RU2671570C1 - Cosmetic asset based on planarian flatworms extracts - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Zoology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Изобретение относится к области косметологии и касается использования косметических активов для приготовления косметических препаратов. Экстракт из регенерирующих плоских червей планарий используется в качестве косметического актива для добавки в различного рода косметические средства - крем, сыворотки, флюиды, маски. Данный актив обладает противозрастными и регенеративными эффектами на клетки кожи человека.The invention relates to the field of cosmetology and relates to the use of cosmetic assets for the preparation of cosmetic preparations. The planaria worm extract is used as a cosmetic asset to supplement various cosmetic products - cream, serums, fluids, masks. This asset has anti-aging and regenerative effects on human skin cells.
Пресноводные плоские черви - планарий обладают уникальной способностью к регенерации за счет наличия большого количества стволовых клеток. Пролиферация и дифференцировка этих клеток обеспечивает восстановление утраченных частей тела и обновление организма животного [1, 2]. Несмотря на то, что эти животные примитивны, основные механизмы, обеспечивающие процесс регенерации планарий, активность стволовых клеток достаточно схожи с таковыми у млекопитающих и человека [3]. Анализ транскриптома и протеома этих животных показывает высокую степень гомологии основных сигнальных белков, регулирующих стволовые клетки и регенерацию [4]. Также в процессе регенерации в теле планарий в большом количестве синтезируются и более простые соединения в частности нейротрансмиттеры - серотонин, мелатонин, аденозин, серии, допамин, эпинефрин, норэпинефрин, октапамин, ацетилхолин, гистамин и др., которые способны в этих физиологически-активных дозах оказывать благотворное воздействие на клетки кожи человека [5]. Особо стоит отметить и наличие сложной системы пептидной регуляции регенерации планарий и их стволовых клеток, причем большинство олигопептидов и их производных (ди, трипетидов, т.д) идентичны пептидам, регулирующим активность стволовых клеток человека [6, 7]. Слизь планарий также в своем составе имеет богатый набор физиологически-активных веществ, способных оказывать воздействие на клетки кожи человека. В частности там присутствуют ферменты-антиоксиданты (каталаза, пероксиредоксин, супероксиддисмутаза), мукополисахариды (гепарансульфат, хондроитинсульфат) [8, 9]. Таким образом, экстракт регенерирующих планарий можно рассматривать как мощный физиологически-активный актив для применения в косметических средствах.Freshwater flatworms - planaria have a unique ability to regenerate due to the presence of a large number of stem cells. The proliferation and differentiation of these cells ensures the restoration of lost parts of the body and the renewal of the animal’s body [1, 2]. Despite the fact that these animals are primitive, the main mechanisms that ensure the regeneration of planaria, the activity of stem cells are quite similar to those in mammals and humans [3]. Analysis of the transcriptome and proteome of these animals shows a high degree of homology of the main signaling proteins that regulate stem cells and regeneration [4]. Also, in the process of regeneration in the body of planarium, simpler compounds are synthesized in large quantities, in particular, neurotransmitters - serotonin, melatonin, adenosine, series, dopamine, epinephrine, norepinephrine, octapamine, acetylcholine, histamine, etc., which are capable of these physiologically active doses have a beneficial effect on human skin cells [5]. Of particular note is the presence of a complex peptide regulation system for the regeneration of planaria and their stem cells, with most oligopeptides and their derivatives (di, tripetides, etc.) identical to peptides that regulate human stem cell activity [6, 7]. Slime planaria also has a rich set of physiologically active substances that can affect human skin cells. In particular, there are antioxidant enzymes (catalase, peroxyredoxin, superoxide dismutase), mucopolysaccharides (heparan sulfate, chondroitin sulfate) [8, 9]. Thus, the extract of regenerating planaria can be considered as a powerful physiologically active asset for use in cosmetics.
