Cultivation and separation method for taxus stem cells

A technology of stem cell culture and separation method, which is applied in the field of plant stem cell separation, and can solve problems such as difficult super-large-volume culture, weak resistance to stress, and cell line degradation.

Inactive Publication Date: 2013-05-08
鹭港生物药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the essence of this kind of cells is derived from differentiated somatic cells, so the ability to divide is limited and the ability to resist stress is not strong
In the process of industrial mass production, cell lines often degenerate and have weak division ability, making it difficult to culture in large volumes

Method used

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  • Cultivation and separation method for taxus stem cells
  • Cultivation and separation method for taxus stem cells
  • Cultivation and separation method for taxus stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1: the method for cultivating and separating the stem cells of Taxus chinensis of the present invention, at first the twigs of the year with a diameter of 1 cm were sterilized with 0.1% mercuric chloride for 10 minutes, and then cut off the twigs containing Sections of exogenous cells including skin, phloem and cambium (see appendix figure 1 ), placed on B5 medium containing 0.5 mg.L-1 picloram and 0.5 mg.L-1 naphthaleneacetic acid, and cultured at 25°C in the dark. Due to the rapid division of cambium stem cells, the cambium stem cell mass naturally separates from the rest of the exogenous body (see appendix figure 2 ), this stem cell mass has a dense texture and a smoother surface. Two weeks later, the explants whose cambium proliferated significantly were taken out, and the cambium stem cells were separated and transferred to the subculture medium for culture (see appendix image 3 ). The subculture medium is B5 medium containing 0.5mg.L-1 picloram and...

Embodiment 2

[0013] Embodiment 2: First, the diameter of 1 centimeter of Taxus chinensis is sterilized with 0.1% mercuric chloride for 10 minutes, and then the exogenous shoots including periderm, phloem and cambium are cut off from the shoots. Body slices were placed on B5 medium containing 0.2 mg.L-1 picloram and 1.5 mg.L-1 naphthalene acetic acid, and cultured at 25°C in the dark; after two weeks, the explants with obvious proliferative cambium were taken out and separated The cambium stem cells were transferred to the subculture medium for culture; the subculture medium was B5 medium containing 0.2 mg.L-1 picloram and 1.5 mg.L-1 naphthalene acetic acid, which was subcultured once every two weeks. A large number of stem cells can be obtained.

Embodiment 3

[0014] Embodiment 3: at first the twig of the year with a diameter of 1 cm was treated with 0.1% mercuric chloride for 10 minutes, and then the exogenous twigs including the periderm, phloem and cambium were cut off from the twig. Body slices were placed on B5 medium containing 2.0 mg.L-1 picloram and 0.2 mg.L-1 naphthaleneacetic acid, and cultured at 25°C in the dark; after two weeks, the explants with obvious proliferating cambium were taken out and separated The cambium stem cells were transferred to the subculture medium for culture; the subculture medium was B5 medium containing 2.0 mg.L-1 picloram and 0.2 mg.L-1 naphthalene acetic acid, which was subcultured once every two weeks. A large number of stem cells can be obtained.

[0015] In addition, micromanipulation can be considered to isolate shoot apex growth points, root apex growth points or stem cells that form layers, and then directly culture the isolated stem cells in vitro. However, the operation of this scheme ...

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Abstract

The invention relates to a cultivation and separation method for taxus stem cells. The method comprises the steps of sterilizing annual epicormic branches of taxus cuspidate, with the diameter of 1 centimeter, for 10 minutes by using 0.1% mercury chloride; cutting off explants including periderm, phloem and cambium from the epicormic branches, cutting into slices; putting the slices on a B5 medium containing 0.2-2.0 mg.L<-1> picloram and 0.2-1.5 mg.L<-1> naphthalene acetic acid, culturing in the dark at a temperature of 25 DEG C; taking the explants with obvious cambium proliferation out after two weeks; separating cambium stem cells and transferring the stem cells to a subculture medium for culture, wherein the subculture medium is a B5 medium containing 0.2-2.0 mg.L<-1> picloram and 0.2-1.5 mg.L<-1> naphthalene acetic acid; and sub-culturing one time every two weeks. A mass of stem cells can be obtained in a short time. The stem cells cultured by the method are cells in an undifferentiated state which have the capacity of infinite division and can obtain a mass of the stem cells, so that a great amount of paclitaxel can be produced, thereby meeting the needs of patients.

Description

technical field [0001] The invention specifically relates to a plant stem cell separation method, in particular to a yew stem cell culture and separation method. Background technique [0002] The medicinal ingredient paclitaxel extracted from the medicinal plant yew has important application value and prospects for the treatment of various cancers. Since wild yew is a rare and protected plant, logging is prohibited by law. In production, a large number of yews are planted to provide raw materials for the separation and extraction of paclitaxel. However, due to the long growth cycle of yew and the occupation of cultivated land; and because only the bark of yew contains paclitaxel, and the content is extremely low, only 1 kg of paclitaxel can be extracted from about 10 tons of bark, so the cost of this traditional production technology is extremely high. high. In addition, in the process of extracting paclitaxel, due to the addition of chemical solvents, there are toxic res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N5/02
Inventor 黄涛
Owner 鹭港生物药业有限公司
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