METHOD FOR ESTIMATING ANTIMYCOBACTERIAL ACTION OF ANTI-TUBERCULOSIS DRUGS USING BIOLOGICAL MATERIAL OF PATIENTS WITH PULMONARY TUBERCULOSIS

摘要

FIELD: biotechnology.;SUBSTANCE: invention is a method for evaluating the anti-mycobacterial effect of anti-tuberculosis drugs using the biological material of patients with pulmonary tuberculosis, including the collection of biological material, cultivation in a nutrient medium, its replacement with fresh nutrient medium in the control culture and with the same nutrient medium in the experimental culture, additionally containing an anti-tuberculosis drug, comparison of the growth of mycobacterium tuberculosis in the control and experimental cultures, where samples of lung tissue from the surgical material are used as the biological material; ex vivo culture of alveolar macrophages is obtained from it; cultivating it for 1 day in a nutrient medium; replace the nutrient medium with fresh in the control cell culture and with the same medium with the addition of an anti-tuberculosis drug in experimental cultures of cells differing in concentrations of the same anti-tuberculosis drug; continue cultivation for three days; fix the cells on the cover glasses; stain according to the Ziehl-Nielsen method; carry out cytological analysis for the presence of signs of cytotoxic effects of the anti-TB drug on alveolar macrophages; determine the proportion of alveolar macrophages containing mycobacterium tuberculosis, of the total number of alveolar macrophages in the control and experimental cell cultures; in the absence of signs of a cytotoxic effect of the anti-TB drug on alveolar macrophages in an experimental culture, the absence of mycobacteria in alveolar macrophages or a decrease in the proportion of alveolar macrophages containing mycobacterium tuberculosis, 2 or more times in the experimental culture relative to the control, a positive conclusion is made about the anti-mycobacterial effect of the anti-tuberculosis drug and its minimum concentration sufficient to achieve the anti-mycobacterial effect, and when conducting a cytological analysis for the presence of signs of a cytotoxic effect of the anti-TB drug on alveolar macrophages, take into account the presence of a nucleus with signs of chromatin condensation, and/or lack of integrity of the plasma membrane, and/or the formation of apoptotic bodies, and/or destruction of cells to separate sections of the cytoplasm, and/or the appearance of cells without nuclei; in the presence of at least one of them make a conclusion about the presence of signs of cytotoxic effects.;EFFECT: invention allows to reduce the duration of the method to 4 days from the date of receipt of biological material, the provision of conditions for evaluating the effect of anti-tuberculosis drugs on Mycobacterium tuberculosis, which are in alveolar macrophages from the patient's lung surgery material, determination of the anti-tuberculosis drug and its concentration sufficient to achieve an anti-mycobacterial effect on the patient's mycobacteria in the absence of a cytotoxic effect on the alveolar macrophages.;1 cl, 6 dwg, 5 tbl, 7 ex