The use of apolipoprotein A-I as a transport form of the green fluorescent protein GFP gene in rat hepatocytes
https://doi.org/10.18699/SSMJ20230409
Abstract
The aim of this study was to investigate the possibility of using apolipoprotein A-I (apo A-I) as a transport form of the green fluorescent protein (GFP) gene into rat hepatocytes.
Material and methods. A culture of isolated rat hepatocytes was used as a model. Apo A-I conjugate with fluorescein isothiocyanate (FITC) was obtained by incubation of apo A-I protein with FITC in carbonate buffer pH 9.5 at a ratio of 12.5 μg FITC per 1 mg of protein. Plasmids for pE-GAG transfection with an integrated GFP gene were enriched in the promoter part with cis-elements of the CC(GCC)3-5 type to enhance complex formation with apo A-I. An inverted fluorescence microscope was used for visual analysis of cell fluorescence.
Results and discussion. The paper presents evidence of FITC-labeled apo A-I penetration into the cytoplasm and nuclei of rat hepatocytes by receptor-mediated endocytosis. On this basis, it is proposed an attempt to use apo A-I as a means of targeted delivery of plasmid DNA with an integrated GFP gene into the cell. According to the results of fluorescence microscopy, the use of apo A-I as a plasmid DNA transfection agent led to the accumulation of the GFP protein in the cytoplasm of hepatocytes. No fluorescent protein was observed in the absence of apo A-I.
Conclusions. The result obtained may indicate the delivery of the GFP gene to the nuclear apparatus of the cell, its expression and GFP protein synthesis.
Supporting agencies: The study was conducted within the framework of the state assignment of the Ministry of Science and Higher Education of the Russian Federation (registration number 1021050400914-1-1.6.4.) using the equipment of the Common Use Centers “Spectrometric Measurements” and “Proteomic Analysis”, supported by funding of the Ministry of Science and Higher Education of the Russian Federation (agreement number 075-15-2021-691).
About the Authors
L. M. Polyakov
Federal Research Center of Fundamental and Translational Medicine
Russian Federation
Russian Federation
Lev M. Polyakov, doctor of medical sciences, professor
630117, Novosibirsk, Timakova st., 2
D. V. Sumenkova
Novosibirsk State Medical University of Minzdrav of Russia
Russian Federation
Russian Federation
Dina V. Sumenkova, doctor of biological sciences
630091, Novosibirsk, Krasny ave., 52
M. V. Kotova
Federal Research Center of Fundamental and Translational Medicine
Russian Federation
Russian Federation
Mariya V. Kotova
630117, Novosibirsk, Timakova st., 2
N. V. Trifonova
Federal Research Center of Fundamental and Translational Medicine
Russian Federation
Russian Federation
Natalia V. Trifonova
630117, Novosibirsk, Timakova st., 2
R. A. Knyazev
Federal Research Center of Fundamental and Translational Medicine
Russian Federation
Russian Federation
Roman A. Knyazev, candidate of biological sciences
630117, Novosibirsk, Timakova st., 2
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Review
For citations:
Polyakov L.M., Sumenkova D.V., Kotova M.V., Trifonova N.V., Knyazev R.A. The use of apolipoprotein A-I as a transport form of the green fluorescent protein GFP gene in rat hepatocytes. Сибирский научный медицинский журнал. 2023;43(4):91-96. (In Russ.) https://doi.org/10.18699/SSMJ20230409
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