RU2482870C1 - Agent inducing hemopoietic stem cell differentiation into thrombocytes - Google Patents
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Изобретение относится к области медицины.The invention relates to the field of medicine.
Применение химиотерапии у онкологических больных приводит к повреждению и гибели кроветворных клеток костного мозга со снижением количества тромбоцитов в периферической крови. Тромбоцитопения является серьезной проблемой в лечении онкологических больных (Lorusso D, Ferrandina G, Greggi S, Gadducci A, Pignata S, Tateo S, Biamonte R, Manzione L, Di Vagno G, Ferrau' F, Scambia G. // Phase III multicenter randomized trial of amifostine as cytoprotectant in first-line chemotherapy in ovarian cancer patients. Ann. Oncology. 2003 Jul; 14(7): 1086-93).The use of chemotherapy in cancer patients leads to damage and death of hematopoietic cells of the bone marrow with a decrease in the number of platelets in the peripheral blood. Thrombocytopenia is a serious problem in the treatment of cancer patients (Lorusso D, Ferrandina G, Greggi S, Gadducci A, Pignata S, Tateo S, Biamonte R, Manzione L, Di Vagno G, Ferrau 'F, Scambia G. // Phase III multicenter randomized trial of amifostine as cytoprotectant in first-line chemotherapy in ovarian cancer patients. Ann. Oncology. 2003 Jul; 14 (7): 1086-93).
Известно ограниченное количество средств, способствующих дифференцировке тромбоцитов. Наиболее близким к заявляемому средству является тромбопоэтин (прототип). Тромбопоэтин - гормон, индуцирующий дифференцировку мегакариоцитарного ростка из стволовых клеток костного мозга и влияющий на пролиферацию, созревание и дифференцировку мегакариоцитов и образование тромбоцитов (Nagahisa H, Nagata Y, Ohnuki T, Osada M, Nagasawa Т, Abe T, Todokoro K. // Bone marrow stromal cells produce thrombopoietin and stimulate megakaryocyte growth and maturation but suppress proplatelet formation. Blood. 1996 Feb 15; 87(4):1309-16).A limited number of agents are known to promote platelet differentiation. Closest to the claimed agent is thrombopoietin (prototype). Thrombopoietin is a hormone that induces the differentiation of a megakaryocytic germ from bone marrow stem cells and affects the proliferation, maturation and differentiation of megakaryocytes and platelet formation (Nagahisa H, Nagata Y, Ohnuki T, Osada M, Nagasawa T, Abe T, Todokoro K. // Bone marrow stromal cells produce thrombopoietin and stimulate megakaryocyte growth and maturation but suppress proplatelet formation. Blood. 1996 Feb 15; 87 (4): 1309-16).
Задачей изобретения является расширение ассортимента средств, индуцирующих дифференцировку тромбоцитов. Задача решается тем, что предлагается новое средство, индуцирующее дифференцировку тромбоцитов, представляющее собой рекомбинантный циклофилин А человека (рчЦфА).The objective of the invention is to expand the range of agents that induce platelet differentiation. The problem is solved by the fact that a new tool is proposed that induces platelet differentiation, which is a recombinant human cyclophilin A (rhtsfA).
