Constructing endothelial-specific hypoxia-regulated vectors

Soluspesifinen geenien ilmentyminen on molekyylibiologian yksi suurista tutkimusalueista. Geenit aktivoituvat eri solutyypeissä eri tavoin, vaikka soluissa on sama geneettinen perimä. Tutkimuksissa on löydetty useita tekijöitä mm. enhanserit ja enhanseri-RNA molekyylit, jotka vaikuttavat mm. solukoh...

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Päätekijä: Laitalainen, Jarkko
Muut tekijät: Matemaattis-luonnontieteellinen tiedekunta, Faculty of Sciences, Bio- ja ympäristötieteiden laitos, Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylän yliopisto
Aineistotyyppi: Pro gradu
Kieli:eng
Julkaistu: 2013
Aiheet:
Linkit: https://jyx.jyu.fi/handle/123456789/42755
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author Laitalainen, Jarkko
author2 Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto
author_facet Laitalainen, Jarkko Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto Laitalainen, Jarkko Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto
author_sort Laitalainen, Jarkko
datasource_str_mv jyx
description Soluspesifinen geenien ilmentyminen on molekyylibiologian yksi suurista tutkimusalueista. Geenit aktivoituvat eri solutyypeissä eri tavoin, vaikka soluissa on sama geneettinen perimä. Tutkimuksissa on löydetty useita tekijöitä mm. enhanserit ja enhanseri-RNA molekyylit, jotka vaikuttavat mm. solukohtaiseen geenin ilmentymiseen. Enhanserit ovat alueita genomissa, jotka sitovat proteiineja ja vuorovaikuttavat transkriptiokompleksin kanssa lisäten geenin transkriptiota. Enhanseri-RNA:t ovat enhaserialueilta tuottettuja RNA-molekyylejä. Geenin ilmentymiseen vaikuttaa todella moniulotteinen säätelyjärjestelmä, kun mukaan lasketaan kaikki transkriptioon ja translaatioon vaikuttavat tekijät. Endoteelisolut ovat veri- ja imusuonten seinämien soluja, jotka muodostavat ohuen kerroksen suonten sisäpinnalle. Solukerroksen tehtävä on säädellä mm. nesteiden ja ravinteiden pääsyä suonesta ympäröivään kudokseen. Endoteelisolut ovat myös suuressa roolissa uusien verisuonten muodostumisessa niin normaalioloissa kuin syövissäkin. Hypoksia on eräs tekijä, joka aktivoi verisuonten muodostumista endoteelisoluista. Hypoksia on fysiologinen tila, jossa solu, kudos tai koko keho kärsii hapen puutteesta. Hypoksia johtaa vakavimmillaan hapenpuutteesta kärsivän kudoksen kuolemaan eli nekroosiin. Hypoksia voi muodostua, mikäli verenkierto häiriintyy esim. verisuonen tukkeuman tms. takia eikä veri kulkeudu normaalisti kudokseen. Työn tarkoitus oli luoda endoteelispesifisiä hypoksia-aktivoituvia vektoreita. Genomista valittiin yksi promoottori, kolme enhanseria ja kolme 3’-UTR-aluetta. Alueet kloonattiin napanuoran verisuonten endoteelisoluista (HUVEC) eristetystä genomista ja liitettiin pGL4.10 pohjaisiin lusiferaasi-vektoreihin. Tuotetut konstruktit transfektoitiin viiteen solulinjaan, joista kaksi oli endoteelisolulinjoja. Transfektion jälkeen soluja kasvatettiin 24 tuntia normaalissa happipitoisuudessa tai 1%-hypoksiassa. Käsittelyiden jälkeen konstruktien lusiferaasi-aktiivisuus mitattiin soluista ja aktiivisuuksia vertailtiin keskenään. Tulokset osoittavat soluspesifisyyden ja hypoksia-aktivoitumisen olevan monen eri tekijän summa. Geenin ilmentyminen oli suurempaa kahdella konstruktilla kahdessa solulinjassa verrattuna kontrolleihin. Konstruktien luciferaasi-geeni aktivoitui endoteelisoluissa, mutta myös muissa solulinjoissa. Hypoksia-aktivoitumisesta oli tilastollisesti merkittäviä tuloksia yhden konstruktin kohdalla, joskin rohkaisevia tuloksia saatiin myös muilla konstrukteilla. Cell specific gene expression is one of the most interesting fields in molecular biology. Genes are expressed differently in different cell types even the cells have same genetic material. Studies have found multiple factors affecting cell specific gene expression, enhancer regions and enhancer-RNA (eRNA) molecules among other things. Enhancers are DNA regions that can bind transcription factors and then interact with transcription complexes enhancing the expression. eRNAs are RNA-molecules transcribed from the enhancer regions. Cell specific gene expression is a complex system when all factors affecting transcription and translation are counted in. Endothelial cells are a thin layer of cells in blood- and lymphatic vessel walls. Main functions of endothelial cells are to regulate access of fluids and nutrients from vessels to the surrounding tissues. Endothelial cells play also a critical role in angiogenesis in normal situations and also in cancers and in cardiovascular diseases. Hypoxia is one factor that can activate angiogenesis. Hypoxia is a physiological condition where a cell, a tissue or the whole body is lacking oxygen. It can lead to cell deaths or even tissue necrosis. Hypoxia can be formed when blood circulation is disturbed for example by partial blockage of a blood vessel so that blood cannot flow freely. The aim of this study was to produce endothelial-specific hypoxia-regulated vectors. One promoter-, three enhancer- and three 3’UTR-regions were selected to be cloned from the genome of human umbilical vein endothelial cells (HUVEC). Cloned regions were inserted into pGL4.10 based vectors containing luciferase gene. The constructs were transfected into five cell lines where two of them were endothelial cells. After transfection cells were subjected to normoxia or 1%-hypoxia. After the treatments luciferase activities were measured and compared. Results indicate that cell specific gene expression and enhancing is sum of a multiple factors. Gene expressions were higher with two of the constructs compared to the controls. All constructs expressed luciferase in endothelial cells but also in other cell types. One construct showed statistically significant hypoxia activation and the other constructs gave promising results of hypoxia activation.