В настоящее время косметическая промышленность практически перестала применять активы на основе стволовых клеток человека, так как после обработок (для устранения возможных патогенов) эти экстракты теряют всю свою физиологическую активность в силу денатурации пептидных компонентов и разрушения низкомолекулярных физиологически-активных веществ [10]. Некоторые производители перешли на применение в качестве активов экстрактов стволовых клеток растений [11-13], но как показывает литературный анализ, регуляция стволовых клеток растений и млекопитающих в корне отличаются друг от друга и в принципе активы на их основе не могут воздействовать должным образом на клетки человека [14-17]. Применение экстрактов планарий в качестве косметического актива позволяет безопасно использовать его в нативном виде, только с предварительной фильтрацией для стерилизации, и использовать в качестве актива для косметических средств. Уникальная способность к регенерации и бесполому размножению, неприхотливость планарий при содержании и культивировании позволяет их размножать в больших количествах в относительно малом объеме воды и за короткий промежуток времени, что было показано заявителем [18]. Это позволяет получать необходимую биомассу планарий для промышленного применения их экстрактов в качестве косметического актива.Currently, the cosmetics industry has practically ceased to use assets based on human stem cells, because after treatments (to eliminate possible pathogens), these extracts lose all their physiological activity due to the denaturation of peptide components and the destruction of low molecular weight physiologically active substances [10]. Some manufacturers switched to the use of plant stem cell extracts as assets [11–13], but as the literature analysis shows, the regulation of stem cells of plants and mammals is fundamentally different from each other and, in principle, assets based on them cannot affect cells properly person [14-17]. The use of planaria extracts as a cosmetic asset allows you to safely use it in its native form, only with preliminary filtration for sterilization, and use it as an asset for cosmetics. The unique ability to regenerate and asexually reproduce, the unpretentiousness of planaria during maintenance and cultivation allows them to be propagated in large quantities in a relatively small volume of water and for a short period of time, which was shown by the applicant [18]. This allows you to get the necessary planarium biomass for industrial use of their extracts as a cosmetic asset.
Целью настоящего изобретения является применение экстракта плоских червей планарий в качестве косметического актива для добавления в косметические кремы, сыворотки, флюиды, маски.The aim of the present invention is the use of planaria worm extract as a cosmetic asset for addition to cosmetic creams, serums, fluids, masks.
Заявителем были проведены исследования биологической активности экстракта планарий на пролиферацию, дифференцировку и жизнеспособность стволовых клеток человека in vitro.The applicant has conducted studies of the biological activity of the planaria extract for the proliferation, differentiation and vitality of human stem cells in vitro.
Пример 1. Исследование биологической активности экстракта планарий.Example 1. The study of the biological activity of the planaria extract.
В данном примере представлено описание полученных результатов исследования биологической активности экстрактов плоских червей планарий на примере культивируемых первичных мезенхимальных стволовых клеток человека. Было определено влияние экстрактов на жизнеспособность рост, пролиферацию и метаболическую активность клеток.This example describes the results of a study of the biological activity of planaria worm extracts using cultured primary human mesenchymal stem cells. The effect of extracts on the viability of cell growth, proliferation, and metabolic activity was determined.
Фиг. 1 показывает результаты исследования цитотоксичности экстрактов планарий (Э1 и Э2) в разведении 1:10, 1:50 и 1:100 на культивируемые мезенхимальные стволовые клетки человека Th при культивировании клеток в течение 4-х суток. Показаны значения среднего и 95% доверительный интервал. Испытания серии разведений полученных экстрактов планарий in vitro на уровне культивируемых мезенхимальных стволовых клеток человека продемонстрировали полное отсутствие цитотоксических эффектов даже высоких концентраций экстракта планарий.FIG. 1 shows the results of a study of the cytotoxicity of planaria extracts (E1 and E2) at a dilution of 1:10, 1:50 and 1: 100 on cultured human Th mesenchymal stem cells during cell cultivation for 4 days. Shown are mean and 95% confidence intervals. Tests of a series of in vitro dilutions of the obtained planarium extracts at the level of cultured human mesenchymal stem cells demonstrated the complete absence of cytotoxic effects of even high concentrations of planaria extract.
