WO2020151097A1 - 人参形成层干细胞分离培养方法 - Google Patents

人参形成层干细胞分离培养方法 Download PDF

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WO2020151097A1
WO2020151097A1 PCT/CN2019/082446 CN2019082446W WO2020151097A1 WO 2020151097 A1 WO2020151097 A1 WO 2020151097A1 CN 2019082446 W CN2019082446 W CN 2019082446W WO 2020151097 A1 WO2020151097 A1 WO 2020151097A1
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ginseng
cambium
stem cells
compound
treatment
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French (fr)
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吴冬
代陈胜
成丽丽
陈怀德
刘民宪
侯丽丽
江灼和
刘雨佳
凌远建
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深圳先声科技发展有限公司
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2521/00—Culture process characterised by the use of hydrostatic pressure, flow or shear forces
    • C12N2521/10—Sound, e.g. ultrasounds

Definitions

  • the invention relates to a method for separating and cultivating ginseng cambium stem cells, the ginseng cambium stem cells obtained therefrom, and their use in preparing products for ginseng suspension culture.
  • Ginseng is a perennial dicotyledonous plant belonging to the genus Araliaceae, and it is a traditional precious Chinese medicinal material in my country. At present, ginseng has been more and more widely used in the fields of medicine, health care products and cosmetics.
  • the sources of ginseng include wild sources, artificial cultivation and tissue culture. Ginseng resources from natural wild sources are limited and cannot meet market demand at all; artificial cultivation is still the main source of ginseng at present, but artificial cultivation involves cutting down trees, occupying arable land, long growth cycle, susceptibility to pests, pesticides and heavy metal residues, etc. Many obvious shortcomings.
  • the method of tissue culture is to use callus or adventitious roots to cultivate and obtain. It can overcome the problems of limited wild sources and many defects of artificial cultivation.
  • plant stem cells are a type of cell with unlimited self-renewal and differentiation into multiple cell and tissue types.
  • plant stem cells are embedded in stem apical meristems, root tip meristems, and vascular cambium meristems. They can self-renew, divide and produce new cells. Some daughter cells differentiate, and the other forms.
  • New stem cells The function of plant stem cells runs through the entire life process of plants, and constantly provides new cell sources for the formation and regeneration of roots, stems and leaves.
  • the cambium of the vascular system of plants contains pluripotent stem cells, which provide a niche for stem cells and maintain the number and function of stem cells.
  • plant stem cells Compared with dedifferentiated cells, plant stem cells retain the full set of genetic information of their mothers, which is conducive to long-term stable culture; plant stem cells have small but many vacuoles, strong shear resistance, and can maintain synchronous growth and high growth rate; plants Stem cells stably synthesize various types of active secondary metabolites for a long time. For the separation of cambium cells and their use in tissue culture, there have been reports in the prior art.
  • CN104920227A discloses a technique for cultivating stem cells using cambium on a separation medium
  • CN103509749A discloses a method for separating and culturing ginseng cambium stem cells, including sterilizing Northeast ginseng and cutting semicircular slices in Cultivation on the culture medium, separation of cambium stem cells and transfer to the subculture medium for culture and other steps.
  • the division activity of ginseng stem cells obtained by these tissue culture methods and seedling techniques is not high enough, and their proliferation ability is not yet satisfactory.
  • An object of the present invention is to provide a method for separating and culturing ginseng cambium stem cells that can increase the activity of telomerase.
  • Treating tissues containing ginseng cambium can significantly increase the activity of telomerase, where R is - ⁇ -D-glucopyranosyl (1-2)- ⁇ -D-glucopyranose (saponin a) or - ⁇ -D-Glucopyranose (saponin b).
  • a method for isolating and/or culturing ginseng cambium stem cells which includes the step of treating tissue containing ginseng cambium with a compound of formula I:
  • R is - ⁇ -D-glucopyranose (1-2)- ⁇ -D-glucopyranose or - ⁇ -D-glucopyranose.