РчЦфА известен как белок с молекулярной массой 18 кД, присутствующий в различных тканях, усиливает миграцию стволовых клеток, предшественников дендритных клеток, гранулоцитов и лимфоцитов из костного мозга на периферию, способствует дифференцировке дендритных клеток и гранулоцитов (Khromykh L.M., Kulikova N.L., Anfalova T.V. et al. // Cyclophilin A produced by thymocytes regulates the migration of murine bone marrow cells. Cell Immunol. - 2007. - vol. 249(1). - p.46-53; Bharadwaj U, Zhang R, Yang H, Li M, Doan LX, Chen C, Yao Q. // Effects of cyclophilin A on myeloblastic cell line KG-1 derived dendritic like cells (DLC) through p38 MAP kinase activation. J Surg Res. 2005 Jul 1; 127(1):29-38; Патент РФ №2370277).RFCfA is known as a protein with a molecular weight of 18 kD, present in various tissues, enhances the migration of stem cells, precursors of dendritic cells, granulocytes and lymphocytes from bone marrow to the periphery, and promotes the differentiation of dendritic cells and granulocytes (Khromykh LM, Kulikova NL, Anfalova TV et al . // Cyclophilin A produced by thymocytes regulates the migration of murine bone marrow cells. Cell Immunol. - 2007. - vol. 249 (1). - p. 46-53; Bharadwaj U, Zhang R, Yang H, Li M, Doan LX, Chen C, Yao Q. // Effects of cyclophilin A on myeloblastic cell line KG-1 derived dendritic like cells (DLC) through p38 MAP kinase activation. J Surg Res. 2005 Jul 1; 127 (1): 29- 38; RF Patent No. 2370277).
РчЦфА усиливает адгезивные свойства тромбоцитов (Патент РФ №2435607). Для выполнения поставленной задачи оценивали влияние рчЦфА на дифференцировку стволовых клеток лейкаферезного концентрата и костного мозга онкологических больных после химио-лучевой терапии и здоровых доноров. Дифференцировку клеток тромбоцитарного ростка оценивали по экспрессии маркера CD61, который присутствует на всех этапах становления тромбоцитов от стволовых до зрелых клеток.RFCfA enhances the adhesive properties of platelets (RF Patent No. 2435607). To accomplish this task, the effect of rhcfA on the differentiation of stem cells from leukapheresis concentrate and bone marrow of cancer patients after chemo-radiation therapy and healthy donors was evaluated. Platelet cell differentiation was evaluated by expression of the marker CD61, which is present at all stages of platelet formation from stem to mature cells.
Для получения рчЦфА использовали клетки Escherichia coli BL21, трансформированные плазмидой pCyPAwt/pGEX-2TK (M.Bukrinsky Albert Einstein College of Medicine of Yeshiva University, США). Качество полученного рчЦфА оценивали с помощью SDS-PAAG по Лэммли (U.K.Laemmli. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 1970; V.227, P.680-685). Концентрацию рчЦфА определяли по методу Бредфорда (Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 72:248-254). Идентификацию рчЦфА проводили с помощью Wetstern-blott анализа (Gershoni JM, Palade GE. Electroblotting of proteins from polyacrylamide gels. Methods Mol Biol. 2004; 244:345-352) с использованием кроличьих антител к рчЦфА (е-Biosciences). Чистота полученного рчЦфА составила более 98%.Escherichia coli BL21 cells transformed with the plasmid pCyPAwt / pGEX-2TK (M. Bukrinsky Albert Einstein College of Medicine of Yeshiva University, USA) were used to obtain rhcfA. The quality of the rhcfA obtained was evaluated using Laemmli SDS-PAAG (U. K. Laemmli. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 1970; V.227, P.680-685). The concentration of rfCfA was determined by the Bradford method (Bradford, M. M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 72: 248-254). The identification of rfcPA was carried out using Wetstern-blott analysis (Gershoni JM, Palade GE. Electroblotting of proteins from polyacrylamide gels. Methods Mol Biol. 2004; 244: 345-352) using rabbit antibodies to rfcA (e-Biosciences). The purity of the obtained rhcfA was more than 98%.
Для исследования использовали лейкаферезный концентрат, представляющий собой фракцию клеток периферической крови с преобладанием мононуклеаров. Лейкаферезный концентрат получали у пяти онкологических больных после химио-лучевой терапии путем сепарации клеток периферической крови.For the study, leukapheresis concentrate was used, which is a fraction of peripheral blood cells with a predominance of mononuclear cells. Leukapheresis concentrate was obtained from five cancer patients after chemo-radiation therapy by separation of peripheral blood cells.
В качестве образцов использовали костный мозг трех онкологических больных и трех здоровых доноров.The bone marrow of three cancer patients and three healthy donors were used as samples.