На фиг. 2 показано воздействие экстрактов планарий (Э1 и Э2) в разведении 1:10, 1:50 и 1:100 на метаболическую активность (фиг. 2, А) и рост клеточной культуры мезенхимальных стволовых клеток человека Th (фиг. 2, Б). Показаны значения среднего и 95% доверительный интервал. Метаболическая активность мезенхимальных стволовых клеток на 2 сутки инкубации после добавления экстрактов в серии разведений 1:10, 1:50 и 1:100 не отличалась от контрольной культуры клеток, не подвергавшихся воздействию экстракта. Добавление экстрактов планарий к культивируемым стволовым клеткам приводило к стимуляции роста культуры. В разведении 1:50 и 1:100 экстракт Э1 на 36 ч и 48 ч способствовал увеличению количества клеток (пролиферации) на 26% (p<0,01) и 33% (p<0,01). Добавление экстракта планарий Э2 в культуру клеток человека также способствовало увеличению пролиферации клеток на 31% (p<0,01) в концентрации 1:50.In FIG. Figure 2 shows the effect of planarium extracts (E1 and E2) at a dilution of 1:10, 1:50 and 1: 100 on metabolic activity (Fig. 2, A) and cell growth of human Th mesenchymal stem cells (Fig. 2, B). Shown are mean and 95% confidence intervals. The metabolic activity of mesenchymal stem cells on the 2nd day of incubation after the addition of extracts in a series of dilutions of 1:10, 1:50 and 1: 100 did not differ from the control culture of cells not exposed to the extract. The addition of planarium extracts to cultured stem cells stimulated culture growth. At a dilution of 1:50 and 1: 100, E1 extract for 36 h and 48 h contributed to an increase in the number of cells (proliferation) by 26% (p <0.01) and 33% (p <0.01). The addition of planarium E2 extract to human cell culture also contributed to an increase in cell proliferation by 31% (p <0.01) at a concentration of 1:50.
Таким образом, полученные экстракты из плоских червей планарий не обладают цитотоксичностью, не влияют на метаболическую культивируемых клеток человека, при этом способны увеличивать их пролиферативную активность. Это указывает на высокий уровень биосовместимости данного препарата и его регенеративной активности на уровне клеток человека.Thus, the obtained extracts from planar worms do not possess cytotoxicity, do not affect the metabolic of cultured human cells, and are able to increase their proliferative activity. This indicates a high level of biocompatibility of this drug and its regenerative activity at the level of human cells.
МетодыMethods
Типы используемых клеток и их культивированиеTypes of cells used and their cultivation
В работе использовали первичные культуры мезенхимальных стволовых клеток (МСК) - пульпы зуба (Th), которые выделяли из зачатка третьего моляра, у здорового пациента 16 лет.Клетки извлекали путем промывки через иглу шприца, вставленную в верхушку канала зуба струей среды DMEM ("ПанЭко", Россия), содержащей 200 Ед/мл пенициллина и 200 мкг/мл стрептомицина ("Life technologies", США,). Далее клетки диссоциировали 0,25% трипсином с 0,02% ЭДТА ("ПанЭко", Россия) в при 37°С течение 30 минут.We used primary cultures of mesenchymal stem cells (MSCs) - tooth pulps (Th), which were isolated from the rudiment of the third molar in a healthy patient 16 years old. Cells were removed by washing through a syringe needle inserted into the top of the tooth canal with a DMEM medium (PanEco ", Russia), containing 200 U / ml of penicillin and 200 μg / ml of streptomycin (" Life technologies ", USA,). Then the cells dissociated with 0.25% trypsin with 0.02% EDTA (PanEco, Russia) at at 37 ° C for 30 minutes.
Полученные клетки центрифугировали 2 минуты при 1500 g, а затем ресуспензировали в среде DMEM/F12 («ПанЭко») в соотношении 1:1 с добавлением 10% фетальной сыворотки крупного рогатого скота (FBS). Далее суспензию переносили в 25 см2 флаконы и культивировали в атмосфере 5% CO2, при 37°С в среде DMEM («ПанЭко»), содержащей 10% FBS (HyClone), 100 ед./мл пенициллина/стрептомицина и добавлением 2 мМ L-глютамина. При достижении субконфлюентного состояния клетки обрабатывали 0,25% раствором трипсин-ЭДТА и пассировали в 75 см2 флаконы в соотношении 1:3. Дальнейшее культивирование проводили в среде α - MEM («ПанЭко»), с 10% FBS, 100 ед./мл пенициллина/стрептомицина и добавлением 2 мМ L-глютамина. При проведении исследований использовали культуры 3 пассажа.The obtained cells were centrifuged for 2 minutes at 1500 g, and then resuspended in DMEM / F12 medium (PanEco) in a 1: 1 ratio with the addition of 10% fetal cattle serum (FBS). Next, the suspension was transferred into 25 cm 2 vials and cultured in an atmosphere of 5% CO 2 at 37 ° C in DMEM (PanEco) containing 10% FBS (HyClone), 100 units / ml penicillin / streptomycin and 2 mM L-glutamine. Upon reaching a subconfluent state, the cells were treated with 0.25% trypsin-EDTA solution and passaged in 75 cm 2 bottles in a ratio of 1: 3. Further cultivation was carried out in α-MEM medium (PanEco), with 10% FBS, 100 units / ml penicillin / streptomycin and 2 mM L-glutamine added. When conducting research used culture 3 passage.
