LITHUANIAN UNIVERSITY OF HEALTH SCIENCES LUHS LIBRARY REPOSITORY

Biologiškai aktyvių molekulių elektropernašos į ląsteles in vitro ir į navikinius audinius efektyvumo tyrimas

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dc.contributor.author Šalomskaitė Davalgienė, Sonata
dc.date.accessioned 2017-08-21T08:07:25Z
dc.date.available 2017-08-21T08:07:25Z
dc.date.issued 2006
dc.identifier.uri http://repository.lsmuni.lt/handle/1/60064
dc.description.abstract ABBREVIATION E – strength of electric field τ – pulse duration HV – high voltage, short pulses LV – low voltage, long pulses MEM - Minimum Essential Medium S – MEM - Spinner Minimum Essential medium LISPB – low ionic strength pulsing buffer ECT – electrochemotherapy LLC – Lewis Lung Carcinoma DC-3F – Chinese hamster lung fibroblastocites LPB – fibrosarkoma cell line BLM – bleomycin CPA – cyclophosphamide LY – Lucifer Yellow 1. INTRODUCTION The delivery of appropriate short and intense electric pulses to living cells, either in suspension or in tissue, results in a transient and reversible alteration of their cell membrane [Mir et al., 1995; Neumann et al., 1989]. The concept widely accepted is that under influence of strong external electric field the potential difference on the cell membrane occurs [Bernhardt and Pauly, 1973]. As a result of it the nanosize transient aqueous pores in the plasma membrane are created [Abidor et al, 1979, Leikin et al., 1986]. The process is called electroporation and initiated pores are named electropores [Neumann et al., 1989]. It is proposed that at higher field strength and pulse duration more and larger pores are initiated [Glaser et al., 1988]. At the tolerant pulse intensities the resealing of pores takes place and membrane came back to the previous state. The process is called reversible electroporation - REP [Weaver and Chizmadzhev, 1966]. Resealing of pores is in a range of milliseconds - minutes and often is going significantly slower than that of initiation [Saulis and Venslauskas, 1991; Benz and Zimmermann, 1981; Kinosita and Tsong, 1977]. When the parameters of electric field are to strong, cells are not recovering after electroporation and the result of this – lysis of cells. It must be noted that direct evidence of electropores at the membrane level is not shown. It’s a reason why some authors prefer to use term electropermeabilization instead of electroporation [Rols and Teissie, 1990]. Method of electroporation was successfully used for: - transfer of small bioactive molecules (such as dyes, sacharides, vitamins, drugs, etc.) into the cells and tissues [Neumann et al., 1989; Belehradek et al., 1994; Mir et al., 1988; ], - transfer of bioactive macromolecules (proteins, DNA) into the cells and tissues [Andre and Mir, 2004]. As a result of that assays the transfection of cells could be achieved and used for practical aims in biotechnology [Satkauskas and Saulis, 2004], - for cell fusion in vitro and in vivo [Neumann et al., 1989; Rols et al., 1989; Grasso et al., 1989; Mekid and Mir, 2000 ], - for the enhanced permeability of drugs into the tumor’s cells and tissues with the therapeutic aims. The method was called – electrochemotherapy [Orlowski and Mir, 1993]. In spite of a lot of credible investigations, cell electroporation (electropermeabilization) mechanism remains still unclear. By using premises based on above mentioned investigations, which were carried out on planar lipid membranes and on cells in suspensions, the theoretical model of cells electroporation was elaborated in our laboratory [Saulis and Venslauskas, 1988 and 1993]. The creation of pores is considered as one step barrier process. The model is enabling to estimate the probability of cell electroporation P as a function of external electric field intensity E and pulse duration. 1.1 Aims of the study The aim of this study was to investigate the effectiveness of transfer of bioactive molecules into the cells in vitro as well as into mice tumors in vivo as a function of applied external electric field parameters. The objectives of the study: 1. To evaluate the effectiveness of transfer of small bioactive molecules (fluorescent dye – Lucifer Yellow and anticancer drug – bleomycin) into the cells in suspension and monolayer by measuring electroporation probability as a function of electric field intensity and pulse duration; 2. To evaluate the effectiveness of electrotransfer of bioactive macromolecules (pDNA) into the cells in vitro as a function of electric field parameters and their combinations; 3. To evaluate the effectiveness of electrochemotherapy on mice tumors as a function of electric field parameters combined with anticancer drugs using histological – morphometrical analysis. 4. To evaluate the intensity of cells electrofusion in vitro and in vivo as a function of electric field strength and cell line. 2. MATERIALS AND METHODS OF THE STUDY Cell treatment in vitro. B16F10 melanoma, LPB fibrosarcoma, transformed Chinese hamster lung fibroblasts (DC - 3F) and hepatoma MH22A cells lines were grown in Minimum Essential Medium (MEM, GIBCO) or in Dulbecco’s modified Eagle’s medium (DMEM, SIGMA) with added fetal bovine serum (8 %, GIBCO) and antibiotics (penicillin and streptomycin, GIBCO). 