Method for stably culturing taxus chinensis plant cell seeds

A plant cell and culture method technology, applied in the field of stable cultivation of cell seeds and yew plant cell seeds, can solve the problems of aging, death, browning, slow growth, low paclitaxel content, etc., to reduce reddening and browning, Increase the difficulty of operation and cultivate the effect of high success rate

Active Publication Date: 2014-12-10
GUANGDONG KELUN PHARMACEUTICAL CO LTD
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  • Description
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Problems solved by technology

[0003] At present, there are three main sources of paclitaxel raw materials: one is to obtain paclitaxel from the branches and leaves of Taxus chinensis, but due to the slow growth of Taxus chinensis, the production cycle is as long as 3 to 5 years, and the content of paclitaxel in the branches and leaves is low, resulting in the extraction of paclitaxel. high cost
The second is to semi-synthesize paclitaxel through precursors such as Baccatin Ⅲ and 10~DAB, but the semi-synthetic precursors required also come from planting branches and leaves, which are also limited by the source of raw materials for extraction, and still cannot meet the large demand for paclitaxel in clinical and experimental studies
[0004] China, the United States, Canada, South Korea, Japan and other countries have conducted a lot of research on the production of paclitaxel using yew plant cell culture technology, but the research hotspots of scientists are mainly concentrated on the high yield of paclitaxel. So far, no set of yew cell seeds has been proposed for long-term stable succession. The technical scheme of subculture, due to the great difference in seed quality and growth state, the growth is slow, red or brown substances will be continuously produced during the growth process, and the technology of subculture often occurs due to allergies, redness or senescence, death and browning, and fails Difficulties, resulting in poor stability of paclitaxel content between batches when plant cells are cultured to produce paclitaxel, which is a common technical problem faced by the large-scale cultivation of yew plant cells in the industrial production of paclitaxel. The technical research on the production of paclitaxel, so far, few research teams have entered the pilot test and industrialization stage, and there is no way to cultivate yew cell seeds with stable quality and growth state, which is one of the main reasons for the failure of cell culture

Method used

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  • Method for stably culturing taxus chinensis plant cell seeds
  • Method for stably culturing taxus chinensis plant cell seeds
  • Method for stably culturing taxus chinensis plant cell seeds

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Seed source and seed subculture conditions of Taxus chinensis plant cells

[0034] 1. Materials and culture conditions

[0035] The test material is the Chinese yew cell line induced and screened by the company's screening laboratory and subcultured. 5 As the basic medium, add 1mg / L NAA, 0.2mg / L 6~BA, 30g / L sucrose, 100mg / L Vc and 292.8mg / L L~glutamine as the subculture medium, and use 1000ml triangular The bottle is filled with 400ml culture volume, and the yew plant cell seeds and B are inserted in a ratio of 2:3. 5 culture medium, and then place the triangular flask on a shaker in a constant temperature room at 23-27°C, and carry out shaking culture at a shaker speed of 120rpm. After 9-14 days of subculture, the cells are used as seeds for orthogonal optimization experiments.

[0036] 2. Detection method of cell culture samples

[0037]① Determination of fresh weight of cells: filter the cell culture with a 120-mesh nylon sieve, rinse the cells with water three ti...

Embodiment 2

[0046] Effects of initial biomass, seed culture time and initial sugar content of culture medium on the growth of Taxus chinensis cells

[0047] Put 60ml of culture medium into a 250ml Erlenmeyer flask, use B 5 The medium is the basic formula, add 1mg / L NAA and 0.2mg / L 6~BA, according to the 4 factors and 3 levels in Table 1 and Table 2 9 (3 4 ) Orthogonal test table is prepared into the different medium formulas of 25g / L, 16g / L, 10g / L sucrose concentration, after inserting the cell seed of 40ml, the initial biomass in the culture is respectively 1.87g / L, 2.81g / L and 3.74g / L, then evenly place the shaker flask on a shaker in a constant temperature room at 25±2°C, shake the shaker at a speed of 120rpm, and take samples from the 7th, 9th, and 11th day of the subculture of the cell seeds. Detect the final cell biomass in the culture, the final pH of the culture solution, the final conductivity of the culture solution, and the final sugar content of the culture solution.

[00...

Embodiment 3

[0061] Initial biomass, seed subculture time and replacement B 5 Effect of medium volume on the growth of Taxus chinensis cells

[0062] 250ml Erlenmeyer flask with a volume of 100ml, add cell seeds and B according to the medium volume ratio mentioned in Table 3 5 Culture medium, after the cell seeds are inserted, the initial biomass in the culture is 2.14g / L, 4.29g / L and 6.43g / L respectively, and then the shaker flask is evenly placed on a shaker in a constant temperature room at 25±2°C , carry out shaking culture with 120rpm shaker speed, cell culture to the 10th day, 14th day, 18th day sampling, detect the final seed biomass in the culture and the final pH of the culture solution, the final conductivity of the culture solution, and the final sugar content of the culture solution.

[0063] From the orthogonal experiments in Table 3 and Table 4, the following conclusions can be drawn:

[0064] 1. Replace B during subculture 5 Effect of medium volume ratio on cell growth: r...

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Abstract

The invention discloses a method for stably culturing taxus chinensis plant cell seeds, and aims to provide a method which is capable of being used for stably culturing the taxus chinensis plant cell seeds; and for the method, the advantages of being economically feasible, simple and controllable in process, and low in cost are realized. The method comprises the steps of: (1) sampling and detecting subculturing seeds; (2) adjusting a final biomass of the seeds to 5-10g / L; (3) increasing the initial sugar degree of a culture solution to 15-25g / L; (4) increasing the initial electric conductivity of the culture solution to 2.5-3.5ms / cm; (5) controlling an initial biomass of the culture solution to be in the range of 2.5-3.5g / L and the initial pH (potential of Hydrogen) to be in the range of 5.0-6.0, stirring and carrying out seed subculturing; and (6) when the subcultured seeds in the step (5) meet the requirements that A, the final sugar degree of the culture solution is greater than 8g / L or B, the final electric conductivity of the culture solution is greater than 2.0ms / cm or C, the final pH of the culture solution is less than 6.0, starting next seed subculturing. The method belongs to the technical field of seed culturing.

Description

technical field [0001] The patent of the present invention relates to a method for stably cultivating cell seeds, in particular to a method for stably cultivating yew plant cell seeds, which belongs to the technical field of seed cultivation. Background technique [0002] Paclitaxel (Taxol) is a diterpene alkaloid isolated and extracted from the bark of the yew plant for the first time. expensive. [0003] At present, there are three main sources of paclitaxel raw materials: one is to obtain paclitaxel from the branches and leaves of Taxus chinensis, but due to the slow growth of Taxus chinensis, the production cycle is as long as 3 to 5 years, and the content of paclitaxel in the branches and leaves is low, resulting in the extraction of paclitaxel. high cost. The second is to semi-synthesize paclitaxel through precursors such as Baccatin Ⅲ and 10-DAB, but the semi-synthesized precursors also come from planting branches and leaves, which are also limited by the source of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
Inventor 李干雄侯云屏古志渊骆雪兰曾腾锋吴永艳肖华
Owner GUANGDONG KELUN PHARMACEUTICAL CO LTD
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