  • the tissue containing ginseng cambium is obtained by a method including the following steps: containing cambium, phloem, cortex And the tissue of the epidermis is peeled from the xylem.
  • the treatment of the tissue containing the ginseng cambium with the compound of formula I is achieved by placing the tissue containing the ginseng cambium in a solution containing the compound of formula I.
  • the solution is preferably an aqueous solution of the compound of formula I.
  • the compound of formula I used to treat ginseng cambium tissue may be a compound, for example, wherein R is - ⁇ -D-glucopyranosyl(1-2)- ⁇ -D-
  • the R is - ⁇ -D-glucopyranosyl (1-2)- ⁇ -D-glucopyranose and the compound of formula I (saponin a) and the R is The molar ratio of the compound of formula I (saponin b) of - ⁇ -D-glucopyranose is 1:10-10:1, preferably 1:5-5:1, more preferably 1:3-3:1. Particularly preferably, the molar ratio of saponin a to saponin b is 2:5.
  • the concentration of the solution of the compound of formula I is 1 ⁇ M-100 ⁇ M, preferably 10 ⁇ M-100 ⁇ M.
  • the compound of formula I is a mixture of saponin a and b in a molar ratio of 2:5, and the concentration of each in the solution is 20 ⁇ M and 50 ⁇ M, respectively.
  • the tissue containing the ginseng cambium is subjected to ultrasonic treatment.
  • the frequency of the ultrasonic treatment is 5 kHz to 100 kHz, preferably 20 kHz to 40 kHz; the treatment time is 0.1 min to 10 min.
  • the treatment is performed with a sucrose solution.
  • a ginseng cambium stem cell obtained according to the method of the present invention is provided.
  • the use of the ginseng cambium stem cells obtained according to the method of the present invention in preparing a product for ginseng suspension culture is provided.
  • the ginseng cambium stem cells obtained by the method of the present invention can be subjected to suspension culture to obtain ginseng plants.
  • the method for isolating and/or culturing ginseng cambium stem cells of the present invention may include the following steps:
  • Anti-browning treatment treat the sterilized ginseng material with a browning inhibiting medium containing antioxidants
  • the peeled tissue is treated with the saponin compound a and/or b of the present invention, and optionally subjected to ultrasonic treatment and sucrose treatment;
  • the disinfection step of step (1) preferably includes firstly using 75% ethanol for surface disinfection, and then using sodium hypochlorite for disinfection.
  • concentration of sodium hypochlorite is preferably 2%, and it is preferably sterilized twice, the first sterilization time is preferably 8 minutes, and the second sterilization time is preferably 4 minutes.
  • the ultrasonic frequency of the ultrasonic treatment in step (4) is preferably 20kHz, and the ultrasonic treatment time is preferably 5min; the concentration of sucrose treatment is preferably 1M, and the sucrose treatment time is preferably 4h; the concentration of saponin a treatment is preferably 20uM, and saponin b treatment The concentration at the time is preferably 50uM, and the treatment time is 5h.
  • the separation medium preferably contains 3.0 mg.L-1IBA and 0.5mg.L-1KT B5 medium.
  • the culturing in step (5) preferably includes preliminary culturing with MS medium or B5 medium, and then performing subculture; the subculture medium is preferably 2,4-two medium containing 3.0 mg.L-1 Chlorphenoxyacetic acid (2,4-D) and 6.0 mg.L-1NAA in MS medium.
  • the technical solutions provided by the embodiments of the present invention have the beneficial effects that the method for separating and cultivating ginseng cambium stem cells provided by the present invention can effectively separate the cambium cells of ginseng, and the cambium cells have unlimited division ability, rapid growth, and stress resistance. Strong and improves telomerase activity. Moreover, ultrasonic and hyperosmotic treatment can effectively cause specific tissue necrosis, and cambium cells are effectively induced due to their strong resistance to stress, which effectively shortens the time of hypertonic treatment and reduces the probability of infection. At the same time, the concentration of hormones in the culture medium is controlled, so that while the cambium cells proliferate, somatic cells will not proliferate. Anti-browning treatment will reduce the browning of cells during the culture process.
