The Studies On Selection Of High Production Of Paclitaxel Cell Line Of Taxus Chinensis (Pilger) Rehd Var. Mairei

Paclitaxel has a better curative effect as new antitumor drug raw material, which has been found from plants, especially from yew branch and bark in the past. But, countries all over the world successively promulgated laws which prohibit deforestation endangered taxus chinensis fossil plants in the end of last century. Therefore, adopt the method of cell engineering production of taxol is one important way for plant synthesis of taxol. Cell lines with high yield of taxol are the basis of the cultivation and synthetic engineering. Therefore, this research focus on collecting the Taxus Chinensis var.mairei cell line from nature, and doing the research and evaluation on the performance of the taxol production, in order to obtain high yield cell line. The main results are as follows:1, Optimizing the cell culture system of Taxus Chinensis var.mairei; which studying on the callus from the stems resource and seeds resource. Cultivation system:the modified type B5medium as the basic culture medium+2.0%sucrose+2.0%phytagar+1.0mg/L6BA+1.0mg/L6mg/L NAA, PH5.8-6.0. Suspension culture:the initial cell density is5%-8%, replace the medium every7d, rotate speed is110r/min, dark culture under25±2℃.It showed that the growth rate of the cell from seeds resource is higher than stem resource, but in the process of suspension culture situation instead.2, To establish an efficient and accurate method to detect the content of paclitaxel in the cell suspension of Taxus Chinensis var.mairei by HPLC. Methods:Unitary C18column(150.0mm×4.6mm,5μm), was used with the mobile phase of acetonitrile and water at1.0mL/min, the UV detection wavelength at227nm, the column temperature30℃and the injection volume20μL. There was a systematic study on Taxus Chinensis var.mairei cell pretreatment method:with methanol, acetic acid (95:5) ultrasonic extraction30min, cell SAP and extraction solvent ratio is1:3.3, To inspect the changes of taxol content in different periods of the same strain of Taxus Chinensis var.mairei and different strains of the same period. By dynamic analysis test on the taxol content of Taxus Chinensis var.mairei, it can determine that the fifth successive transfer was the optimal harvesting time of suspended cell lines.4, To use the suspension cell culture method and testing method to evaluate and select the existing materials. Finally, it determined that there were four cell lines’taxol content higher than15mg/L:A26, B8, B35and B85.