Úvod a cieľ práce: Exozómy, ktoré sú generované endozomálnou cestou, sa exocytózou uvoľňujú do extracelulárneho priestoru vrátane slín. Obsahujú nukleové kyseliny, proteíny a lipidy, ktoré transportujú do rôznych častí tela. Podieľajú sa na medzibunkovej komunikácii, či už podporujú alebo narúšajú rôzne fyziologické procesy. Cieľom práce bolo overiť efektívnosť ultracentrifugačnej metódy izolácie exozómov zo slín, izolovať a identifikovať proteíny v nich obsiahnuté, s dôrazom na exozomálne proteíny súvisiace s ochoreniami ústnej dutiny alebo systematickými ochoreniami s prejavmi v ústnej dutine. Metódy: Exozómy boli z plných nestimulovaných ľudských slín izolované opakovanými centrifugačnými krokmi s premývaním vo fosfátovom tlmivom roztoku s následnou metanolovo/chloroformovou precipitáciou proteínov. Proteíny boli identifikované bottom-up prístupom pomocou hmotnostnej spektrometrie s predradenou separáciou na kvapalinovom chromatografe. Výsledky: Identifikované proteíny boli klasifikované podľa proteínových tried, molekulových funkcií a biologických procesov. Najviac identifikovaných proteínov bolo z proteínových tried: cytoskeletálne proteíny, obranné/imunitné proteíny a štrukturálne proteíny, a proteínov zodpovedajúcich za katalytickú aktivitu a štrukturálnu molekulovú aktivitu. Závery: Proteíny boli klasifikované do skupín na základe ich molekulovej funkcie a biologických procesov, na ktorých sa v ľudskom organizme zúčastňujú. Boli identifikované niektoré proteíny/skupiny proteínov, ktoré môžu byť zaujímavé z hľadiska skúmania etiológie niektorých orálnych ochorení, napr. anexín A1, zymogén granulárny proteín 16 homológ B, mucín-5B, oblasti Ig lambda-3 reťazca C, oblasť Ig kapa reťazca C, oblasť Ig alfa-2 reťazca C a oblasť Ig alfa-1 reťazca C.
Introduction, aim: Exosomes generated by the endosomal pathway are released by the exocytosis into the extracellular space, including saliva. They contain nucleic acids, proteins and lipids that are transported to different parts of the body. They participate in intercellular communication, whether they support or disrupt various physiological processes. The aim of this work was to verify the efficiency of the ultracentrifugation method of isolation of exosomes from saliva, to exctract and identify proteins contained therein, with an emphasis on exosomal proteins related to diseases of the oral cavity or systemic diseases with manifestations in the oral cavity. Methods: Exosomes were isolated from full unstimulated human saliva by repeated centrifugation steps with washing in phosphate buffer followed by methanol/chloroform protein precipitation. Proteins were identified by a bottom-up approach using mass spectrometry with pre-separation by liquid chromatography. Results: The identified proteins were classified according to protein classes, molecular functions and biological processes. The most identified proteins were of the protein classes: cytoskeletal proteins, defense/immune proteins and structural proteins and proteins responsible for catalytic activity and structural molecular activity. Conclusions: Proteins have been classified into groups based on their molecular function and biological processes in which they participate in the human body. Some proteins/family of proteins have been identified that may be of interest in investigating the etiology of some oral diseases e.g. annexin A1, zymogen granular protein 16 homologue B, mucin-5B, Ig lambda-3 chain C region, Ig kappa chain C region, Ig alpha-2 chain C region and Ig alpha-1 chain C region.
- MeSH
- exozómy MeSH
- klinická studie jako téma MeSH
- lidé MeSH
- nemoci úst * diagnóza MeSH
- proteomika MeSH
- sliny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Endometrial cancer is one of the most frequent gynecological malignancies present in more than 95 % of all uterine cancers. In spite of that, screening of such disease is not commonly performed in clinical practice due to enormous costs and relatively low sensitivity. Therefore, developing an effective screening test to diagnose endometrial cancer at early stages is of great importance for the clinical area of investigation. In this work, we applied urinary proteomics (i.e., bottom-up proteomic approach followed by nano HPLC-ESI-MS/MS) in patients with endometrial cancer, with respect to find proteins aimed for the early diagnostics and screening. According to the results, the significant semi-quantitative changes were observed in urinary proteome of treated patients. The proteins that may be pivotal in pathogenesis of endometrial cancer, like cadherin-1 (CDH1), vitronectin (VTN) and basement membrane specific-heparan sulphate proteoglycan core protein (HSPG2) were down-regulated, when compared to the control group. Ultimately, it can be stated that urinary proteomics has a potential for the searching of cancer protein biomarkers based on their altered concentration.