Использованные клетки были фенотипированы по наличию антигенов CD44, CD90, CD105 и отсутствию CD34, CD45 и HLA-DR.Used cells were phenotyped by the presence of antigens CD44, CD90, CD105 and the absence of CD34, CD45 and HLA-DR.
Исследование скорости роста культуры клетокStudy of cell culture growth rate
Скорость роста клеточной культуры оценивали по динамике изменения конфлюентности (занимаемой площади) с помощью планшетного ридера Clone Pix Imager (Molecular Devices, США). Съемку проводили каждые 24 часа в течение 5 суток. На каждую экспериментальную точку приходилось не менее 4 измерений (по 96 лункам каждое). Затем, на основе полученных значений строили кривые роста плотности клеточной культуры.The cell culture growth rate was estimated by the dynamics of changes in confluency (occupied area) using a Clone Pix Imager plate reader (Molecular Devices, USA). The survey was carried out every 24 hours for 5 days. Each experimental point had at least 4 measurements (96 wells each). Then, on the basis of the obtained values, density growth curves of the cell culture were built.
Определение метаболической активности клетокDetermination of metabolic activity of cells
Оценку жизнеспособности клеток проводили с помощью колориметрического МТТ-теста. Данный тест основан на принципе восстановления бесцветной соли тетразолия (3-[4,5-диметилтиазол-2-ил]-2,5-дифенилтетразолия бромид, МТТ) до формазана цитоплазматическими и митохондриальными дегидрогеназами живых метаболически активных клеток. Для контроля фоновой активности использовалась культуральная среда, подвергшаяся воздействию условий и процедур исследования. Для проведения теста исходную культуральную среду удаляли и в каждую лунку вносили по 100 мкл раствора МТТ 0,5 мг/мл в бессывороточной культуральной среде DMEM/F12. Далее смесь инкубировали в течение 3 часов при 37°С в увлажненной атмосфере 5% CO2 затем удаляли жидкость и вносили по 100 мкл диметилсульфоксида (ДМСО) и при постоянном встряхивании планшетов в течение 10 мин при комнатной температуре добивались растворения солей формазана. Окраску в лунках 96 луночного планшета регистрировали по измерению оптической плотности при длине волны 540 нм фотометром (модель 680 BIO-RAD, США).Evaluation of cell viability was performed using a colorimetric MTT test. This test is based on the principle of restoring the colorless tetrazolium salt (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide, MTT) to formazan by cytoplasmic and mitochondrial dehydrogenases of living metabolically active cells. To control the background activity, a culture medium was used that was exposed to the conditions and procedures of the study. For the test, the initial culture medium was removed and 100 μl of a 0.5 mg / ml MTT solution in serum-free DMEM / F12 culture medium was added to each well. The mixture was then incubated for 3 hours at 37 ° C in a humidified atmosphere of 5% CO 2, then the liquid was removed and 100 μl of dimethyl sulfoxide (DMSO) was added and, with constant shaking of the plates, the dissolution of formazan salts was achieved for 10 min at room temperature. Staining in the wells of a 96-well plate was recorded by measuring the optical density at a wavelength of 540 nm with a photometer (model 680 BIO-RAD, USA).
