Oslikavanje živih živčanih matičnih stanica fluorescencijom

master's thesis

• Title (english): Visualization of live neural stem cells using fluourescence signal
• Author: Željka Vujin
• Mentor: Dinko Mitrečić (mentor)
• Committee member: Marija Ćurlin (predsjednik povjerenstva)
• Committee member: Ana Katušić (član povjerenstva)
• Committee member: Dinko Mitrečić (član povjerenstva)
• Granter: University of Zagreb; School of Medicine; (Department of Histology and Embryology); Zagreb
• Defense date and country: 2017-07-14, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Basic Medical Sciences; Cytology, Histology and Embryology

Abstract

Istraživanja temeljena na matičnim stanicama doživljavaju veliki zamah početkom 21. stoljeća. Mogućnost primjene pluripotentnih matičnih stanica dobivenih iz pacijenta predstavlja jednu od glavnih pokretačkih poluga personalizirane medicine, medicine prilagođene pojedinačnom pacijentu. Medicinsko područje u kojem se očekuje velika primjena matičnih stanica jesu bolesti živčanog sustava. Budući da je za analizu učinaka matičnih stanica potrebno razviti pouzdane metode praćenja transplantata, označavanje matičnih stanica je jedna od kritičnih točaka u razvoju novih terapijskih pristupa. Cilj ovog istraživanja bio je analizirati i usporediti dvije metode označavanja i oslikavanja stanica: metodu bojanja stanica pomoću egzogene boje PKH26 te fluorescentni signal dobiven genetskom preinakom stanica pomoću konstrukta Thy1-YFP. Krajnji cilj rada bio je istražiti mogu li se ove dvije metode kombinirati na istoj staničnoj kulturi. Kultura živčanih matičnih stanica dobivena je izolacijom iz moždanog tkiva mišjih zametaka soja Thy1-YFP, nakon čega su stanice označene egzogenom bojom PKH26. Stanice su praćene 14 dana. Tijekom tog perioda, boja PKH26 je dobro oslikavala cijelu staničnu populaciju, a signal YFP se pojavio samo u živčanim stanicama. Kako je diferencijacija neurona napredovala, tako je YFP signal jačao. Konstrukt Thy1-YFP je omogućio prepoznavanje svih dijelova neurona te vizualizaciju svih njihovih nastavaka. Rezultati ovog rada pokazuju kako je moguće kombinirati egzogenu boju i genetski konstrukt. Takva kombinacija staničnih označivača omogućuje prepoznavanje subpopulacije živčanih stanica u cijeloj staničnoj kulturi te doprinosi istraživanjima u području matičnih stanica.

Abstract (english)

Stem cell based research is rapidly developing since the beginning of the 21st century. The possibility of applying patient’s own pluripotent stem cells is one of the main driving forces of the personalized medicine, medicine that is tailored to the individual patient. Medical field in which we expect benefits from stem cell applications is the field of neurological diseases. Since the analysis of possible effects of stem cell application requires reliable transplant tracing method, stem cell labeling is one of the crucial points in developing new therapeutic strategies. The main objective of this study was to analyze and compare two cell labeling and visualizing methods: exogenous staining method with PKH26 dye, and fluorescent signal obtained by genetic modification using the Thy1-YFP construct. The final objective of this study was to determine whether these two methods can be combined in the same cell culture. Neural stem cells obtained from the brains of Thy1-YFP embryos were in addition stained by PKH26. Cells were monitored for 14 days. During that period it was possible to visualize the entire cell population with PKH26 dye, and the YFP signal was present only in neurons. The intensity of YFP signal was increased with the progression of neuronal differentiation. The construct Thy1-YFP enabled the recognition of all parts of neurons and visualization of their processes. The results of this study showed that it is possible to combine exogenous dies and genetic constructs. This combination of cell markers enables recognition of the neuron subpopulation in the entire cell culture, and contributes to the field of stem cell research.

• Keywords: živčane matične stanice; stanična diferencijacija; Thy1-YFP; PKH26; fluorescencija
• Keywords (english): neural stem cells; cell differentiation; Thy1-YFP; PKH26; fluorescence
• Language: croatian
• URN:NBN: urn:nbn:hr:105:015316
• Study programme: Title: Medicine; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: doktor/doktorica medicine (doktor/doktorica medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-11-07 10:49:25