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Titas Petruša 6 Kursas, 21 Grupė BESIVYSTANČIŲ JUTIMINIŲ

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Titas Petruša 6 Kursas, 21 Grupė BESIVYSTANČIŲ JUTIMINIŲ LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA MEDICINOS FAKULTETAS FIZIKOS, MATEMATIKOS IR BIOFIZIKOS KATEDRA Titas Petruša 6 kursas, 21 grupė BESIVYSTANČIŲ JUTIMINIŲ NEURONŲ ELEKTRINĖS SAVYBĖS Baigiamasis magistro darbas (Vientisųjų studijų programa- medicina) Darbo vadovas: Doc. Artūras Grigaliūnas Kaunas, 2020 1 Turinys 1. SANTRAUKA ..................................................................................................................................................... 4 2. SUMMARY ......................................................................................................................................................... 6 3. PADĖKA ............................................................................................................................................................. 8 4. INTERESŲ KONFLIKTAS ................................................................................................................................ 8 5. ETIKOS KOMITETO LEIDIMAS .................................................................................................................... 8 6. SANTRUMPOS ................................................................................................................................................... 9 7. SĄVOKOS ......................................................................................................................................................... 10 8. ĮVADAS ............................................................................................................................................................. 11 9. DARBO TIKSLAS IR UŽDAVINIAI ............................................................................................................... 12 10. LITERATŪROS APŽVALGA ......................................................................................................................... 13 10.1 TRIŠAKIO NERVO MAZGO JUTIMINIŲ NEURONŲ KILMĖ .................................................................. 13 10.2 TRIŠAKIO MAZGO MORFOLOGIJA. .......................................................................................................... 13 10.3 NERVŲ AUGIMO FAKTORIAUS VAIDMUO NEURONO AUGIMO METU. ........................................... 14 10.4 TRIŠAKIO MAZGO ANATOMIJA IR INERVUOJAMOS SRITYS. ........................................................... 15 10.5 NEURONŲ PLAZMINĖS MEMBRANOS SAVYBĖS. RAMYBĖS BEI VEIKIMO POTENCIALAI. ........ 16 10.6 NEURONŲ PLAZMINĖS MEMBRANOS SAVYBĖS. PLAZMINĖS MEMBRANOS VARŽA IR ELEKTRINĖ TALPA. .................................................................................................................................................. 16 10.7 JONINIŲ KANALŲ SAMPRATA, APIBŪDINIMAS. .................................................................................... 17 10.8 JONINIŲ NATRIO KANALŲ SAVYBĖS. ...................................................................................................... 18 10.9 JONINIŲ KANALŲ INAKTYVACIJA. .......................................................................................................... 18 10.10 KALCIO KANALŲ STRUKTŪRA BEI VEIKIMAS. ..................................................................................... 19 10.11 MEDŽIAGOS, KEIČIANČIOS JONINIŲ KANALŲ VEIKIMĄ NEURONUOSE. ...................................... 20 11. TYRIMO METODIKA ..................................................................................................................................... 23 11.1 TRIŠAKIO MAZGO NEURONŲ PARUOŠIMAS .......................................................................................... 23 11.2 ELEKTROFIZIOLOGINIAI MATAVIMAI ................................................................................................... 23 11.3 ATVIRKŠTINĖS TRANSKRIPCIJOS POLIMERAZĖS GRANDININĖ REAKCIJA (RT-PCR) ............... 24 2 11.4 DUOMENŲ ANALIZĖ ..................................................................................................................................... 26 12. REZULTATAI .................................................................................................................................................. 27 12.1 NEURONŲ MORFOLOGIJA .......................................................................................................................... 27 12.2 ELEKTROFIZIOLOGINĖS NEURONŲ SAVYBĖS ...................................................................................... 28 12.3 TRYLIKTOS (E13) IR ŠEŠIOLIKTOS (E16) PAROS EMBRIONŲ TRIŠAKIO NERVO MAZGO ELEKTROFIZIOLOGINIŲ SAVYBIŲ PALYGINIMAS ........................................................................................... 