Proteomika je stále viac používanou metódou vo viacerých odvetviach medicíny, avšak jej aplikácia v psychiatrii je stále ve3⁄4mi obmed-zená. V našej pilotnej štúdii sme identifikovali alterovanú skupinu proteínov s použitím proteomickej analýzy mozgovomiechovéhomoku a krvných doštièiek u suicidálnych jedincov v rámci metabolických dráh glykolýzy/glukoneogenézy, 14-3-3 sprostredkovanej sig-nalizácie a komplementovej a koagulaènej kaskády. Na základe našich výsledkov predpokladáme, že zmeny metabolizmu glukózy spoluso zmenami komplementovej a koagulaènej kaskády a 14-3-3 sprostredkovanej signalizácie môžu hraś úlohu v neurobiológii samovrážd v zmysle zníženej utilizácie glukózy a zhoršenej odpovede na oxidatívny stres. Skupinu proteínov identifikovaných v našej pilotnej štúdii, tzv. potencionálnych kandidátov biomarkerov suicidality, je však nevyhnutné overiś ïalšími výskumami na väèšom súbore jedincov.
Despite the fact that proteomic analysis is becoming widely used in various medical fields its use in psychiatry is still very limited. We decided to study suicidal behaviour via cerebrospinal fluid and platelets with the use of proteomics. We identified a group of alte-red proteins in suicidal patients in the metabolic pathways of glycolysis/gluconeogenesis, complement and coagulation cascade and14-3-3 mediated signaling pathway. Based on these findings we suppose that alterations of glucose metabolism (especially utilisation of glucose and altered response to oxidative stress) together with alterations in complement and coagulation cascade and 14-3-3 mediated signaling pathway may play a role in the neurobiology of suicide. However, further research is needed to clarify whether the identified group of proteins can be used as a potential peripheral biomarker for suicidal behaviour.
- MeSH
- biologické markery krev MeSH
- glukoneogeneze MeSH
- glykolýza MeSH
- hemokoagulace MeSH
- komplement MeSH
- lidé středního věku MeSH
- lidé MeSH
- pilotní projekty MeSH
- pitva MeSH
- proteiny 14-3-3 MeSH
- proteiny v mozkomíšním moku * MeSH
- proteiny izolace a purifikace klasifikace MeSH
- proteomika * MeSH
- sebevražda * MeSH
- trombocyty * chemie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
PURPOSE: Doxorubicin is an anthracycline drug which inhibits the growth of breast cancer cell lines. However, a major factor limiting its use is a cumulative, dose-dependent cardiotoxicity, resulting in a permanent loss of cardiomyocytes. Vitamin C was found to potentiate the cytotoxic effects of a variety of chemotherapeutic drugs including doxorubicin. The aim of the study was to describe the changes in protein expression and proliferation of the MCF-7 cells induced by the vitamin C applied with doxorubicin. METHODS: Label-free quantitative proteomics and real-time cell analysis methods were used to search for proteome and cell proliferation changes. These changes were induced by the pure DOX and by DOX combined with vitamin C applied on the MCF-7 cell line. RESULTS: From the real-time cell analysis experiments, it is clear that the highest anti-proliferative effect occurs with the addition of 200 µM of vitamin C to 1 µM of doxorubicin. By applying both the label-free protein quantification method and total ion current assay, we found statistically significant changes (p ≤ 0.05) of 26 proteins induced by the addition of vitamin C to doxorubicin on the MCF-7 cell line. These differentially expressed proteins are involved in processes such as structural molecule activity, transcription and translation, immune system process and antioxidant, cellular signalling and transport. CONCLUSION: The detected proteins may be capable of predicting response to DOX therapy. This is a key tool in the treatment of breast cancer, and the combination with vit C seems to be of particular interest due to the fact that it can potentiate anti-proliferative effect of DOX.
- MeSH
- antioxidancia farmakologie MeSH
- chemorezistence MeSH
- doxorubicin farmakologie MeSH
- kyselina askorbová farmakologie MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- nádory prsu farmakoterapie metabolismus MeSH
- proliferace buněk MeSH
- proteom metabolismus MeSH
- proteomika MeSH
- protinádorová antibiotika farmakologie MeSH
- screeningové testy protinádorových léčiv MeSH
- synergismus léků MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
