CN103087974A

CN103087974A - Cultivation and separation method for taxus stem cells

Info

Publication number: CN103087974A
Application number: CN 201110330833
Authority: CN
Inventors: 黄涛
Original assignee: LUGANG BIOLOGICAL PHARMACEUTICAL CO Ltd
Current assignee: LUGANG BIOLOGICAL PHARMACEUTICAL CO Ltd
Priority date: 2011-10-27
Filing date: 2011-10-27
Publication date: 2013-05-08

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

The Ramulus et folium taxi cuspidatae stem cell is cultivated, separation method

Technical field

The present invention is specifically related to a kind of plant stem cell separation method, particularly a kind of Ramulus et folium taxi cuspidatae stem cell cultivation, separation method.

Background technology

The medicinal component taxol that extracts from the medicinal plant Ramulus et folium taxi cuspidatae has important using value and prospect for the treatment of kinds cancer.Because wild Ramulus et folium taxi cuspidatae is rare protective plant, law is forbidden felling.Adopting the way of a large amount of plantation Ramulus et folium taxi cuspidatae in production is that the separation and Extraction taxol is supplied raw materials.But because the growth of taxol cycle is long, occupy cultivated land; Just contain taxol in bark of Ramulus et folium taxi cuspidatae owing to only having again, and content is extremely low, could extracts 1 kilogram of taxol in about 10 tons of barks, so the cost of this traditional production technology is high.In addition, refine in the process of taxol, due to the chemical solvents that adds being arranged, cause refining the poisonous resistates that rear remainder can not be removed, exceed the dosage of appointment and affect the effect for the treatment of.

Prior art is paclitaxel produced next life by the cell of the suspension culture Ramulus et folium taxi cuspidatae of exsomatizing, and this method is not subjected to the restriction in growth of taxol season just can obtain taxol by large-scale production.Usually get stem or the blade of Ramulus et folium taxi cuspidatae and cultivate on defined medium for growing body outward, the process dedifferentiation stage is induced into the healing cell with splitting ability, and is further that this cell suspension culture is paclitaxel produced next life.

But present existing technology all adopts the histoorgan that has broken up for growing body outward when separating yew cell, by the cultivation (Dedifferentiation) in isolated culture stage, it is induced as having the healing cell of differentiation capability.But the essence of this cell is to derive from the somatocyte that has broken up, so splitting ability is limited, anti-adversity ability is not strong.In industrialization volume production process, clone often can occur and degenerate, a little less than splitting ability, be difficult to carry out the cultivation of ultra-large volume.

Summary of the invention

The purpose of this invention is to provide a kind of Ramulus et folium taxi cuspidatae stem cell cultivation, separation method, be used for solving the problems of the prior art, a kind of cultivation, separation method with unlimited splitting ability and the strong Ramulus et folium taxi cuspidatae stem cell that is in undifferentiated state of splitting ability is provided, this stem cell is due to the character of itself, can carry out the cultivation of ultra-large volume by the suspension culture method that exsomatizes, obtain a large amount of stem cells, thus can mass production of taxinol.

The present invention's technical scheme that adopts of dealing with problems is:

The Ramulus et folium taxi cuspidatae stem cell is cultivated, separation method, it is characterized in that: the spray of giving birth to then that with the diameter of taxus chinensis in northeast is at first 1 centimetre was disinfected 10 minutes through 0.1% mercuric chloride, then comprising perithelium, phloem and form layers from the spray cutting-out cuts into slices at the interior outer body of growing, be placed on the B5 medium that contains 0.2-2.0mg.L-1 picloram and 0.2-1.5mg.L-1 naphthylacetic acid, cultivate under 25 ℃ of dark; The outer body of growing with the form layers had significant proliferation after two weeks takes out, and separates the form layers stem cell and transfers on subculture medium and cultivate; Subculture medium is to contain on the B5 medium of 0.2-2.0mg.L-1 picloram and 0.2-1.5mg.L-1 naphthylacetic acid, and every two all subcultures once can obtain a large amount of stem cells in a short time.

Beneficial effect of the present invention: the stem cell of cultivating in the present invention is the cell that is in undifferentiated state, ability with unlimited division, if so adopt stem cell can avoid the somatic shortcoming of dedifferentiation in suspension culture, can obtain a large amount of stem cells, thereby production taxol that can be a large amount of satisfies patient's needs.

Description of drawings

Fig. 1 is Ramulus et folium taxi cuspidatae explant segment figure of the present invention;

Fig. 2 is that Ramulus et folium taxi cuspidatae form layers stem cell of the present invention is induced vegetative map;

Fig. 3 is Ramulus et folium taxi cuspidatae stem cell shoot proliferation figure of the present invention.

