| الوصف: |
V magistrski nalogi smo preverjali odziv celic in protoplastov, pridobljenih iz kalusnega tkiva koruze, na dodatek acetilholina in agonistov acetilholinskih receptorjev. Razvoj kalusa smo inducirali z nacepom mladih poganjkov na gojišče Murashige-Skoog z dodatkom sintetičnega avksina 2,4-D [3 mg/L]. Odzivanje celic in protoplastov s povišanjem [Ca2+]i ob izpostavitvi 60 µM acetilholin-jodidu, nikotinu ali pilokarpin-hidrokloridu smo spremljali prek sprememb v intenziteti fluorescence celic po stimulaciji. Za zaznavo sprememb v [Ca2+]i smo uporabili fluorescenčni indikator za Ca2+ ione, Fluo-4-AM. Ne glede na uporabljeni stimulans v izbrani koncentraciji, pri protoplastih in kalusnih celicah nismo opazili odziva, tj. spremenjene [Ca2+]i, ki bi se statistično značilno razlikoval od negativne kontrole (celice in protoplasti stimulirani s hranilno raztopino). Povišanja [Ca2+]i v času stimulacije so sicer bila prisotna, a so bila najverjetneje posledica mehanskega vzdraženja ob dodatku raztopin. Rezultati torej kažejo na odsotnost acetilholinskih receptorjev pri nediferenciranih celicah koruze oziroma, v primeru njihovega morebitnega obstoja, njihovo neodzivnost na uporabljeno koncentracijo 60 µM acetilholin-jodida, nikotina ali pilokarpin-hidroklorida. Odziv celic in protoplastov koruze na ionomicin je bil neznačilen. Kot pozitivna kontrola za so bile v eksperimentu uporabljene celice HUVEC. Ob izpostavitvi celic acetilholin-jodidu, nikotinu ali pilokarpin-hidrokloridu smo opazili odzive, tj. povišanje [Ca2+]i, ki so bili statistično značilno različni od negativne kontrole (HUVEC stimulirane z zunajcelično raztopino z 10 mM glukozo). Rezultati torej potrjujejo zaznavo 60 µM acetilholin-jodida, nikotina in pilokarpin-hidroklorida ter odzivnost celic HUVEC. Sprememba [Ca2+]i po izpostavitvi HUVEC ionomicinu je bila statistično značilna. In this master thesis we checked the response of the callus-derived maize cells and protoplasts to acetylcholine and acetylcholine receptor agonists. Callus formation was induced by inoculation of young shoots of maize on Murashige-Skoog medium with addition of synthetic auxin 2,4-D [3 mg/L]. We followed the responses of cells and protoplasts to stimulation by 60 µM acetylcholine-iodide, nicotine or pilocarpine-hydrochloride, by alterations in fluorescence intensity. Fluorescent calcium indicator, Fluo-4-AM was used to follow the alterations in [Ca2+]i. We did not observe statistically significant differences between responses of cells and protoplasts to stimulants and negative control (plant cells and protoplasts stimulated by nutrient solution). Increased [Ca2+]i was observed at the time of stimulation, but probably as a consequence of mechanical stimulation when solutions were added to cells and protoplasts. Results show absence of achetilcholine receptors on the dediferentiated maize callus cells or, if expressed, their unresponsiveness to used concentration – 60 µM of acetylcholine-iodide, nicotine or pilocarpine-hydrochloride. The response to ionomycin was uncommon. HUVEC cells were used as positive control. When cells were exposed to acetylcholine-iodide, nicotine and pilocarpine-hydrochloride, statistically significant differenences from negative control (HUVEC stimulated by extracellular solution with 10 mM glucose) were observed in responses, thus increased [Ca2+]i. Results confirm sensing of acetylcholine-iodide, nicotine and pilocarpine-hydrochloride, and responsiveness of HUVEC cells. Alteration in [Ca2+]i after addition of ionomycine to HUVEC was statistically significant. |
