Primerjava lipofekcije in elektroporacije pri vnosu plazmida pEGFP-N1 v človeške mioblaste in vitro : diplomska naloga

Kotnik, Nejc (author), Grubič, Zoran (mentor), Marš, Tomaž (comentor)

Z vpogledom v človeški genom se je odprla tudi moţnost genskega zdravljenja. Kljub nespornim prednostim, ki bi ga prineslo tako zdravljenje, pa so se, vsaj za zdaj, visoka pričakovanja glede učinkovitosti tega pristopa izkazala za pretirana. Ključni problem pri razvoju genskega zdravljenja predstavlja pot, po kateri bi na ciljan, učinkovit, in varen način dostavili in vnesli genski material v celice ţivega organizma. Danes je sicer ţe v rabi nekaj pristopov, ki omogočajo vnos tujih molekul v notranjost ţivih celic, kot tudi metod za trajno vnašanje genov, vendar pa njihova primernost za rešitev zgoraj omenjenega problema še ni dorečena in je predmet intenzivnih raziskav. V okviru diplomske naloge sem se namenil primerjati učinkovitost in citotoksičnost dveh nevirusnih transfekcijskih metod: lipofekcije in elektroporacije. Lipofekcija je metoda za vnos tujih molekul v celice s pomočjo liposomov - v našem primeru smo uporabili komercialno dostopen kationski lipid Lipofectamine 2000, ki tvori kationske liposome. Pri elektroporaciji pa s pomočjo električnih pulzov povečamo permeabilnost celične membrane, kar posledično olajša vnos tujih molekul v celice. Kot poskusni model smo uporabili primarno kulturo človeških mioblastov, saj skeletna mišičnina predstavlja obetavno tarčno tkivo za in vivo in ex vivo gensko zdravljenje. Mioblaste smo transfecirali z reporterskim plazmidom pEGFP-N1, ki nosi zapis za ojačan zeleno fluorescentni protein. Učinkovitost transfekcije je bila pri obeh metodah pribliţno enaka: največji deleţ uspešno transfeciranih celic pri lipofekciji je znašal 40,9 ± 9,7 %, pri elektroporaciji pa 41,4 ± 14,4 %. Večje razlike smo opazili na ravni citotoksičnosti obeh metod. Citotoksičnost elektroporacijskega protokola, s katerim smo dosegli najvišjo stopnjo transfekcije, je bila skoraj trikrat višja od citotoksičnosti najučinkovitejšega lipofekcijskega kompleksa (21,8 ± 7,8 % pri lipofekciji proti 63,9 ± 8,4 % pri elektroporaciji). Glede na naše rezultate lahko sklepamo, da je lipofekcija zaradi niţje citotoksičnosti primernejša metoda za transfekcijo človeških miolastov in vitro. Elektroporacija pa ima, predvsem zaradi večjih tehničnih moţnosti uporabe, prednost pred lipofekcijo v razmerah in vivo.

• Language: Slovenian
• Keywords: gensko zdravljenje lipofekcija elektroporacija mioblasti metode dela analiza podatkov
• Work type: Undergraduate thesis
• Typology: 2.11 - Undergraduate Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [N. Kotnik]
• Year: 2011
• Number of pages: VIII, 45 f.
• PID: 20.500.12556/RUL-71127
• UDC: 542:577.2
• COBISS.SI-ID: 2947953
• Publication date in RUL: 10.07.2015

Secondary language

• Language: English
• Title: Comparison of lipofection and electroporation for transfer of plasmid pEGFP-N1 into human myoblasts in vitro

Abstract

With increasing knowledge of human genome in recent years, gene therapy has become a promising strategy in the treatment and prevention of several diseases. Although the theoretical advantages of gene therapy are undisputable, it has so far not delivered the promised results. The clinical success of gene therapy is critically dependent on the development of efficient and safe delivery methods. Genetic material and foreign molecules can be introduced into eukaryotic cells by various ways. The aim of this study was to compare the efficiency and toxicity of two different methods for the transfer of plasmid DNA in vitro: lipofection and electroporation. Lipofection is a method where liposomes are used as a delivery system for DNA transfer into the cells - in this study we used a transfection reagent Lipofectamine 2000, a cationic lipid that forms cationic liposomes. Electroporation is a technique where electric pulses are applied in order to permeabilize cell membrane, which therefore enables transfer of plasmid DNA into cells. Since each of these methods has its own advantages and disadvantages, it is important to know which parameters determine the efficiency of both methods in vitro as well as to understand the mechanism involved in gene transfer in order to optimize in vivo applications of gene electrotransfer or lipofection. Plasmid pEGFP-N1, coding for enhanced green fluorescent protein, was transfected into primary human myoblasts either with lipofection or with application of high-voltage pulses. The highest rate of transfection, achieved by lipofection was 40.9 ± 9.7 % and by electroporation 41.4 ± 14.4 %. However, cytotoxicity of electroporation was significantly higher (63.9 ± 8.4 % of dead cells) compared to lipofection (21.8 ± 7.8 % of dead cells). In conclusion our results show no significant difference in efficiency of both methods, while cytotoxicity proved to be higher by electroporation. Our results showed that, due to lower cytotoxicity, lipofection is more suitable method for transfection studies on myoblasts in vitro. Electroporation has advantage over lipofection in in vivo environment due to simplicity of protocol.