С целью мобилизации стволовых кроветворных клеток пациентам предварительно проводили стимуляцию кроветворения гранулоцитарным колониестимулирующим фактором (Г-КСФ) в стандартной дозе 5 мкг/кг в течение 3-14 дней. Количество CD34+ стволовых клеток в аликвотах образцов ленкаферезного концентрата варьировало от 0,25% до 1,3%, в аликвотах образцов костного мозга от 2,1% до 5,8%.In order to mobilize hematopoietic stem cells, patients were previously stimulated with hematopoiesis with granulocyte colony stimulating factor (G-CSF) in a standard dose of 5 μg / kg for 3-14 days. The number of CD34 + stem cells in aliquots of samples of lenkapheresis concentrate varied from 0.25% to 1.3%, in aliquots of bone marrow samples from 2.1% to 5.8%.
Клетки крови и костного мозга человека культивировали при концентрации 1×106/мл в питательной среде альфа-МЕМ (для клеток крови, Gibco) и IMDM (для клеток костного мозга, Gibco) с добавлением 20% эмбриональной телячьей сыворотки, 2-меркаптоэтанола 5×10-5 M, L-глютамина 2 мМ и цифрана 100 мкг/мл. Культивирование клеток проводили при температуре 37°C в присутствии 5% CO2 в пластиковых 24-луночных планшетах (Corning) в течение 5 дней. Концентрация рчЦфА составила 10,0 мкг/мл.Human blood and bone marrow cells were cultured at a concentration of 1 × 10 6 / ml in alpha-MEM (for blood cells, Gibco) and IMDM (for bone marrow, Gibco) nutrient medium supplemented with 20% fetal calf serum, 2-mercaptoethanol 5 × 10 -5 M, L-
Анализ клеточных маркеров проводили при помощи проточного цитофлюориметра Faxcanto II (Beckton Dickenson) с использованием флюоресцентномеченных антител анти-CD61Fitc (E-Bioscience). Контрольные маркеры были установлены в соответствии с изотипическим контролем: анти-IgG Fitc. Для исключения неспецифического окрашивания до окраски с флюоресцентными антителами клетки человека инкубировали с донорской сывороткой крови (IV группа) в разведении 1:10. Погибшие клетки исключали путем гейтирования в параметрах прямого и бокового рассеивания (FSC/SSC), а также включения 7-амино-актиномицина D (Calbiochem, CA). Данные анализировали при помощи программ BD FACSDiva (версия 6.1.2, Beckton Dickinson) и WinMDI 2.8 (J.Trotter, http://facs.scripps.edu/).Cell marker analysis was performed using a Faxcanto II flow cytometer (Beckton Dickenson) using fluorescently labeled anti-CD61Fitc antibodies (E-Bioscience). Control markers were set according to isotypic control: anti-IgG Fitc. To exclude nonspecific staining, before staining with fluorescent antibodies, human cells were incubated with donor blood serum (group IV) at a 1:10 dilution. Dead cells were excluded by gating in forward and side scatter parameters (FSC / SSC), as well as inclusion of 7-amino-actinomycin D (Calbiochem, CA). Data was analyzed using the BD FACSDiva (version 6.1.2, Beckton Dickinson) and WinMDI 2.8 (J.Trotter, http://facs.scripps.edu/) programs.
Изобретение иллюстрировано фиг.1 и 2.The invention is illustrated in figures 1 and 2.
На фиг.1(А-Г) представлено влияние рчЦфА на дифференцировку тромбоцитов в лейкаферезном концентрате.Figure 1 (A-D) shows the effect of rhcfA on platelet differentiation in leukapheresis concentrate.
