Določanje ravni mRNA bioloških označevalcev holinergičnega protivnetnega sistema v levkocitih periferne krvi pri človeku

Štrancar, Urša (author), Kovačič, Uroš (mentor)

Zdrav organizem v telesu ves čas vzdržuje ravnovesje vnetnih procesov s protivnetnimi, s čimer zmanjšuje možnosti škodljivih učinkov prekomernega sistemskega vnetja. Med pomembne dejavnike uravnavanja vnetja in imunosti sodita endokrina odziva preko hipotalamo-hipofizo-suprarenalne poti in simpatično-medularno-adrenalne poti ter nevrogeni odziv preko vagusa. 25 let raziskovanja je vodilo do podrobnejšega razumevanja delovanja in funkcije novo odkritega holinergičnega protivnetnega odziva. Njegovo aktivacijo v nucleus tractus solitarius sprožijo začetni mediatorji vnetja, čemur sledi aktivacija eferentnega vagusa in vraničnega živca, iz katerega se v belo pulpo vranice sprošča noradrenalin. Slednji preko adrenergičnih receptorjev aktivira specifične limfocite T v vranici, ki sintetizirajo in sproščajo acetilholin, ki se veže na podenote α7 nikotinskega receptorja na bližnjih monocitih. V njih se sprožijo signalne poti, ki zmanjšajo nastajanje in izločanje vnetnih citokinov. Določanje izražanja nikotinskega (CHRNA7) in adrenergičnega (ADRB2) receptorja ter encimov holin acetiltransferaze (CHAT) in acetilholinesteraze (ACHE) odpira nove možnosti za razumevanje poteka holinergičnega protivnetnega odziva in zdravljenje bolezni. V eksperimentalnem delu je preizkušen in analiziran potek dela, ki zajema izolacijo imunskih celic iz periferne človeške krvi, izolacijo RNA z dodatkom DNaze in merjenje izražanja različnih genov holinergičnega sistema s qPCR v realnem času. Izražanje CHRNA7 smo potrdili v vseh vrstah levkocitov, pri čemer je raven izražanja najvišja v izoliranih monocitih in praktično zanemarljiva v nevtrofilcih. Zaradi slednjega menimo, da granulociti, prisotni v izolatih monocitov, ne vplivajo pomembno na določanje ravni izražanja tega gena v monocitih. Uspešno smo določili izražanje ACHE v limfocitih T, zanesljivih rezultatov izražanja CHAT in ADRB2 v limfocitih T pa nismo dobili. Na manjši skupini bolnikov z zmerno hudim potekom akutnega COVID-19 smo potrdili trend znižanega izražanja CHRNA7 v monocitih in značilno povečano izražanje ACHE v limfocitih T. To nakazuje, da bi bil lahko pri teh bolnikih oslabljen vagalni protivnetni odziv. Kljub temu, da določanje izražanja CHAT in ADRB2 ni dovolj zanesljivo za nadaljnjo kvantitativno analizo, smo z vzpostavitvijo metodologije postavili temelj za nadaljnje raziskave in spremljanje izražanja bioloških markerjev v levkocitih periferne krvi, ki dajejo vpogled v stanje in delovanje protivnetnega holinergičnega sistema.

• Language: Slovenian
• Keywords: holinergični protivnetni odziv, alfa-7 nikotinski receptor, acetilholinesteraza, PBMC
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-140165
• COBISS.SI-ID: 130961923
• Publication date in RUL: 12.09.2022

Secondary language

• Language: English
• Title: Determination of mRNA levels of biological markers of the cholinergic anti-inflammatory system in human peripheral blood leukocytes

Abstract

Healthy organisms constantly maintain a balance between inflammatory and anti-inflammatory processes, thus preventing the harmful effects of excessive systemic inflammation. Important factors in the regulation of inflammation include the endocrinic hypothalamic-pituitary-suprarenal pathway and the sympathetic-medullary-adrenal pathway, as well as the neurogenic vagal. 25 years of research has led to a more detailed understanding of the action and function of the newly discovered cholinergic anti-inflammatory response. Its activation in the nucleus tractus solitarius is triggered by initial mediators of inflammation, followed by activation of the efferent vagus and the splenic nerve, from which noradrenaline is released into the white pulp of the spleen. Noradrenaline activates specific T lymphocytes in the spleen, which synthesize and release acetylcholine. It binds to α7 subunits of the nicotinic receptor on nearby monocytes and affects signal pathways in monocytes to reduce inflammation. Determining the expression of the nicotinic (CHRNA7) and adrenergic (ADRB2) receptors, as well as the choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) opens new possibilities for understanding the course of the cholinergic anti-inflammatory response. The experimental process, that has been critically tested and analysed, includes the isolation of immune cells from peripheral human blood, the isolation of RNA with DNase and the measurement of the expression of various genes of the cholinergic system by qPCR in real time. The expression of CHRNA7 was confirmed in all types of leukocytes; it is the highest in monocytes and negligible in neutrophils. We successfully determined the expression of ACHE in T lymphocytes, but we did not get reliable results of the expression of CHAT and ADRB2 in T lymphocytes. In a small group of patients with a moderately severe acute COVID-19, we confirmed decreased expression of CHRNA7 in monocytes and characteristically increased expression of ACHE in T lymphocytes. This is in accordance with the results of other studies and suggests weakened anti-inflammatory system in these patients. Even though the measurement of expression of CHAT and ADRB2 is not reliable enough for further quantitative analysis, we laid the foundation for further research and monitoring the expression of biological markers in peripheral blood leukocytes, which provide insight into the state and function of the anti-inflammatory cholinergic system.

• Keywords: cholinergic anti-inflammatory response, alpha7 nicotinic receptor, acetylcholinesterase, PBMC