Определение жизнеспособности клетокDetermination of cell viability
Для определения цитотоксических эффектов экстрактов планарий использовали набор для определения доли живых и мертвых клеток (Invitrogen, Life Technologies, США). После воздействия экстрактов на клетки производили окраску клеток в 96 луночном планшете согласно прилагаемому протоколу. После 25 мин. инкубации клетки визуализировали путем фотографирования с помощью инвертированного флуоресцентного микроскопа Axiovert 200 (Carl Zeiss, Германия) с использованием фотокамеры Canon А620 (Canon, США). Зеленая флуоресценция (краситель SYTO 9 λ=485/498 nm) характеризовала живые клетки, красная флуоресценция (йодистый пропидий λ=535/617 nm) характеризовала мертвые клетки. Производили подсчет количества живых и мертвых клеток.To determine the cytotoxic effects of planaria extracts, a kit was used to determine the proportion of living and dead cells (Invitrogen, Life Technologies, USA). After the extracts were exposed to the cells, cells were stained in a 96 well plate according to the attached protocol. After 25 minutes cell incubations were visualized by photographing with an Axiovert 200 inverted fluorescence microscope (Carl Zeiss, Germany) using a Canon A620 camera (Canon, USA). Green fluorescence (dye SYTO 9 λ = 485/498 nm) characterized live cells, red fluorescence (propidium iodide λ = 535/617 nm) characterized dead cells. Counted the number of living and dead cells.
Статистический анализStatistical analysis
Каждый эксперимент повторяли не менее 3-х раз, в каждой серии разведений экстрактов. Статистический анализ производили с использованием методов вариационной статистики, с определением среднего и стандартного отклонения среднего. Для проверки значимости отличий использовали программу "Sigma-Plot 9.11" (Systat Software Inc., Германия) и алгоритм ANOVA с применением t-теста Стьдента.Each experiment was repeated at least 3 times in each series of dilutions of the extracts. Statistical analysis was performed using methods of variation statistics, with the determination of the mean and standard deviation of the mean. To check the significance of differences, the Sigma-Plot 9.11 program (Systat Software Inc., Germany) and the ANOVA algorithm using the Student t-test were used.
ЛитератураLiterature
1. Karami A., Tebyanian Н., Goodarzi V. et al. Planarians: an In Vivo Model for Regenerative Medicine, International journal of stem cells, 8 (2015) 128-133.1. Karami A., Tebyanian N., Goodarzi V. et al. Planarians: an In Vivo Model for Regenerative Medicine, International journal of stem cells, 8 (2015) 128-133.
2. Sahu S., Dattani A., Aboobaker A.A. Secrets from immortal worms: What can we learn about biological ageing from the planarian model system?, Seminars in cell & developmental biology, 70 (2017) 108-121.2. Sahu S., Dattani A., Aboobaker A.A. Secrets from immortal worms: What can we learn about biological ageing from the planarian model system ?, Seminars in cell & developmental biology, 70 (2017) 108-121.
3. Zhu S.J., Pearson B.J. (Neo)blast from the past: new insights into planarian stem cell lineages, Current opinion in genetics & development, 40 (2016) 74-80.3. Zhu S.J., Pearson B.J. (Neo) blast from the past: new insights into planarian stem cell lineages, Current opinion in genetics & development, 40 (2016) 74-80.
4. Labbe R.M., Irimia M., Currie K.W. et al. A comparative transcriptomic analysis reveals conserved features of stem cell pluripotency in planarians and mammals, Stem cells, 30 (2012) 1734-1745.4. Labbe R.M., Irimia M., Currie K.W. et al. A comparative transcriptomic analysis reveals conserved features of stem cell pluripotency in planarians and mammals, Stem cells, 30 (2012) 1734-1745.
5. Rangiah K., Palakodeti D. Comprehensive analysis of neurotransmitters from regenerating planarian extract using an ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring method, Rapid communications in mass spectrometry: RCM, 27 (2013) 2439-2452.5. Rangiah K., Palakodeti D. Comprehensive analysis of neurotransmitters from regenerating planarian extract using an ultrahigh-performance liquid chromatography / mass spectrometry / selected reaction monitoring method, Rapid communications in mass spectrometry: RCM, 27 (2013) 2439-2452.