Embodiment

Embodiment 1: the cultivation of Ramulus et folium taxi cuspidatae stem cell of the present invention, separation method, at first the spray of giving birth to then that with the diameter of taxus chinensis in northeast is 1 centimetre was disinfected 10 minutes through 0.1% mercuric chloride, then downcut from spray and comprise perithelium, phloem and form layers in the interior outer body section (seeing accompanying drawing 1) of growing, be placed on the B5 medium that contains 0.5mg.L-1 picloram and 0.5mg.L-1 naphthylacetic acid, cultivate under 25 ℃ of dark.Because cambial stem cell division speed is fast, cause form layers stem cell group's nature and grow the division of body rest part outward and come (seeing accompanying drawing 2), it is fine and close that this stem cell is rolled into a ball quality, and the surface is more smooth smooth.The outer body of growing with the form layers had significant proliferation after two weeks takes out, and separates the form layers stem cell and transfers to and cultivate (seeing accompanying drawing 3) on subculture medium.Subculture medium is the B5 medium that contains 0.5mg.L-1 picloram and 0.5mg.L-1 naphthylacetic acid, and every two all subcultures once can obtain a large amount of stem cells in a short time.

Embodiment 2: the spray of giving birth to then that with the diameter of taxus chinensis in northeast is at first 1 centimetre was disinfected 10 minutes through 0.1% mercuric chloride, then comprising perithelium, phloem and form layers from the spray cutting-out cuts into slices at the interior outer body of growing, be placed on the B5 medium that contains 0.2mg.L-1 picloram and 1.5mg.L-1 naphthylacetic acid, cultivate under 25 ℃ of dark; The outer body of growing with the form layers had significant proliferation after two weeks takes out, and separates the form layers stem cell and transfers on subculture medium and cultivate; Subculture medium is to contain on the B5 medium of 0.2mg.L-1 picloram and 1.5mg.L-1 naphthylacetic acid, and every two all subcultures once can obtain a large amount of stem cells in a short time.

Embodiment 3: the spray of giving birth to then that with the diameter of taxus chinensis in northeast is at first 1 centimetre was disinfected 10 minutes through 0.1% mercuric chloride, then comprising perithelium, phloem and form layers from the spray cutting-out cuts into slices at the interior outer body of growing, be placed on the B5 medium that contains 2.0mg.L-1 picloram and 0.2mg.L-1 naphthylacetic acid, cultivate under 25 ℃ of dark; The outer body of growing with the form layers had significant proliferation after two weeks takes out, and separates the form layers stem cell and transfers on subculture medium and cultivate; Subculture medium is to contain on the B5 medium of 2.0mg.L-1 picloram and 0.2mg.L-1 naphthylacetic acid, and every two all subcultures once can obtain a large amount of stem cells in a short time.

Can consider in addition to separate by micrurgy the stem cell that shoot tip meristem, tip of a root vegetative point or form layers go out, then directly in vitro culture of isolated stem cell out.But the operation easier of this scheme is larger, and successful probability is very low.

For somatic shortcoming in existing Taxus chinesis var. mairei, attention to training of the present invention and the stem cell that separates Ramulus et folium taxi cuspidatae.Because stem cell is the cell that is in undifferentiated state, ability with unlimited division, and splitting ability is strong, if can avoid the somatic shortcoming of dedifferentiation so adopt stem cell to cultivate in suspension culture, and can obtain a large amount of stem cells in a short time, be conducive to a large amount of productions of taxol, satisfy patient's demand.

Claims

1. Ramulus et folium taxi cuspidatae stem cell cultivation, separation method, it is characterized in that: the spray of giving birth to then that with the diameter of taxus chinensis in northeast is at first 1 centimetre was disinfected 10 minutes through 0.1% mercuric chloride, then comprising perithelium, phloem and form layers from the spray cutting-out cuts into slices at the interior outer body of growing, be placed on the B5 medium that contains 0.2-2.0mg.L-1 picloram and 0.2-1.5mg.L-1 naphthylacetic acid, cultivate under 25 ℃ of dark; The outer body of growing with the form layers had significant proliferation after two weeks takes out, and separates the form layers stem cell and transfers on subculture medium and cultivate; Subculture medium is to contain on the B5 medium of 0.2-2.0mg.L-1 picloram and 0.2-1.5mg.L-1 naphthylacetic acid, and every two all subcultures once can obtain a large amount of stem cells in a short time.