  • Anti-browning treatment Place the sterilized ginseng roots in a browning inhibiting medium containing antioxidants (see Table 1 below), and incubate in a shake flask for about 30 minutes to 1 hour. Then, sterile filter paper is used to remove moisture from the tissue.
  • Saponin treatment The stripped tissue after pre-culture is placed in an aqueous solution containing a saponin compound (the molar ratio of saponin a and b is 2:5, and the concentration of each in the solution is 20 ⁇ M and 50 ⁇ M, respectively). 5min; Put the saponin-treated peeled tissue in a 1M sucrose aqueous solution, first use 20kHz frequency and 20W ultrasonic wave treatment for 5min, then low temperature treatment for 4 hours; then put the ultrasonically treated peeled tissue into 0.05M sucrose aqueous solution Treat it for 5 minutes, and finally place it in a 0.1M sucrose aqueous solution for 5 minutes. Use a sterile pipette to absorb the solution to remove the sucrose and necrosis of specific tissues (phloem, xylem, pith, etc.), and only induce cambium (meristem).
  • Cultivation The tissue obtained after the above treatment is inoculated to contain 30g/L sucrose, 0.7g/L agarose, 3mg.L -1 2,4-dichlorophenoxyacetic acid, and 3.0mg.L -1 IBA And 0.5mg.L -1 KT B5 medium, culture at 20°C in the dark.
  • the secondary medium was 2, containing 30g/L sucrose and 3.0mg.L -1 , MS medium containing 4-dichlorophenoxyacetic acid and 6.0 mg.L -1 NAA. Subcultured once every two weeks, a large number of cambium cells were obtained in a short period of time.
  • Telomerase activity detection method Testing purpose: To test the telomerase activity of the cell clusters obtained under different treatment conditions, and compare the effects of different treatment conditions on telomerase activity.
  • telomerase Take 1g of vigorously growing ginseng cell clusters, add liquid nitrogen to grind them into uniform powders, and quickly transfer them to a 50mL centrifuge tube. Add 10mL pre-cooled lysis buffer (Tris-HCl, pH7.4, 50mM, MgCl 2 15mM, KCl 1M, EGTA 0.25M, DTT 0.1M, PMSF 12mM, PVP 7.5%, glycerol 50%, DEPC water constant volume) treatment , Shake and mix in ice bath for 5 min, 4°C, 16000 ⁇ g, centrifuge for 20 min, transfer the supernatant to a centrifuge tube, add 4% (v/v) PEG6000, mix in ice bath at 100 rpm for 30 min; 2ml aliquoted and 16000 ⁇ Centrifuge at g for 15 minutes to remove the supernatant; add 1/4 of the original amount of lysis buffer to the pellet, resuspend and lyse again. 4°C
  • telomerase Enzymatic reverse transcription of the above-mentioned extracted protein is performed to synthesize telomerase DNA fragments.
  • the enzymatic reaction solution was Tris-HCl (pH 8.3) 15uM, KCl 15 ⁇ M, EGTA 3 ⁇ M, MgCl 2 1.5 ⁇ M, BSA 0.01%, dNTP 0.015 ⁇ M, Triton x-100 0.01%, DTT 0.3 ⁇ M, primer 0.36 ⁇ M, protein extraction Several materials, 300 ⁇ L system.
  • the upstream primer GG CACTATCGACTACGCGATCGG, 21bp
  • the downstream primer ACX GCGCGGCTATACCCTATACCCTAAACC, 27bp.
  • TRAP method to measure telomerase activity The reaction system is 25 ⁇ L of rTaq enzyme, 2 ⁇ L of primers, 15 ⁇ L of enzymatic reaction solution, and 8 ⁇ L of ddH 2 O.
  • the PCR program was 95°C for 5min, 95°C for 30sec, 47°C for 30sec, 72°C for 40sec, 30 cycles.
