www.ksmf.org/arhimed   arhimed@ksmf.org   [5/10/2025 7:53:15 AM] http://www.ksmf.org/arhimed/poglej.asp?id=83 Dolocanje tirozinazne mRNK z RT-PCR postopkom v periferni krvi bolnikov z malignim melanomom Avtor: Nebojša Glumac Mentor: prof. dr. Marko Snoj, dr. med. Somentor: dr. Marko Hocevar, dr. med., doc. dr. Srdjana Novakovic, dipl. biol IZHODIŠCA: Maligni melanom je tumor kože, cigar incidenca narašca z vrednostjo 10% na leto. Je tudi med najbolj malignimi tumorji nasploh ter visokim potencialom metastaziranja še zelo majhnih primarnih lezij. Razlikovanje med lokalizirano in metastatsko napredovalo boleznijo trenutno temelji na citološkem dokazu malignih celic v klinicno že makroskopsko sumljivih podrocnih bezgavkah. Omenjena metoda je omejena v smislu zgodnjega odkrivanja mikrometastatske ali celo potencialno metastatske bolezni. Cilj naše raziskave je razviti obcutljiv RT-PCR postopek za dolocanje tirozinazne mRNK in tako dokazati cirkulirajoce melanomske celice v polni venski krvi bolnikov z malignim melanomom. Izkorišcamo dejstvo, da je tirozinaza tkivno specificen encim, ki se izraža le v normalnih kožnih melanocitih in v celicah malignega melanoma, ki ob metastaziranju prodirajo v kri. MATERIALI IN METODE: V raziskavo smo vkljucili 18 bolnikov v III ali IV stadiju malignega melanoma po American Joint Committee on Cancer klasifikaciji in 8 zdravih prostovoljcev kot kontrolno skupino. Vsem smo odvzeli 10 ml venske krvi in iz nje s kislim gvanidin izotiocianatom, fenolom in kloroformom izolirali skupno RNK. Nato smo s pomocjo encima reverzne transkriptaze celtno RNK prepisali v cDNK. Kot primerje za to reakcijo smo uporabili nakljucne heksamere. Produkt reakcije smo s tirozinaza specificnim kompletom primerjev pomnožili v nested verižni reakciji s polimerazo. Kot kontrola nam je služilo izražanje v vseh celicah prisotnega gena za GAPDH. Koncne nested RT-PCR produkte smo dolocili z elektroforezo na 2% agaroznem gelu obarvanem s etidijevim bromidom. REZULTATI: Celoten postopek smo najprej optimatizirali in dosegli obcutljivost 2500 celic malignega melanoma v kulturi ter 10 celic malignega melanoma podtaknjenih v 1 ml venske krvi. Tirozinazno mRNK smo dolocili pri 5 bolnikih in pri nobenemu zdravemu prostovoljcu. ZAKLJUCKI: Opisana metoda je visoko obcutljiva in specificna v dolocitvi za cirkulirajoce melanomske celice specificnega gena za tirozinazo. V naši predklinicni raziskavi smo dobili zacetne pozitivne rezultate, ki opravicujejo nadaljnje delo na tem podrocju in ugotavljanju možnosti izboljšanja dolocitve stadija bolnikov ter uspešnosti adjuvantnega zdravljenja z interferonom a2b. «» [Abstract / English version] Detection of tyrosinase mRNA by nested RT-PCR in the peripheral blood of patients with malignant melanoma Author: Nebojša Glumac Mentor: prof. dr. Marko Snoj, dr. med. Co-mentor: dr. Marko Hocevar, dr. med., doc. dr. Srdjana Novakovic, dipl. biol OUTLINE: Malignant melanoma is a skin tumor with increasing incidence of 10% per year. It is also one of the most malignant tumors and even small primary lesions have a high metastatic potential. The differentiation between localized and advanced disease is currently based on cytologic verification of malignant cells in clinically suspectious regional lymph nodes. This method is limited in early detection of micrometastatic or even potentially metastatic disease. The objective of of our study is to develop a sensitive RT-PCR assay for detection of tyrosinase mRNA and hence to detect circulating melanoma cells in full venous blood of patients with malignant melanoma. We are exploiting the fact that tyrosinase is a tissue specific enzyme which is only expressed in normal skin melanocytes and malignant melanoma cells that invade blood during metastazing. MATERIALS AND METHODS: Eighteen patients with American Joint Committee on Cancer stage III and IV disease and 8 healthy voulenteers were included in our study. Ten ml of peripheral blood were taken from all subjects and total RNA was isolated using acid guanidium isothiocyanate, phenol and chlorophorm. Total RNA was transcribed into cDNA using enzyme reverse transcriptaze and random hexameres as primers. Product of previous reaction was multiplied in nested polymerase chain reaction using tyrosinase specific set of primers. Expression of ubiquitous present gene for GAPDH was used as a control for the reaction. Final products were analysed on 2% agarose gels and visualized using ethidium bromide. RESULTS: The whole procedure was first optimized. Sensitivity of 2500 malignant melanima cells in cell culture and of 10 malignant melanoma cells in 1 ml of venous blood was achieved. Tyrosinase mRNA was detected in 5 patients and none of the healthy voulenteers. CONCLUSIONS: The method described is highly sensitive and specific in detecting for circulating melanoma cells specific tyrosinase gene. Our preclinical trial has provided us with positive initial results. We propose further to explore the method, to determine the chance of improving the staging system and the success of adjuvant treatment with interferon a2b.