Ispitivanje citotoksičnosti kompleksa zlata i platine na ćelijskim linijama adenokarcinoma pluća in vitro i in vivo

Abstract

Lečenje tumora cisplatinom je povezano sa brojnim neželjenim efektima i relativno brzim razvojem rezistencije. Hemioterapija NSCLC - (non small cell type) tumora pluća podrazumeva primenu derivata platine samostalno ili u kombinaciji sa drugim grupama hemioterapeutika kao što su antimetaboliti folata. Ovo istraživanje ima za cilj da ispita citotoksičnost novih kompleksa zlata i platine na epitelnim ćelijama adenokarcinoma pluća in vitro (ćelijske linije A549 i LLC1). Na mišjem modelu eksperimentalnih metastaza tumora pluća ispitivano je koji kompleksi izazivaju smrt tumorskih ćelija in vivo i koji bi kompleks mogao dalje da se ispita kao potencijalni terapeutik. Ciljevi: U skladu sa osnovnim ciljem postavljeni su sledeći specifični ciljevi: 1. MTT i LDH testom ispitati potencijalnu citotoksičnost kompleksa. 2. Ispitati relativni odnos nekrotične i apoptotske smrti ćelija izazvane ipitivanim kompleksima. 3. Ispitati da li kompleksi utiču na smanjenje već uspostavljenih metastaza karcinoma pluća u mišjem modelu. Metode: In vitro metodama ovog istraživanja je ispitivana citotkosičnost derivata zlata i platine u laboatorijskim uslovima pomoću MTT (3-(4,5-dimetiltiazol-2-il)-2,5- difeniltetrazolium bromid) testa i LDH (laktat dehidrogenaza) testa. Procenat ćelija umrlih apoptozom nakon izlaganja komleksima ispitana je flow citometrijskom analizom ćelija bojenih Annexin-om V i propidijum jodidom. Da bi se ispitalo da li postoji korelacija između rezultata dobijenih u kulturi ćelija i efekata u živom sistemu, urađeno je in vivo ispitivanje na animalnom modelu za metastaze adenokarcinoma pluća. Eksperimentalne životinje su bili C57BL/6 miševi, mužjaci uzrasta 6 do 10 nedelja kojima je intravenski aplikovano 5 x 105 LLC1 ćelija u repnu venu. Nakon žrtvovanja miševima su uklonjena pluća, jetra, slezina i desni bubreg od kojih su načinjeni parafinski isečci i obojeni hematoksilin/eozinom. Određivaće se prisustvo i broj metstazau parenhimu pluća i upoređivaće se broj metastaza po grupama. Takođe ispitaće se postojanje oštećenja tkiva jetre i bubrega, kako u smislu sekundarnih depozita, tako i u smislu oštećenja nastalih dejstvom kompleksa. Miševima su takođe uklonjene slezine i, nakon adekvatne pripreme, učinjena je protočna citofluorometrija kako bi se odredila relativna zastupljenost ćelija specifične i nespecifične imunosti odgovornih za antitumorksu imunost: Natural kiler (NK), NKT, CD8+ citotoksičnih limfocita kao i CD4+ T lifocita i makrofaga u slezini miševa eksperimentalnih i kontrolne grupe.

Cisplatin treatment of tumors is associated with a number of adverse effects and a relatively rapid development of resistance. Chemotherapy of NSCLC - (non- small cell type) lung tumor involves the application of platinum derivatives alone or in combination with other groups of chemotherapeutic anti-metabolites such as folate. This study aims to examine the cytotoxicity of novel gold and platinum complexes in epithelial cells of lung adenocarcinoma in vitro(cell lines A549 and LLC1). In a mouse model of experimental lung metastasis was examined that the complexes cause the death of tumor cells in vivo and that the complex may be further examined as potential therapeutic agents. Objectives: In accordance with the main objective set out the following specific goals: 1. MTT and LDH cytotoxicity assay аre performed to examine the potential of the complex 2. To examine the relative ratio of necrotic and apoptotic cell death induced by the tested complexes 3. To examine whether the complexes affect the reduction of the already established lung metastases in a mouse model. Methods : The in vitro methods of this study tested the cytotoxicity of a derivative of gold and platinum in the laboratory using the MTT (3 -(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay , and LDH (lactate dehydrogenase) test . The percentage of dead cells by apoptosis after exposure to the complex was investigated by flow cytometric analysis of cells stained Annexin-V machine and propidium iodide. In order to investigate whether a correlation exists between the results obtained in cell culture and the effects of the mercury in the system , has been done in vivo examination of an animal model for metastatic adenocarcinoma of the lung. The experimental groups were C57BL / 6 mice, males aged from 6 to 10 weeks was intravenously injected with 5 x 105 LLC1 cells into the tail vein. After sacrificing the mice were removed from the lung, liver, spleen and left kidney of which are made from paraffin embedded and stained with hematoxylin / eosin. Will determine the presence and number metstazau lung parenchyma and comparative of the number of metastases per group. You will investigate the existence of tissue damage liver and kidneys, both in terms of secondary deposits, and in terms of damage caused by the activity of the complex. The mice were also removed by the spleen, and after appropriate preparation was made flow citofluorometrija to determine the relative representation of the cell -specific and nonspecific immunity responsible for antitumorksu immunity: natural killer ( NK ) , NKT , CD8 + cytotoxic lymphocytes and CD4 + T lymphocytes and macrophages in the the spleen of the mice of the experimental and control groups.