  • the PCR products were collected by ethanol precipitation, and 12% polyacrylamide gel electrophoresis was performed. The electrophoresis bands were stained by silver staining, and the telomerase activity was judged according to the number of bands.
  • the test results show that: when treated with low concentration of ginsenosides, the telomerase activity does not change significantly; when the concentration of ginsenosides is too high, such as higher than 200 ⁇ M, the telomerase activity decreases instead.
  • the test results show that the optimum concentration is about 10 ⁇ M to 100 ⁇ M; when the saponin is a mixture of the two, it is preferably a mixture of saponin a and saponin b in a molar ratio of 2:5, and the most preferred concentrations are respectively 20 ⁇ M and 50 ⁇ M. .
  • the method for separating and culturing ginseng cambium cells can effectively separate the cambium cells of ginseng.
  • the cambium cells have unlimited division ability, rapid growth, strong resistance to stress, and provide a basis for super-large liquid culture, which can significantly Reduce production costs.
  • ultrasonic and hyperosmotic treatment can effectively cause specific tissue necrosis.
  • Cambium cells are effectively induced due to their strong resistance to stress, which effectively shortens the time of hypertonic treatment and reduces the probability of infection.
  • the concentration of hormones in the culture medium is controlled, so that while the cambium cells proliferate, somatic cells will not proliferate. Anti-browning treatment will reduce the browning of cells during the culture process.
  • Example 3 The effect of treatment and hypertonic treatment time on the induction rate of ginseng stem cells
  • the saponin compound is a mixture of saponin a and saponin b in a molar ratio of 2:5, and their concentrations are respectively 20 ⁇ M and 50 ⁇ M.
