University of Rijeka. Department of Biotechnology.
Abstract
U neuroznanosti i virologiji Herpes simpleks virusa tipa 1 postoji težnja ka idealnom in vitro modelnom sustavu u istraživanjima koja se bave proučavanjem latentne infekcije HSV-1 virusa. Mnoge studije su se usredotočile na diferencijaciju stanica neuroblastoma u stanice nalik neuronima. Diferencijacijom pomoću retinoične kiseline i neurotrofnog faktora BDNF mogu se dobiti stanice promijenjene morfologije i biokemije. Homogena kultura takvih diferenciranih stanica rutinski se koristi u raznim istraživanjima, kao na primjer kod istraživanja neurodegenerativnih bolesti kao što su Parkinsonova i Alzheimerova bolest.
Cilj ovog diplomskog rada jest optimizirati protokol diferencijacije stanične kulture neuroblastoma SH-SY5Y u homogenu kulturu stanica nalik neuronima, kako bi mogli proučavati uspostavu latencije i reaktivacije HSV-1 virusa. Diferencijacija je provedena tretmanom stanica s retinoičnom kiselinom i neurotrofnim faktorom, a sam proces diferencijacije je praćen kroz detekciju ekspresije dva proteina karakteristična za stanice neurona, NeuN i MAP-2. Nakon diferencijacije stanice su imale morfološke karakteristike neuralnih stanica, a također je došlo do povećanja ekspresije MAP-2 neuralnog markera.
Rezultati ukazuju da se pomoću retinoične kiseline i BDNF-a može potaknuti diferencija stanica neuroblastoma te je, uz dodatna istraživanja i optimizacije protokola, moguće dobiti homogenu kulturu stanica nalik neuronima potrebnih za in vitro istraživanje latentne infekcije Herpes simpleks virusa tipa 1.In neuroscience and the virology of Herpes simplex virus type 1 there is a great need for the ideal in vitro model system for researching HSV-1 latency infection. A lot of studies are focused on differentiating neuroblastoma cell line SH-SY5Y to neuron-like cells. With help of retionic acid and BDNF factor, cells can differentiate and change their morphology and biochemistry. Completely homogenous cell culture can be used in a variety of research, and also there have been findings that those cells are used in researching neurodegenerative diseases such as Parkinson and Alzheimer's disease.
Main goal of this Master's Thesis is to optimize the differentiation protocol to get homogenous cell culture for studying latency and reactivation of HSV-1. Differentiation was carried out by using retinoic acid and neurotrofic factor, and the process of differentiation is monitored by detecting the expression of two characteristic neuronal proteins, NeuN and MAP-2. After the treatment cells had morphological characteristics of neurons and the expression of MAP-2 neuronal marker was increased.
The results show that retinoic acid and BDNF can induce differentiation of SH-SY5Y neuroblastoma cell line and also, with additional research and protocol optimization, it is possible to get homogenous neuron-like cell culture that can be used for in vitro research of HSV-1 latent infection