На фиг.1 (А и Б) по оси абсцисс обозначен уровень экспрессии маркера CD34, по оси ординат (SSC) - уровень клеточной гранулярности. На фиг.1А представлена идентификация стволовых клеток, несущих маркер CD34+, в контроле, и на фиг.1Б - в присутствии рчЦфА (очерченный регион).In Fig. 1 (A and B), the level of expression of the CD34 marker is indicated on the abscissa axis, and the level of cell granularity on the ordinate axis (SSC) is indicated. On figa presents the identification of stem cells bearing the marker CD34 + , in the control, and figb - in the presence of rftsfA (outlined region).
На фиг.1(В и Г) по оси абсцисс показан уровень экспрессии маркера CD61, по оси ординат - уровень экспрессии маркера CD34. На фиг.1В представлено количество стволовых клеток, несущих маркер CD61 в контроле (24,69%), и на фиг.1Г - под влиянием рчЦфА (65,81%).Figure 1 (B and D) shows the expression level of the CD61 marker along the abscissa axis, and the expression level of the CD34 marker along the ordinate axis. Figure 1B shows the number of stem cells bearing the CD61 marker in the control (24.69%), and Figure 1G shows the effect of rhCfa (65.81%).
На фиг.2 (А-Г) показано влияние рчЦфА на дифференцировку тромбоцитов в костном мозге.Figure 2 (A-D) shows the effect of rhcfA on platelet differentiation in the bone marrow.
На фиг.2 (А и Б) по оси абсцисс обозначен уровень экспрессии маркера CD34, по оси ординат - уровень клеточной гранулярности (SSC).In Fig. 2 (A and B), the level of expression of the CD34 marker is indicated on the abscissa axis, and the level of cell granularity (SSC) on the ordinate axis.
На фиг.2А представлена идентификация стволовых клеток костного мозга в контроле и на фиг.2Б - в присутствии рчЦфА (очерченный регион).On figa presents the identification of stem cells of the bone marrow in the control and figb - in the presence of rftsfA (outlined region).
На фиг.2 (В и Г) по оси абсцисс показан уровень экспрессии маркера CD61, по оси ординат - уровень экспрессии маркера CD34.Figure 2 (B and D) shows the expression level of the CD61 marker along the abscissa axis, and the expression level of the CD34 marker along the ordinate axis.
На фиг.2 В представлено количество стволовых клеток, несущих маркер CD61 в контроле (1,11%) и на фиг.2Г - под влиянием рчЦфА (3,83%).Figure 2B shows the number of stem cells bearing the CD61 marker in the control (1.11%), and Figure 2G shows the effect of rhCfa (3.83%).
Таким образом, показано, что под влиянием рчЦфа наблюдается увеличение количества стволовых клеток, экспрессирующих маркер CD61, в лейкаферезном концентрате и костном мозге - в 3 раза относительно контрольных значений.Thus, it was shown that, under the influence of rccf, there is an increase in the number of stem cells expressing the CD61 marker in leukapheresis concentrate and bone marrow - 3 times relative to control values.
Технический результат изобретенияThe technical result of the invention
Средство, индуцирующее дифференцировку тромбоцитов, представляет собой рекомбинантный циклофилин А человека.The platelet differentiation inducing agent is human recombinant cyclophilin A.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2606764C2 (en) * | 2011-05-13 | 2017-01-10 | Университет Токио | Method for producing polyploidized megakaryocyte and platelets |
| RU2756000C2 (en) * | 2015-09-15 | 2021-09-24 | Мегакарион Корпорейшн | Method for the production of platelets by a method for rotational mixing of the culture |
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| US20090324583A1 (en) * | 2008-06-30 | 2009-12-31 | Ewha University-Industry Collaboration Foundation | Methods for inducing the differentiation of hematopoietic stem cells into megakaryocytes and platelets, and gene controlling the differentiation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2606764C2 (en) * | 2011-05-13 | 2017-01-10 | Университет Токио | Method for producing polyploidized megakaryocyte and platelets |
| RU2756000C2 (en) * | 2015-09-15 | 2021-09-24 | Мегакарион Корпорейшн | Method for the production of platelets by a method for rotational mixing of the culture |
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