  • the factors and levels of the orthogonal test are shown in Table 3 below.
  • Stem cell induction rate refers to the induction of loose stem cells, no obvious callus cells, and no infectious bacteria.
  • the preferred treatment mode of the present invention is as follows: ultrasonic frequency is 20kHz, ultrasonic treatment time is 5min, ginsenoside a and b concentration are respectively 20 ⁇ M and 50 ⁇ M, and 1M sucrose hypertonic treatment time is 4h.

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Abstract

本发明涉及人参形成层干细胞的分离培养方法，所述方法包括用本发明的式I化合物处理含有人参形成层的组织的步骤。本发明还涉及根据本发明的方法获得的人参形成层干细胞及其在制备用于人参悬浮培养的产品中的用途。本发明中提供的人参形成层细胞分离及培养方法可以有效地分离人参的形成层细胞，形成层细胞具有无限分裂能力，生长快速，抗逆能力强，为超大体积的液体培养提供基础，可以显著减少生产成本。

Description

人参形成层干细胞分离培养方法 技术领域
本发明涉及人参形成层干细胞的分离培养方法、由此获得的人参形成层干细胞及其在制备用于人参悬浮培养的产品中的用途。
背景技术
人参是五加科人参属多年生双子叶植物，是我国传统的名贵中药材。目前人参已经越来越广泛地应用于药品、保健品以及化妆品领域。人参的来源包括野生来源、人工栽培和组织培养。由天然野生来源的人参资源有限，根本无法满足市场需求；人工栽培仍然是目前人参的主要来源，但人工栽培存在砍伐林木、占用耕地、生长周期长、易受病虫害影响、存在农药及重金属残留等诸多明显缺点。组织培养方式是利用愈伤组织或不定根进行培养获取，可以克服野生来源有限、人工栽培诸多缺陷等问题，不受外部环境约束且在最佳条件下能够获得植物主要成分，是提供人参资源的最有前景的优选方式。然而，愈伤组织和不定根存在分裂能力有限、易退化、抗逆能力弱等缺陷，并不适合进行大规模连续培养。
人们发现，植物干细胞是一类具有无限的自我更新和分化成多种细胞和组织类型能力的细胞。研究表明，植物干细胞嵌入在茎顶端分生组织、根尖分生组织和维管形成层分生组织中，它们能自我更新，能够分裂和产生新的细胞， 一部分子细胞进行分化，另一部分形成新的干细胞。植物干细胞的功能贯穿植物的整个生命过程，并为根、茎和叶等器官的形成和再生不断提供新的细胞来源。植物的维管系统形成层包含有多能的干细胞，为干细胞提供小生境，并维持干细胞的数量和功能。为解决脱分化细胞因遗传信息缺失导致长期培养不稳定及次生代谢产物产量低等问题，数十年来，科学家们致力于植物干细胞的分离及离体培养，并获得成功。因此，来自植物干细胞的组织培养成为提供珍贵药用植物来源的重要渠道。
与脱分化细胞相比，植物干细胞保留了母本全套的遗传信息，有利于长期稳定培养；植物干细胞具有小而多的液泡，抗剪切力强，并且可保持同步生长和高生长率；植物干细胞长期稳定合成多种类型的活性次生代谢产物。对于分离形成层细胞并用于组织培养，现有技术已有报道。例如，CN104920227A公开了一种利用形成层在分离培养基上培养的干细胞组培育苗技术；CN103509749A公开了一种人参形成层干细胞分离及培养方法，包括将东北人参消毒处理、将半圆形切片在培养基上培养、分离形成层干细胞并转移到转继代培养基上培养等步骤。然而，这些组织培养方法和育苗技术所获得的人参干细胞的分裂活性还不够高，其增殖能力还不能让人满意。
为了获取更高效的人参干细胞，提高组织培养的效率，克服已有方法存在的缺陷，现有技术迫切需要新的更高效的人参干细胞的分离培养方法。
发明内容
