://www.medenosrce.net/arhimed/poglej.asp?id=97 Izvor acetilholinesteraze v živcnomišicnem stiku sesalca Avtor: Marko Jevšek Mentor: prof.dr. Zoran Grubic Somentor: dr. Tomaž Marš Izhodišce: Molekularni mehanizmi nastajanja sinaps so ena od osrednjih tem modernih nevrobioloških znanosti. Acetilholinesteraza (AChE) je encim, ki katalizira hidrolizo acetilholina in tako prekine holinergicen prenos signala v živcno-mišicnem stiku (ŽMS). Velika vecina AChE je v tej sinapsi pricvršcena na bazalno lamino oz. membrano. Ta se nahaja med presinapticno membrano, ki je del živcnega koncica in postsinapticno membrano, ki je del mišicnega vlakna. Ker poteka sinteza AChE tako v motoricnem nevronu, kot v mišicnem vlaknu, je teoreticno možno, da je izvor AChE tako v enem, kot v drugem tkivu. Dosedanje raziskave mocno podpirajo mišicni izvor, a dopušcajo tudi možnost živcnega izvora AChE v ŽMS. Namen: V tej nalogi smo preucevali izvor sinapticne AChE v zgodnejši fazi nastajanja sesalskega ŽMS v pogojih in vitro. Hipoteza: V preucevani zgodnji fazi razvoja ŽMS prispeva motoricni nevron vsaj del AChE, ki jo najdemo v sinapticni špranji. Pristop in metode: V poskusih smo uporabili in vitro sistem, v katerem smo cloveške mišicne cevcice gojili v kokulturi z eksplanti, izoliranimi iz podganje hrbtenjace. V teh kokulturah se med mišicnimi cevcicami in motoricnimi nevroni, ki izrastejo iz eksplantov, razvijejo visoko diferencirani ŽMS. Z uporabo vrstno specificnih protiteles proti cloveški in podganji AChE, smo z imunofluorescencno metodo ugotavljali ali je sinapticna AChE cloveškega (mišicnega) ali podganjega (živcnega) izvora. Vrstno specificnost protiteles smo preverili z metodo Western blot. ŽMS smo identificirali s kombiniranjem fazno kontrastne mikroskopije mišicnih in živcnih vlaken in flourescencne mikroskopije skupkov acetilholinskih receptorjev. Slednje smo opazovali, prek vezave a-bungarotoksina, oznacenega z rodaminom. Rezultati: Po barvanju cloveške AChE smo dobili mocan imunofluorescencni signal na mestu ŽMS, kar kaže na visok delež mišicne AChE v ŽMS. Signal v podrocju ŽMS smo dobili tudi po barvanju podganje AChE. To kaže, da je del AChE v opazovani fazi razvoja ŽMS vendarle prispeval tudi živec, vendar je ta delež, sodec po intenziteti signala, manjši od prispevka mišice. Po kvantitativni oceni intenzitete signala je bil ta v primeru cloveške AChE pozitiven v 62%, v primeru podganje AChE pa le v 32% od vseh opazovanih ŽMS. Zakljucki: Omenjeni rezultati potrjujejo našo hipotezo. Vecina AChE v zgodnji fazi razvoja ŽMS je sicer mišicnega izvora, manjši delež pa prispeva tudi motoricni nevron. «» [Abstract / English version] Origin of acetylcholinesterase in mammalian neuromuscular junction Author: Marko Jevšek Mentor: prof.dr. Zoran Grubic Co-mentor: dr. Tomaž Marš Background: Molecular mechanisms underlying synaptic formation are one of the central themes of modern neuroscience. 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 nerve ending and postsynaptic membrane belonging to the skeletal muscle fiber. Both cells can therefore be a source of synaptic AChE. Present evidence strongly supports muscular origin of synaptic AChE, however the possibility of neural origin has never been eliminated and is supported by another line of evidence. AIM. The aim of this work was to study the origin of synaptic AChE at the earlier stages of the formation of mammalian NMJ under the in vitro conditions. Hypothesis. At least part of the synaptic AChE, found in the NMJ at the earlier stages of its formation is contributed by the motor neuron. Approach and methods: We employed an in vitro model in which human muscle was co-cultured with the explants isolated from the 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 by Western blotting. Phase-contrast microscopy and fluorescent identification of acetylcholine receptors were used for the visualization of the NMJs. Results: A strong signal was observed after the staining with anti-human AChE antibodies indicating relatively strong muscular AChE contribution in the NMJ. Signal was observed at the NMJ also after the staining of rat AChE suggesting neural origin of a part of synaptic AChE. Lesser intensity of this signal suggests that neural contribution was smaller in comparison to muscular one. Quantitative analysis based on the intensities of the signals revealed that 62% of the examined NMJ were human (muscle) AChE - positive and 32% of them was rat (nerve) AChE - positive. Conclusion: The above results support our hypothesis. Majority of AChE at the earlier stages of NMJ formation is of muscular origin; however a small part is also contributed by the motor neuron.