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Določanje izražanja mRNA alfa-7 nikotinskih receptorjev v krvi pri človeku
ŠTRANCAR, URŠA (Author), Kovačič, Uroš (Mentor) More about this mentor... This link opens in a new window

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Abstract
Za uspešno uravnavanje vnetnih reakcij pri poškodbah ali infekcijah je pravilno delovanje vnetnih in protivnetnih mehanizmov imunskega sistema ključnega pomena. Vnetje je pri zdravem človeku uravnavano s pravilnim razmerjem v količini vnetnih in protivnetnih mediatorjev, saj tako ne pride do prekomernega sistemskega vnetja ali sepse. Izločanje vnetnih in protivnetnih molekul nadzorujeta humoralni sistem (kortizol) preko hipotalamo-hipofizo-suprarenalne osi in živčni sistem preko živca vagusa. V zadnjih dvajsetih letih so znanstveniki iz različnih laboratorijev po svetu poročali o najrazličnejših odkritjih, ki so privedle do razumevanja mehanizma in pomena novo odkritega holinergičnega protivnetnega odziva, ki ga nadzira živec vagus. Holinergični protivnetni odziv temelji na aktivaciji živca vagusa ter prenosu signala na vranični živec, pri čemer se iz perifernih živčnih končičev slednjega v vranici sprošča noradrenalin, ta pa stimulira podvrsto limfocitov T, da v vranici začnejo sintetizirati in izločati acetilholin. Acetilholin se veže na α7 nikotinske receptorje na makrofagih in preko različnih mehanizmov preprečuje izločanje vnetnih citokinov. Določanje izražanja mRNA alfa-7 nikotinskih receptorjev v periferni krvi bi bila lahko dokaj enostavna metoda, s katero bi pri človeku lahko določali aktivnost holinergične protivnetne poti v različnih bolezenskih stanjih. V metodologiji smo predlagali protokol dela za vzpostavitev rutinskega merjenja izražanja mRNA α7 nikotinskih receptorjev, ki zajema izolacijo PBMC iz človeške krvi, izolacijo RNA in dvostopenjski RT-PCR. To bi lahko postalo uporabno metodološko orodje za klinične raziskave, s časom pa potencialno tudi kot rutinski diagnostični biomarker stanja holinergičnega protivnetnega odziva. Ugotovitve glede postopkov izolacije PBMC in RT-PCR smo primerjali z metodologijo izolacije, ki so jo opisali v raziskavah, kjer so raven izražanja mRNA nikotinskih acetilholinskih receptorjev v celicah iz periferne krvi pri bolnikih že določali. Nova in do sedaj redko uporabljena je metoda imunomagnetne negativne selekcije z EasySep, ki temelji na označevanju krvnih celic z monoklonskimi protitelesi in njihovo izolacijo s pomočjo magnetnih delcev. Ta metoda se zdi še posebej uporabna, saj hitro in v zadostni količini omogoča izolacijo specifičnih podvrst limfocitov kot tudi monocitov. S predlagano metodologijo qPCR želimo ugotavljati razlike v izražanju gena za α7 nikotinski receptor pri bolnikih z različnimi oblikami in jakostjo vnetja v primerjavi z zdravimi.

Language:Slovenian
Keywords:holinergični protivnetni odziv, alfa-7 nikotinski receptor, PBMC
Work type:Bachelor thesis/paper (mb11)
Tipology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2020
COBISS.SI-ID:29796867 Link is opened in a new window
Views:82
Downloads:32
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Secondary language

Language:English
Title:Determination of alpha-7 nicotinic receptor mRNA expression in human blood
Abstract:
Normal activity of inflammatory and anti-inflammatory mechanism of immune system is crucial for successful regulation of inflammation caused by infections and injuries. Inflammation is normally regulated by the right proportion of inflammatory and anti-inflammatory cytokines, so excessive systemic inflammation and sepsis are excluded. Secretion of inflammatory and anti-inflammatory cytokines is regulated by humoral system (cortisol) through hypothalamic-pituitary-adrenal axis and nervous system through vagus nerve. In the last 20 years it has been reported about many different discoveries by laboratories around the world, which lead to understanding of the mechanism and meaning of the newly discovered cholinergic anti-inflammatory pathway, which is supervised by vagus nerve. Cholinergic anti-inflammatory pathway is based on activation of vagus nerve which transducts the signal to splenic nerve, which secretes noradrenaline, the activator of specific subset of T lymphocytes in spleen. T lymphocytes then start secreting acetylcholine, which binds to α7 nicotinic receptors on macrophages and prevents the secretion of inflammatory cytokines through different molecular mechanisms. Determination of alpha-7 nicotinic receptor mRNA expression in human peripheral blood could present a simple method to determine the activity of human cholinergic anti-inflammatory response during different diseases. We have suggested methodological protocol to establish routine measurement of alpha-7 nicotinic receptor mRNA expression, which includes isolation of PBMC from human peripheral blood, isolation of whole RNA and two-level RT-PCR. This could become a useful methodological tool for clinical researches and in time also a routine diagnostic biomarker for determining the condition of cholinergic anti-inflammatory response. Findings about protocols for isolation of PBMC and RT-PCR were compared to the methodology of isolation of PMBC, which was used for routine measurement of alpha-7 nicotinic receptor mRNA expression in peripheral blood of different patients, described in different researches. Newly discovered and rarely used method for isolation of specific cells of PBMC is immunomagnetic negative selection with EasySep, which is based on labelling blood cells with monoclonal antibodies and suspension of magnetic particles. This method seems to be very useful, because it sufficiently and quickly enables isolating of specific subsets of lymphocytes and monocytes. With the suggested method and qPCR the differences between alpha-7 nicotinic receptor mRNA expression in patients with different forms and intensities of inflammation and healthy people would like to be found.

Keywords:cholinergic anti-inflammatory pathway, alpha-7 nicotinic receptor, PBMC

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