1. To determine the effectiveness of electroporation cells were grown in monolayers on glass coverslips in Petri dishes (40 mm Ø) at 37 °C and 5 % CO2 in incubator. When monolayer of cells has covered the slides, culture medium was discarded and electrical pulses were delivered to the cells on coverslips with presence (for electropermeabilization) or absence (for electrofusion) of Lucifer Yellow (LY, MW 522 g/mol, Sigma) at 2 mM. Two parallel plate stainless steel electrodes, 8 mm apart, were placed onto the monolayer and 8 electric pulses of either 800, 1000 or 1200 V/cm and 0.1 ms of duration were applied at a 1 Hz frequency using a Cliniporator TM (IGEA, Italy). After the delivery of electric pulses cells were left for 5 min incubation at room temperature. Thereafter they were put in culture medium. Two hours later cells were examined using a light microscope (Leica DMIL 520804, Germany). Experiments were repeated two times for each pulse strength. From each experiment four images were taken in the electroporated area and one image from the non-electroporated area. For the analysis of fusion the total number of fused cells in each image was counted. The results are expressed as the percentage of fused cells ± SEM (standard error of the mean). Statistical differences between experimental groups were calculated using the Student’s t-test. Statistically significant difference was accepted when p<0.05. In case of electropermeabilization analyzes using LY, cells after incubation after pulsing were washed with sterile 0.9 % NaCl and then examined under fluorescence microscope (Axiovert 135, Zeiss, Germany). 2. To determine the uptake of LY into permeabilized hepatoma MH22A cells depending on the electric pulse strength and pulse duration, cells were grown in monolayer in flasks (75 cm2) at 37 °C and 5 % CO2 in incubator. After treatment with trypsin-EDTA (SIGMA) cells were centrifuged for 10 min. at 1000 r. p. m. and resuspended in Spinner Minimum Essential medium (S-MEM, SIGMA). A 50 μl droplet of cells suspension (1 x 106) with LY at 2 mM was placed between two parallel plate stainless steel electrodes 2 mm apart and 1 electrical pulse of different strength and 0.1, 0.5 and 1 ms of duration was delivered. After pulse delivery cells were incubated 20 min. at room temperature and then centrifuged 5 min. at 1000 r. p. m. Thereafter supernatant was removed and pellet was diluted in 5 ml of S-MEM. The washing procedure was repeated twice to remove the extracellular Lucifer Yellow. After the washing cells were put on the hematocytometer and 8 images were taken: 4 under the simple light and 4 under the UV light. Cells were counted in all pictures. Percentage of electropermeabilized cells was obtained from the total number of cells in simple light and fluorescent cells samples Values are presented as means ± SEM. 3. To determine the electroporation using anticancer drug cells were grown in monolayer in flasks (75 cm2) at 37 °C and 5 % CO2 in incubator. After trypsination with trypsin-EDTA (GIBCO) cells were centrifuged for 10 min. at 1000 r. p. m. and resuspended in S-MEM (GIBCO). A 50 μl droplet of cells suspension (1 x 106) with bleomycin (BLM, MW 1487.49 g/mol, Nypon Kayaku Ltd., Japan) at 30 nM of concentration was exposed to electric pulses. After pulse delivery cells were incubate for 5 min. at room temperature and then diluted in the culture medium and were transferred into Petri dishes (60 mm Ø). Cells were grown for five days. Then cells were fixed 20 min. in 2 ml of 5% of formalin solution (SIGMA) at room temperature and stained for 15 min. with 1% crystal violet (Sigma). Colonies of cells were counted and normalized to the control (no pulses, just bleomycin) to get the percentage of cells after surviving the exposure to electric pulses with BLM. By subtracting this percentage from 100 %, the percentage of electroporated cells was obtained. Values are presented as means ± SEM (n = 3). 4. For DNA electrotransfer cells were grown in monolayer in flasks (75 cm2) at 37 °C and 5 % CO2 in incubator. After trypsination with trypsin-EDTA (GIBCO) cells were centrifuged for 10 min. at 1000 r. p. m. and resuspended in S-MEM (GIBCO) or in a low ionic strength pulsing buffer (LISPB: 0.25 M sucrose, 10mM Tris HCl, 1mM MgCl2). A 50 μl droplet of cells suspension (1 x 106) contained 8 μg of DNA (pCMV Luc coding for the firefly luciferase under the control of the CMV promoter). Plasmid DNA was prepared using the EndoFree Plasmid Giga Kit (QIAGEN, France). The droplet was placed between two parallel plate stainless steel electrodes 2 mm apart and different combinations of high - voltage (HV) and low - voltage (LV) pulses were applied with Cliniporator TM (IGEA, Italy). After pulse delivery cells were incubated for 5 min. at room temperature and then cultured for two days in Petri dishes (100 mm Ø). Two days after DNA electrotransfer cells were trypsinised and lysed in 100 μl of cell culture lysis reagent solution (Promega, USA). The lusiferase activity was assessed in 20 μl of the supernatant using Berthold luminometer (Lumat LB 9507). The determination of total protein in every sample
dc.language.iso lit
dc.subject Electroporation of cells and tissues
dc.subject Histological analysis
dc.subject Ląstelių ir audinių elektroporacija
dc.subject Histologinė analizė
dc.title Biologiškai aktyvių molekulių elektropernašos į ląsteles in vitro ir į navikinius audinius efektyvumo tyrimas
dc.title.alternative Investigation of the effectiveness of biologically active molecules electrotransfer into cells in vitro and into the malignant tumors
dc.type Daktaro disertacija


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