【作者】 潘学武；

【导师】 吕应堂；

【作者基本信息】 武汉大学 ， 发育生物学， 2004， 博士

【摘要】 喜树碱（Campothecin）,一种色氨酸衍生的水不溶性萜烯类吲哚生物碱,它首先是从我国特有树种.喜树（Campototheca acuminata Decaisne）茎的提取物中分离得到的。喜树碱类药物通过抑制肿瘤细胞内拓扑异构酶I的活性而具抗癌效应,这一作用机理为肿瘤细胞的治疗开辟了又一新的途径,拓扑异构酶I抑制剂喜树碱类药物已成为高选择性抗肿瘤药物研究的一个主攻方向,成为又一新的世界性热门研究课题。喜树碱的两种衍生物,irinothecan和topotecan已于1996年得到美国食品与药品监督管理局（FDA）的批准用于临床,分别治疗乳腺癌和结肠癌。同时,9-硝基喜树碱已完成针对晚期胰腺癌的Ⅲ期临床研究,正向美国FDA提出新药申请,其它的如10-羟基喜树碱,exatecan,lurtotecan等正处于各级临床实验。 随着喜树碱类药物临床研究和开发的不断升温,世界市场上喜树碱类药物的需求不断加大,可持续获取喜树碱及其类似物必定是喜树碱类药物研究的重点之一。单纯靠从植株中提取喜树碱已不能满足市场的需要,利用植物细胞培养法生产喜树碱是一有益的新探索。在这方面,虽然国外的一些研究小组率先进行了研究,但相比其它植物如紫草,人参,黄连,长春花,红豆杉等而言,喜树细胞培养还仅仅处于初始阶段,因此,深入详细地研究喜树细胞悬浮培养生产喜树碱类药物无疑将具有极其重要的应用价值和理论意义,同时为以后生物反应器放大培养提供试验参数,本研究的主要结果如下: 1.对不同外植体愈伤组织的诱导表明,以MS为基本培养基,0.2-2.0mgl^-1的2.4-D为激素,25℃暗培养,可以在2-3周内获得高达100%的喜树不同外植体来源的愈伤组织发生率,愈伤组织在附加0.5mgl^-12.4-D的MS培养基中继代生长良好,胚轴来源的愈伤组织喜树碱产量最高,达到0.034mg flask^-1。 胚轴来源的细胞悬浮培养体系的建立显示,下胚轴诱导的愈伤组织在MS培养基中细胞生长良好,最佳激素组合为0.2mgl^-12.4-D+0.5mgl^-1NAA+0.5mgl^-16-BA,细胞继代周期以21d左右为宜,悬浮细胞适合在22℃-26℃,120rpm下生长。悬浮培养细胞在第20天生物量达到最大,为27.44gl^-1DW。在细胞对数生长期内,喜树碱积累与细胞生长呈线形正相关性,第20天喜树碱含量达到最大值0.009319%,喜树碱产量为2.56mgl^-1。 2.通过改变MS培养基中氨态氮与硝态氮的不同摩尔比例以及初始总氮源来提高喜树悬浮培养细胞的生长和喜树碱的生物合成。在MS培养基中,70mM的硝酸盐而去掉铵盐,悬浮培养细胞的生长最快;而当氨态氮与硝态氮的摩尔比为5:1,总氮源为40mM时,悬浮培养细胞喜树碱的生物合成能力最强。通过改变氮源的两步更多还原