6. Collins J.J. 3rd, Hou X., Romanova E.V. et al. Genome-wide analyses reveal a role for peptide hormones in planarian germline development, PLoS biology, 8 (2010) e1000509.6. Collins J.J. 3rd, Hou X., Romanova E.V. et al. Genome-wide analyses reveal a role for peptide hormones in planarian germline development, PLoS biology, 8 (2010) e1000509.
7. Ong Т.Н., Romanova E.V., Roberts-Galbraith R.H. et al. Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea, The Journal of biological chemistry, 291 (2016) 8109-8120.7. Ong T.N., Romanova E.V., Roberts-Galbraith R.H. et al. Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea, The Journal of biological chemistry, 291 (2016) 8109-8120.
8. Bocchinfuso D.G., Taylor P., Ross E. et al. Proteomic profiling of the planarian Schmidtea mediterranea and its mucous reveals similarities with human secretions and those predicted for parasitic flatworms, Molecular & cellular proteomics: MCP, 11 (2012) 681-691.8. Bocchinfuso D.G., Taylor P., Ross E. et al. Proteomic profiling of the planarian Schmidtea mediterranea and its mucous reveals similarities with human secretions and those predicted for parasitic flatworms, Molecular & cellular proteomics: MCP, 11 (2012) 681-691.
9. Yamada S., Sugahara K., Ozbek S. Evolution of glycosaminoglycans: Comparative biochemical study, Communicative & integrative biology, 4 (2011) 150-158.9. Yamada S., Sugahara K., Ozbek S. Evolution of glycosaminoglycans: Comparative biochemical study, Communicative and integrative biology, 4 (2011) 150-158.
10. Na Y.K., Ban J.J., Lee M. et al. Wound healing potential of adipose tissue stem cell extract, Biochemical and biophysical research communications, 485 (2017) 30-34.10. Na Y. K., Ban J. J., Lee M. et al. Wound healing potential of adipose tissue stem cell extract, Biochemical and biophysical research communications, 485 (2017) 30-34.
11. Morus M., Baran M., Rost-Roszkowska M. et al. Plant stem cells as innovation in cosmetics, Acta poloniae pharmaceutica, 71 (2014) 701-707.11. Morus M., Baran M., Rost-Roszkowska M. et al. Plant stem cells as innovation in cosmetics, Acta poloniae pharmaceutica, 71 (2014) 701-707.
12. WO 2010067212 A2, дата приоритета 20081212.12. WO 2010067212 A2, priority date 20081212.
13. CN 102477411 А, дата приоритета 20101130.13. CN 102477411 A, priority date 20101130.
14. Rahni R., Efroni I., Birnbaum K.D. A Case for Distributed Control of Local Stem Cell Behavior in Plants, Developmental cell, 38 (2016) 635-642.14. Rahni R., Efroni I., Birnbaum K.D. A Case for Distributed Control of Local Stem Cell Behavior in Plants, Developmental cell, 38 (2016) 635-642.
15. Tucker M.R., Laux Т., Connecting the paths in plant stem cell regulation, Trends in cell biology, 17 (2007) 403-410.15. Tucker M.R., Laux T., Connecting the paths in plant stem cell regulation, Trends in cell biology, 17 (2007) 403-410.
16. Drummond-Barbosa D. Regulation of Stem Cell Populations, in: Reviews in Cell Biology and Molecular Medicine, Wiley-VCH Verlag GmbH & Co. KGaA, 2006.16. Drummond-Barbosa D. Regulation of Stem Cell Populations, in: Reviews in Cell Biology and Molecular Medicine, Wiley-VCH Verlag GmbH & Co. KGaA, 2006.
17. Strauss R., Hamerlik P., Lieber A. et al., Regulation of stem cell plasticity: mechanisms and relevance to tissue biology and cancer, Mol Ther, 20 (2012) 887-897.17. Strauss R., Hamerlik P., Lieber A. et al., Regulation of stem cell plasticity: mechanisms and relevance to tissue biology and cancer, Mol Ther, 20 (2012) 887-897.
18. Ermakov, A.M., Ermakova, O.N., Kudravtsev, A.A. et al. Mol Biol Rep, 39 (2012) 3073.18. Ermakov, A.M., Ermakova, O.N., Kudravtsev, A.A. et al. Mol Biol Rep, 39 (2012) 3073.
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