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Geenit aktivoituvat eri solutyypeiss\u00e4 eri tavoin, vaikka soluissa on sama geneettinen perim\u00e4. Tutkimuksissa on l\u00f6ydetty useita tekij\u00f6it\u00e4 mm. enhanserit ja enhanseri-RNA molekyylit, jotka vaikuttavat mm. solukohtaiseen geenin ilmentymiseen. Enhanserit ovat alueita genomissa, jotka sitovat proteiineja ja vuorovaikuttavat transkriptiokompleksin kanssa lis\u00e4ten geenin transkriptiota. Enhanseri-RNA:t ovat enhaserialueilta tuottettuja RNA-molekyylej\u00e4. Geenin ilmentymiseen vaikuttaa todella moniulotteinen s\u00e4\u00e4telyj\u00e4rjestelm\u00e4, kun mukaan lasketaan kaikki transkriptioon ja translaatioon vaikuttavat tekij\u00e4t.\n\nEndoteelisolut ovat veri- ja imusuonten sein\u00e4mien soluja, jotka muodostavat ohuen kerroksen suonten sis\u00e4pinnalle. Solukerroksen teht\u00e4v\u00e4 on s\u00e4\u00e4dell\u00e4 mm. nesteiden ja ravinteiden p\u00e4\u00e4sy\u00e4 suonesta ymp\u00e4r\u00f6iv\u00e4\u00e4n kudokseen. Endoteelisolut ovat my\u00f6s suuressa roolissa uusien verisuonten muodostumisessa niin normaalioloissa kuin sy\u00f6viss\u00e4kin. Hypoksia on er\u00e4s tekij\u00e4, joka aktivoi verisuonten muodostumista endoteelisoluista. Hypoksia on fysiologinen tila, jossa solu, kudos tai koko keho k\u00e4rsii hapen puutteesta. Hypoksia johtaa vakavimmillaan hapenpuutteesta k\u00e4rsiv\u00e4n kudoksen kuolemaan eli nekroosiin. Hypoksia voi muodostua, mik\u00e4li verenkierto h\u00e4iriintyy esim. verisuonen tukkeuman tms. takia eik\u00e4 veri kulkeudu normaalisti kudokseen.\n\nTy\u00f6n tarkoitus oli luoda endoteelispesifisi\u00e4 hypoksia-aktivoituvia vektoreita. Genomista valittiin yksi promoottori, kolme enhanseria ja kolme 3\u2019-UTR-aluetta. Alueet kloonattiin napanuoran verisuonten endoteelisoluista (HUVEC) eristetyst\u00e4 genomista ja liitettiin pGL4.10 pohjaisiin lusiferaasi-vektoreihin. Tuotetut konstruktit transfektoitiin viiteen solulinjaan, joista kaksi oli endoteelisolulinjoja. 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spellingShingle Laitalainen, Jarkko Constructing endothelial-specific hypoxia-regulated vectors hypoxia endothelial cells promoter enhancer UTR gene therapy Solu- ja molekyylibiologia Cell and molecular biology 4013 hypoksia endoteeli geeniterapia solut
title Constructing endothelial-specific hypoxia-regulated vectors
title_full Constructing endothelial-specific hypoxia-regulated vectors
title_fullStr Constructing endothelial-specific hypoxia-regulated vectors Constructing endothelial-specific hypoxia-regulated vectors
title_full_unstemmed Constructing endothelial-specific hypoxia-regulated vectors Constructing endothelial-specific hypoxia-regulated vectors
title_short Constructing endothelial-specific hypoxia-regulated vectors
title_sort constructing endothelial specific hypoxia regulated vectors
title_txtP Constructing endothelial-specific hypoxia-regulated vectors
topic hypoxia endothelial cells promoter enhancer UTR gene therapy Solu- ja molekyylibiologia Cell and molecular biology 4013 hypoksia endoteeli geeniterapia solut
topic_facet 4013 Cell and molecular biology Solu- ja molekyylibiologia UTR endoteeli endothelial cells enhancer geeniterapia gene therapy hypoksia hypoxia promoter solut
url https://jyx.jyu.fi/handle/123456789/42755 http://www.urn.fi/URN:NBN:fi:jyu-201401041022
work_keys_str_mv AT laitalainenjarkko constructingendothelialspecifichypoxiaregulatedvectors