本发明一个目的在于提供一种可提高端粒酶的活性的人参形成层干细胞分离培养方法。
本发明人经过大量研究，通过筛选试验后意外地发现，用下述式I的皂苷化合物
Figure PCTCN2019082446-appb-000001
处理含有人参形成层的组织可以显著提高端粒酶的活性，其中R为-β-D-葡萄吡喃糖基(1-2)-β-D-葡萄吡喃糖(皂苷a)或-β-D-葡萄吡喃糖(皂苷b)。
基于上述发现，在本发明的第一个方面，提供了一种分离和/或培养人参形成层干细胞的方法，包括用式I化合物处理含有人参形成层的组织的步骤:
Figure PCTCN2019082446-appb-000002
其中R为-β-D-葡萄吡喃糖基(1-2)-β-D-葡萄吡喃糖或-β-D-葡萄吡喃糖。
优选地,在本发明的所述分离和/或培养人参形成层干细胞的方法中，其中所述含有人参形成层的组织是通过包括下述步骤的方法获得的：将含有形成层、韧皮部、皮质和表皮的组织从木质部剥离。
在本发明的一些优选的实施方案中，用式I化合物处理含有人参形成层的组织是通过将含有人参形成层的组织置于含有式I化合物的溶液中实现的。所述溶液优选为式I化合物的水溶液。
在本发明的一些实施方案中，用于处理人参形成层组织的式I化合物可以为 一种化合物，例如其中R为-β-D-葡萄吡喃糖基(1-2)-β-D-葡萄吡喃糖的式I化合物(皂苷a)，或者其中R为-β-D-葡萄吡喃糖的式I化合物(皂苷b)；也可以为皂苷a和b的混合物，即其中R为-β-D-葡萄吡喃糖基(1-2)-β-D-葡萄吡喃糖的式I化合物与其中R为-β-D-葡萄吡喃糖的式I化合物的任意比例的混合物。在一些特别优选的实施方案中，所述R为-β-D-葡萄吡喃糖基(1-2)-β-D-葡萄吡喃糖的式I化合物(皂苷a)与所述R为-β-D-葡萄吡喃糖的式I化合物(皂苷b)的摩尔比例为1:10～10:1，优选为1:5～5:1，更优选为1:3～3:1。特别优选地，皂苷a与皂苷b的摩尔比例为2:5。
在本发明的方法中，所述式I化合物的溶液的浓度为1μM-100μM，优选为10μM-100μM。在一个特别优选的实施方案中，式I化合物为皂苷a与b的摩尔比例2:5的混合物，且各自在溶液中的浓度分别为20μM和50μM。
在本发明的一些优选实施方案中，在用式I化合物处理后，将含有人参形成层的组织进行超声处理。所述超声处理的频率为5kHz至100kHz，优选20kHz至40kHz；处理时间为0.1min至10min。
在本发明的一些优选实施方案中，在式I化合物处理和/或超声处理后，采用蔗糖溶液进行处理。
在本发明的另一方面，提供了根据本发明的方法获得的人参形成层干细胞。
在本发明的又一方面，提供了根据本发明的方法获得的人参形成层干细胞在制备用于人参悬浮培养的产品中的用途。例如，可将本发明方法获得的人参形成层干细胞进行悬浮培养，以获得人参植株。
在本发明的一些优选的实施方案中，本发明的分离和/或培养人参形成层干细胞的方法，可以包括以下步骤：
(1)消毒：将洗净的人参药材用消毒剂消毒；
(2)防褐变处理：将消毒的人参材料用含有抗氧化剂的褐变抑制培养基处理；
(3)分离：将防褐变处理的人参材料置于含抗氧化剂的切削液中，将含有形成层、韧皮部、皮质和表皮的组织从木质部剥离；
(4)皂苷处理：将剥离的组织用本发明的皂苷化合物a和/或b处理，并任选地进行超声处理和蔗糖处理；
(5)培养：将皂苷处理后的组织进行组织培养，分离并获得形成层细胞。
在上述分离和/或培养人参形成层干细胞的方法中，步骤(1)的消毒步骤优选包括先采用75％的乙醇进行表面消毒，然后再采用次氯酸钠进行消毒。采用次氯酸钠的浓度优选为2％,并且优选消毒两次，第一次消毒时间优选为8min，第二次消毒时间优选为4min。
步骤(4)中进行超声处理的超声波频率优选为20kHz，超声波处理时间优选为5min；蔗糖处理的浓度优选为1M，蔗糖处理时间优选为4h；皂苷a处理时的浓度优选为20uM，皂苷b处理时的浓度优选为50uM，处理时间为5h。分离培养基优先含有3.0mg.L-1IBA与0.5mg.L-1KT的B5培养基。
步骤(5)中的培养优选包括先用MS培养基或B5培养基的初步培养，然后再进行继代培养；所述继代培养基优选为含有3.0mg.L-1的2,4-二氯苯氧乙酸(2,4-D)和6.0mg.L-1NAA的MS培养基。