31 12.4 ATVIRKŠTINĖS TRANSKRIPCIJOS POLIMERAZĖS GRANDININĖS REAKCIJŲ REZULTATAI ..... 35 13. REZULTATŲ APTARIMAS ............................................................................................................................ 36 14. IŠVADOS .......................................................................................................................................................... 38 15. LITERATŪROS SĄRAŠAS ............................................................................................................................. 40 3 1. SANTRAUKA Titas Petruša. „Besivystančių jutiminių neuronų elektrinės savybės“. Magistro darbo vadovas Doc. Artūras Grigaliūnas. Lietuvos Sveikatos Mokslų Universitetas, Medicinos akademija, Medicinos fakultetas, fizikos, matematikos ir biofizikos katedra. Kaunas, 2020 metai. Darbo tikslas - Išanalizuoti trylikos parų žiurkės embriono trišakio nervo mazgo neuronų elektrines savybes, bei palyginti gautus rezultatus su šešiolikos parų žiurkės embriono trišakio nervo mazgo neuronų elektrinėmis savybėmis. Darbo uždaviniai – 1) Aprašyti trišakio nervo mazgo neuronų elektrinių savybių kitimo dinamiką, bręstant embrionui. 2) Nustatyti trišakio nervo mazgo neuronų membranose esančių joninių kanalų savybes, panaudojant joninių kanalų blokatorius. 3) Palyginti trylikos bei šešiolikos parų embrionų trišakio nervo mazgo neuronų elektrines savybes. Analizuoti skirtumų kilmę, esmę, bei panaudojimo medicinoje galimybes. Tyrimo metodas – tyrimams buvo naudojamos trylikos parų kontroliuojamo nėštumo Sprague Dawley žiurkės, buvo išpreparuoti embrionų trišakio nervo mazgai. Mazgai buvo perpjauti pusiau ir patalpinti į auginimo terpę, kurioje buvo kultivuojami 5-8 dienas, kiekvieną dieną keičiant terpę bei atliekant matavimus. Elektrofiziologiniai matavimai buvo atliekami naudojant visos ląstelės srovės fiksavimo metodiką, duomenys buvo apdoroti naudojant pCLAMP programinę įrangą. RT-PCR buvo atlikta naudojant RNeasy protokolą, RT-PCR duomenys buvo patvirtinti naudojant DNR sekoskaitą. Tyrimo metu naudoti tiriamieji – trylikos parų kontroliuojamo nėštumo Sprague Dawley žiurkių embrionai. Tyrimo rezultatai – buvo ištirti 48 trišakio mazgo neuronai. Morfologiškai tirti 15 neuronų iš kurių 2 buvo vienpoliai, 8 – dvipoliai, 5 – pseudounipoliniai. Kultivuojant 5-8 dienas, statistiškai reikšmingai pakito šios trylikos parų embrionų trišakio mazgo neuronų elektrinės savybės: membranos talpa, hiperpoliarizacijos amplitudė, sujaudinimo slenkstis, matuojamas pikoamperais (pA). Šie dydžiai statistiškai reikšmingai padidėjo. Kultivuojant 5-8 dienas, šios trylikos parų embrionų trišakio mazgo neuronų elektrinės savybės statistiškai reikšmingai nepakito: membranos ramybės potencialas, įėjimo varža, laiko konstanta, veikimo potencialo amplitudė, laikas, per kurį pasiekiama pusė amplitudės, priekinio ir galinio veikimo potencialo frontų maksimalios reikšmės, sujaudinimo slenkstis, matuojamas 4 milivoltais (mV), hiperpoliarizacijos po veikimo potencialo trukmė. Taip pat iki 6 kultivavimo dienos hiperpoliarizacijos atsako kreivėje nebuvo jokių depoliarizuojančių įlinkimų (depolarizing sag). Tačiau nuo septintos kultivavimo paros šis įlinkimas jau buvo pastebimas daugelyje trišakio mazgo neuronų. Veikiant neuronus tetrodotoksinu, pastebėta, jog tryliktos paros neuronai generuoja TTX atsparius veikimo potencialus. Duomenis patikslinus RT-PCR metodu, E13 neuronuose NaV1.9 kanalų nebuvo rasta, jie atsirado nuo E16 paros, o NaV1.8 buvo tiek E13, tiek E16 neuronuose. Išvados – 1) Išsiaiškinta, jog tarp tryliktos ir šešioliktos embriogenezės parų keičiasi tam tikros morfologinės bei elektrofiziologinės neuronų savybės. Auga neuronų kūnai, didėja membranos elektrinė talpa. Taip pat keičiasi pats veikimo potencialas – frontai plokštėja, veikimo potencialo trukmė ilgėja. Taip pat atsiranda skirtumų po veikimo potencialo vykstančios hiperpoliarizacijos metu. 2) Kultivuojant trišakio mazgo neuronus 5-8 dienas, hiperpoliarizacijos amplitudė padidėjo, tačiau nekito ramybės potencialas, įėjimo varža, laiko konstanta. 3) Veikiant neuronus tetrodotoksinu, pastebėta, jog tryliktos paros neuronai turi mažiau TTX atsparių Na joninių kanalų 4) Palyginus skirtumus tarp E13 ir E16 trišakio mazgo neuronų elektrinių savybių, nustatyta, jog šiuo embriogenezės periodu keičiasi neuronų dydis, bei su juo susiję elektriniai parametrai. Kinta ir kitos, labiau su veikimo potencialu ir hiperpoliarizacija susijusios elektrinės savybės. E16 neuronų veikimo potencialo forma tapo šiek tiek plokštesnė, prailgėjo veikimo potencialo repoliarizacijos trukmė. Taip pat išaugo veikimo potencialo amplitudė. 5 2. SUMMARY Titas Petruša. „Electrical properties of developing sensory neurons“. Supervisor of master thesis - Doc. Artūras Grigaliūnas. Lithuanian University of Health Sciences, Academy
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