Pykhtina Mariya, Miroshnichenko Svetlana, Romanov Vladimir, Grazhdantseva Antonina, Kochneva Galina, Beklemishev Anatoly
Institute of Biochemistry of Federal Research Center of Fundamental and Translational Medicine (FRC FTM), Novosibirsk, 2 Timakova Street, Novosibirsk 630117, Russia.
Federal Budgetary Research Institution State Research Center of Virology and Biotechnology "Vector", Koltsovo, Novosibirsk 630559, Russia.
Pharmaceuticals (Basel). 2021 May 13;14(5):459. doi: 10.3390/ph14050459.
In this study, two strains of the yeast were constructed, one of which produced authentic recombinant human granulocyte-macrophage colony-stimulating factor (ryGM-CSF), and the other was a chimera consisting of ryGM-CSF genetically fused with mature human apolipoprotein A-I (ApoA-I) (ryGM-CSF-ApoA-I). Both forms of the cytokine were secreted into the culture medium. The proteins' yield during cultivation in flasks was 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Both forms of recombinant GM-CSF stimulated the proliferation of human TF-1 erythroleukemia cells; however, the amount of chimera required was 10-fold that of authentic GM-CSF to induce a similar proliferative effect. RyGM-CSF exhibited a 2-fold proliferative effect on BFU-E (burst-forming units-erythroid) at a concentration 1.7 fold less than non-glycosylated -derived GM-CSF. The chimera together with authentic ryGM-CSF increased the number of both erythroid precursors and BMC granulocytes after 48 h of incubation of human bone marrow cells (BMCs). In addition, the chimeric form of ryGM-CSF was more effective at increasing the viability of the total amount of BMCs, decreasing apoptosis compared to the authentic form. ryGM-CSF-ApoA-I normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. The chimera form of GM-CSF exhibited the properties of a multilinear growth factor, modulating the activity of GM-CSF and, perhaps, it may be more suitable for the normalization of granulopoiesis.
在本研究中，构建了两株酵母菌株，其中一株产生天然重组人粒细胞巨噬细胞集落刺激因子（ryGM-CSF），另一株是由ryGM-CSF与成熟人载脂蛋白A-I（ApoA-I）基因融合而成的嵌合体（ryGM-CSF-ApoA-I）。两种形式的细胞因子均分泌到培养基中。在摇瓶培养过程中，ryGM-CSF和ryGM-CSF-ApoA-I的蛋白产量分别为100和60 mg/L。两种形式的重组GM-CSF均刺激人TF-1红白血病细胞的增殖；然而，诱导相似增殖效应所需的嵌合体数量是天然GM-CSF的10倍。RyGM-CSF在浓度比非糖基化衍生的GM-CSF低1.7倍时，对BFU-E（红系爆式集落形成单位）表现出2倍的增殖效应。将嵌合体与天然ryGM-CSF一起孵育人骨髓细胞（BMC）48小时后，红系前体细胞和BMC粒细胞的数量均增加。此外，与天然形式相比，ryGM-CSF的嵌合形式在提高BMC总量的活力、减少细胞凋亡方面更有效。ryGM-CSF-ApoA-I在生理范围内使中性粒细胞的增殖、成熟和分叶正常化，在粒细胞生成受损的情况下保留原始细胞池。GM-CSF的嵌合形式表现出多系生长因子的特性，调节GM-CSF的活性，也许它可能更适合使粒细胞生成正常化。