【Abstract】 Camptothecin, a water-insoluble tryptophan- and monoterpene-derived indole alkaloid, was initially isolated from the stems of Camptotheca acuminata Decaisne （Nyssaceae）, an unique Chinese tree. Camptothecin has been of real pharmaceutical interest since it was found that camptothecin exhibited anti-cancer activity by blocking the eukaryotic topoisomerase I. Until now, camptothecin and its analogs are the sole secondary metabolites known to inhibit topoisomerase I and thus appears to be the prototype of a new class of high selective cancer chemotherapeutic agents, therefore, camptothecin（s） have been one of the most up-to-date objectives all over the world. Two camptothecin derivatives, topotecan and irinotecan, were approved by the U.S. Food and Drug Administration （FDA） in 1996 for the treatment of ovarian and colorectal cancers and several other analogs such as 10-hydroxycamptothecin, rubitecan and lurtotecan are currently under clinical development at various stages. Now, the finish of phase ш clinical trial on pancreatic tumor of 9-nitrocamptothecin is leading to its waiting for market admittance from FDA.With more and more attention paid to camptothecin and its analogs under clinical and marketable development, one focus is to produce them with other alternative ways. Limits to the supply of camptothecin（s） from C. acuminata and other plants and the rapid increasing pharmaceutical market and economic value of these alkaloids have prompted efforts to produce camptothecin and its analogs by plant cell cultures as a potential tool, which have attained great succeed in producing useful secondary metabolites in other medicinal plants, but until now, only a few studies addressing this possibility have been carried out. Compared with other species such as Lithospermum erythrorhiznm, Panax ginseng, Coptis japanica, Cantharanthus rosues, Taxus sp., cell cultures of C. acuminata is now only beginning. Therefore, it’s no doubt that studies on cell suspension cultures of C. acumunata to produce theses alkaloids in details will be of significant application and theoretics values. In addition, these will be a prelude to produce them by scale-up. The main results of this paper are as follows.1. Callus induction rate of various explants could be as high as 100% within 2-3 weeks using MS basic medium with 0.2-2.0 mg l^-1 2.4-D without light at 25℃. The cells were developed well in MS medium with 0.5 mg l^-1 2.4-D and the growth rates and the camptothecin yields of cell lines initiated from hypocotyls of C. acuminata seeds attained0.034 mg flask"1, the highest among all cell lines.Callus initiated from hypocotyls grew well in MS liquid medium supplied with 0.2 mg I’1 2.4-D+0.5 mg I’1 NAA+0.5 mg I’1 6-BA, proper subculture time was about 21 days and suspension cells developed well within 22 °C and 26 °C at 120 rpm. At day 20 the maximum dry weight, camptothecin content and yield were 27.44 g I"1, 0.009319% and 2.56 mg I"1 respectively. Camptothecin content was positively linear correlative to cell growth during growth stage until day 20.2. Effects of the molar ratio of NO3" fN$U+ and the total amount of initial nitrogen on the growth rate and camptothecin accumulation were investigated in Murashige & Skoog medium. Increasing nitrate to 70 mM and without ammonium achieved the highest biomass and with initial nitrogen concentration of 40 mM at a NH/ / NO3’ molar ratio of 5:1, maximum camptothecin yield was achieved. A two-stage flask culture system was established by altering nitrogen source supply and the results showed that cell dry weight, camptothecin content and yield in suspension culture cells by such a process increased by 29.68%, 282.55% and 349.70%, respectively when compared with those of control, reaching up to 35.59 g I"1, 0.03565%, and 11.51 mg I"1, respectively.3. Eight microelements （I", BO33", MoO42’, Co2+, Cu2+, Mn2+, Fe2+, Zn2+） have marked effects on the biosynthesis of camptothecin and the growth of suspension cultures of C. acuminata. The increase of I" to 25 uM, Cu2+ to 1 uM, Co2+ to 2 uM and MOO42" to 10 ^M in MS medium resulted in 1.66,2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I’ （25 um）, Cu2+ （1 um）, Co2+ （2 um） and MOO42" （10 um） improved cell dry weight, camptothecin content, and camptothecin yield to 30.56 g I"1, 0.0299%, and 9.15 mg I"1, respectively, which were 20.2%, 208.9% and 273.8% increment respectively when compared with those of control.4. When suspension cells of C. acuminata was supplemented with a rare earth element, cerium at day 0, Ce3+ below 0.075 mM had positive influences on cell growth and Ce3+ 0.05 mM gave the highest biomass, 33.03 g I"1 dry wt, which was 1.25-folds of that of the control. Cerium could make the camptothecin release and the release rate was positively linear correlative to Ce3+ dosage. Ce3+ at 0.01 mM was the most favorable to camptothecin biosythesis and peculiarly effective when added at day 10, the early stage of cell logarithmic growth stage. About 32.31% of total camptothecin was secreted into the medium and total camptothecin yields attained 13.02 mg I"1, which was 6.39-, 1.63-, 1.36-and 1.11-folds of that of the control, that of the treated with Ce3+ at day 0, that at day 5, and that at day 15, respectively.5. Enhancement of camptothecin production and excretion by two biotic elicitors,fungal （Aspergillus niger） elicitor and chitosan elicitor, were investigated respectively in C. acuminata cell suspension cultures. When, on the 20th day of growth, Aspergillus niger elicitor was added to cells, the maximum intercellular and extracellular camptothecin yields were achieved at 60 mg I"1 and 80 mg I"1 of the elicitors after 48hr elicitation, respectively. However, the highest camptothecin yields was 7.86 mg I"1 by adding with elicitor at 60 mg I"1 after 48hr elicitation, 2.84 times of that of the unelicited cells.Similarly, on the 20th day of growth, with chitosan at 80 mg I"1 added to cells, the maximum intercellular and extracellular camptothecin yields were achieved after 48hr elicitation and the total was 11.18 mg I"1,4.52 times of that of the untreated cells. Subsequent experiments shown that camptothecin yields reached the highest after 36 hr elicitation with chitosan at 80 mg I"1, and the total was 15.21 mg I"1 （the intercellular and extracellular was 10.31 and 4.91 mg I"1, respectively）, which achieved 5.92 times higher than that of the control.6. Studies were conducted on the cultivation of C. acuminata cell suspension in two-phase systems for the release of intercellular camptothecin. It was established that during cultivation with hexadecane as a second phase, the maximum camptothecin was 5.72 mg I’1 after 20 days of cultivation by adding hexadecane with 20% （v/v） on the first cultivation day, which was 2.06-folds of that of the control.While two-phase culture cells were elicited by 0.1 mM CeCl3 on the 1st day, the enhancement effects of hexadecane to secondary metabolites acumination and release was rather obvious and the highest yields were achieved with 10% hexadecane after 20 days of cultivation, reaching up to 18.77 mg I"1, which was 6.5 time of that of the untreated cell and 66% higher than that of the elicited cells without hexadecane.The most significant results were accomplished with two-stage culture by altering nitrogen sources supply in two-phase cultured cells elicited by 0.1 mM CeCb on the 1st day, and after 28 days of cultivation, the total alkaloid was 48.83 mg I"1 （the intercellular and extracellular was 19.85 and 18.89 mg I"1, respectively）, which achieved 18.15, 3.14 and 2.28 times of that of the cell by one-step culture （the control）, the cells by two-step cultures with hexadecane and the cell by two-step cultures with elicitation, respectively.更多还原