本发明实施例提供的技术方案带来的有益效果是：本发明提供的人参形成层干细胞分离培养方法可以有效地分离人参的形成层细胞，形成层细胞具有无限分裂能力，生长快速，抗逆能力强，提高了端粒酶活性。而且通过超声波和 高渗透处理可以有效的使特异组织坏死，形成层细胞由于抗逆能力强而被有效诱导,且有效的缩短了高渗处理的时间，减少了染菌概率。同时控制培养基中激素浓度，使形成层细胞增殖的同时体细胞不会进行增殖。防褐变处理又会减少培养过程中细胞褐化的产生。
具体实施方式
实施例1分离与培养方法
⑴清洗与消毒：①将健康、表面无破损的人参储存根用自来水冲洗30分钟，然后置于超净工作台的灭菌烧瓶中用75％的乙醇表面消毒1分钟，然后用灭菌蒸馏水漂洗3～5次。②将上述消毒后的人参储存根用0.5％～10％的次氯酸钠水溶液消毒5～10分钟，弃去消毒液，将组织用灭菌蒸馏水漂洗3～5次。③再次使用0.5％～10％的次氯酸钠水溶液消毒3～5分钟，弃去消毒液，将处理后的人参储存根用灭菌蒸馏水漂洗3～5次。
⑵防褐变处理：将上述灭菌的人参储存根置于含有抗氧化剂的褐变抑制培养基(参见下表1)中，摇瓶培养大约30分钟～1小时。然后，使用灭菌滤纸从组织中除去湿气。
表1 褐变抑制培养基
Figure PCTCN2019082446-appb-000003
Figure PCTCN2019082446-appb-000004
(3)分离：将上述进行过灭菌和防褐变处理的人参储存根放入含抗氧化剂(抗坏血酸)的切削液(见下表2)的灭菌盘中，将含有形成层、韧皮部、皮质和表皮的组织轻轻地从木质部用无菌手术刀切开,剥离，将剥离的组织接种到WPM预培养基中培养30min。
表2 切削液组成
Figure PCTCN2019082446-appb-000005
(4)皂苷处理：将上述预培养后的剥离组织置于含有皂甙化合物(皂苷a与b的摩尔比例2:5的混合物，且各自在溶液中的浓度分别为20μM和50μM)的水溶液中处理5min；将皂苷处理后的剥离组织置于1M蔗糖水溶液中，先用频率为20kHz、功率为20W的超声波处理5min，然后低温处理4小时；之后将超声处理后的剥离组织放入0.05M蔗糖水溶液中处理5min，最后置于0.1M蔗糖水溶液中处理5min，用无菌吸管吸取溶液，去除蔗糖，使特异组织(韧皮部、木质部、髓部等)坏死，只诱导形成层(分生组织)。
(5)培养：将上述处理后获得的组织接种到含有30g/L蔗糖、0.7g/L琼脂糖、3mg.L -12,4-二氯苯氧乙酸、以及3.0mg.L -1IBA与0.5mg.L -1KT的B5培养基 中，20℃黑暗下培养。
培养两周后，将形成层明显增殖的外植体取出，分离形成层细胞并转移到继代培养基上培养，继代培养基为含有30g/L蔗糖与3.0mg.L -1的2,4-二氯苯氧乙酸和6.0mg.L -1NAA的MS培养基。每两周继代一次，短期内获得大量的形成层细胞。
实施例2端粒酶活性检测
端粒酶活性检测方法：检测目的：对不同处理条件下得到的细胞团分别进行端粒酶活性检测，比较不同处理条件对于端粒酶活性的影响。
检测步骤：
端粒酶检测步骤：
1、端粒酶的提取:取1g生长旺盛的人参细胞团，分别加液氮研磨成均匀的粉末，快速转移至50mL的离心管中。加10mL预冷的裂解液(Tris-HCl,pH7.4,50mM,MgCl 2 15mM,KCl 1M,EGTA 0.25M,DTT 0.1M,PMSF 12mM,PVP 7.5％,甘油50％，DEPC水定容)处理，冰浴振荡混匀5min，4℃，16000×g，离心20min，将上清转移至离心管中，加入4％(v/v)PEG6000，冰浴100rpm混匀30min；2ml分装后16000×g离心15min，去除上清；加原添加量1/4的裂解液至沉淀，重悬后再次裂解。4℃冰浴100rpm，30min；16000×g，2min，取上清，加RNase抑制剂(40U/ul)，-20℃待用；
2、端粒酶的酶促反应:将上述提取的蛋白进行酶促反应逆转录合成端粒酶DNA片段。酶促反应液为Tris-HCl(pH 8.3)15uM,KCl 15μM,EGTA 3μM,MgCl 21.5μM,BSA 0.01％,dNTP 0.015μM,Triton x-100 0.01％,DTT 0.3μM,引物0.36μM, 蛋白提取物若干，300μL体系。其中上游引物GG：CACTATCGACTACGCGATCGG，21bp，下游引物ACX：GCGCGGCTATACCCTATACCCTAAACC，27bp。
