Fachbereich Veterinärmedizin



    Entwicklung und Erprobung einer Versuchsanordnung zur Quantifizierung des 3H-Cholin-Einbaus in die Lungenorganoidkultur embryonaler Mäuse (1990)

    Maaß, Gunhild
    Berlin, 1990 — 65 Seiten
    Institut für Tierschutz und Tierverhalten

    Königsweg 67
    Gebäude 21, 1. OG
    14163 Berlin
    Tel.: +49 30 838 62901 (Sekretariat)
    email: tierschutz@vetmed.fu-berlin.de

    Abstract / Zusammenfassung

    The aim of this study was the development and test of experimental conditions, that quantify the input of 3H - Choline into the lung - organoid - culture of fetal NMRI - mice. This method was developed in a series of experiments. One important question was the specific binding of Choline within the cells instead of simple adsorption. Therefore the concentration of 3H- Choline was compared in cultures, cultivated at 37° C and those that had been cultivated in the refrigerator (4° C). 12.2 % of 3H - Choline remained in the cultures cultivated in the refrigerator and could not be eliminated even by several washings with TCA. To settle the same question a dose - response experiment with an increasing amount of " cold " Choline and a constant amount of " hot " Choline was made. The result was a competitive displacement of " hot " Choline by the "cold ". A maximum concentration of 3H - Choline was achieved on the 4th day which terefore was chosen as the point of time for adding the substances that were to be investigated. The concentration of Choline as a mixture of " cold" and " hot " Choline was determined by the curve that was the result of the dose - response experiment. First levels of 3H - Choline in the cultures were also set up by the dose - effect - test mentioned above; with regard to this experiment the optimum dose consisting of a mixture of 180µg Choline base / ml + 0,2 mCi 3H - Choline was established by the curve.In the following tests the influence of various substances on this method was examined. Out of the glucocorticoid group, Dexamethasone was chosen first and tested in 5 series of experiments which contained 7 various doses ( 1, 10, 30, 60, 100, 300, 1000 ng / ml culture medium ) The purpose of the experiments was to determine the effect of the size of the dose and the substance in question on the Choline concentration. The expected increase in the concentration could not be confirmed in spite of the large differences in concentration of the substances; the values were scattered around the control - value on the diagram. There were similar results when the substances Betamethasone and Hydrocortisone were used. Both experiments with doses varying from 1 to 100 ng / ml culture medium coincided with the conclusions of the Dexamethasone -experiments. The reason may be that there was a sufficient supply of glucocorticoids within the medium, so that further stimulation was not possible. On the contrary the tests with Ambroxol show clear deviations from the control - value. In the 4 experiments with doses from 10 - 100 µg Ambroxol / ml culture medium there was an increase that was dependent an dose. When the supply was increased from 10 to 30 µg Ambroxol / ml culture medium, the differences in the values were extremely large with 35 - 40 %. On the contrary the increase from 30 to 100 µg Ambroxol /ml culture medium did not result in values that differed significantly.The substances Retinoic Acid and Actinomycin D were examined next. Retinoic Acid at a concentration of 50 µg / ml medium were used to investigate the inhibition of the absorption as well as the incorporation of Choline in the cell. This concentration of the two substances almost caused the absorption rate ro be cut in half compared to the control values. The lung cell cultures reacted much more sensitively to the substance Actinomycin D. In a series of 8 cultures and with a dose of 1 µg Actinomycin D / ml culture medium the Choline absorption was reduced to a quarter of the control values.In the present investigation this method for quantification of the Choline absorption in lung cell cultures has been developed and examined. It can only be used under certain conditions. The tested substances may not be reagents that are part of the medium or have been added. The method shows distinct deviations from the control values when "medium - strange" substances are used in this investigation.