3、TRAP法测定端粒酶活力。反应体系为rTaq酶25μL，引物2μL，酶促反应液15μL，ddH 2O 8μL。PCR程序95℃5min，95℃30sec，47℃30sec，72℃40sec，30个循环。乙醇沉淀法收集PCR产物，进行12％的聚丙烯酰胺凝胶电泳，银染法对电泳条带染色，根据条带多寡判断端粒酶活性。
测试结果表明：用低浓度人参皂甙处理时，端粒酶活性变化不明显没有变化；当人参皂苷浓度过高时，例如高于200μM时，端粒酶活性反而降低。测试结果表明最适的浓度为约10μM至100μM；当所述皂苷为两者混合物时，其优选为皂苷a与皂苷b的摩尔比例为2:5的混合物，其最优选浓度分别为20μM和50μM。
本发明中提供的人参形成层细胞分离及培养方法可以有效地分离人参的形成层细胞，形成层细胞具有无限分裂能力，生长快速，抗逆能力强，为超大体积的液体培养提供基础，可以显著减少生产成本。而且通过超声波和高渗透处理可以有效的使特异组织坏死，形成层细胞由于抗逆能力强而被有效诱导,且有效的缩短了高渗处理的时间，减少了染菌概率。同时控制培养基中激素浓度，使形成层细胞增殖的同时体细胞不会进行增殖。防褐变处理又会减少培养过程中细胞褐化的产生。
实施例3处理和高渗处理时间对人参干细胞诱导率的影响试验
以超声波的频率、处理时间以及高渗处理时间为影响因素，对干细胞诱导率进行正交试验分析，以探索最佳的处理方式，提高人参干细胞的诱导效率，降低染菌风险，其中所用于处理的皂苷化合物为皂苷a和皂苷b的摩尔比例为 2:5的混合物，其浓度分别为20μM和50μM。正交试验的因素和水平如下表3所示。
表3 正交试验因素水平表
Figure PCTCN2019082446-appb-000006
试验结果如下表4所示。
表4 试验方案和试验数据分析表
Figure PCTCN2019082446-appb-000007
Figure PCTCN2019082446-appb-000008
注： *干细胞诱导率：指只诱导出松散干细胞，无明显愈伤组织细胞，无感染杂菌。
从试验数据可看出，适当增加超声频率和超声时间可有效地缩短高渗处理时间，而超声频率过大或超声时间太长反而会降低干细胞的诱导率。本发明优选的处理方式为：超声波频率20kHz，超声波处理时间5min，人参皂甙a、b浓度分别为20μM和50μM，1M蔗糖高渗处理时间4h。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种分离和/或培养人参形成层干细胞的方法,其特征在于，包括用式I化合物处理含有人参形成层的组织的步骤:
    Figure PCTCN2019082446-appb-100001
    其中：R为-β-D-葡萄吡喃糖基(1-2)-β-D-葡萄吡喃糖或-β-D-葡萄吡喃糖。
  2. 根据权利要求1所述的方法，其特征在于，所述含有人参形成层的组织是通过下式方法获得的：
    将含有形成层、韧皮部、皮质和表皮的组织从木质部剥离。
  3. 根据权利要求1或2所述的方法，其特征在于，用式I化合物处理含有人参形成层的组织是通过将含有人参形成层的组织置于含有式I化合物的溶液中实现的。
  4. 根据权利要求3所述的方法，其特征在于，所用式I化合物的溶液的浓度是10μM-100μM。
  5. 根据权利要求1至4中任一项所述的方法，其特征在于，所述式I化合物为其中R为-β-D-葡萄吡喃糖基(1-2)-β-D-葡萄吡喃糖的式I化合物(皂苷a)与其中R为-β-D-葡萄吡喃糖的式I化合物(皂苷b)的摩尔比例为2:5的混合物。
  6. 根据权利要求1至5中任一项所述的方法，其特征在于，还包括在式I化合物处理后将含有人参形成层的组织进行超声处理的步骤。
  7. 根据权利要求6所述的方法，其特征在于，所述超声处理的频率为20kHz至40kHz，处理时间为0.1min至10min。
  8. 根据权利要求1至7中任一项所述的方法，其特征在于，还包括在式I化合物处理和/或超声处理后采用蔗糖溶液处理的步骤。
  9. 一种产品，其包含根据权利要求1至8中任一项的方法获得的人参形成层干细胞。
  10. 权利要求8的产品在制备用于人参悬浮培养的产品中的用途。
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