摘要
喜树碱是一种色氨酸-萜类生物碱,具有抗癌、清热、杀虫功能,是继紫杉醇之后的第二个从植物提取的衍生抗癌药物,具有积极的开发前景。但目前的研究情况和国内外的研究趋势来看,喜树碱及类似物主要是从喜树中分离获得,其产量必受到喜树生长发育和环境因素的影响,因此通过高等植物细胞培养生产次生代谢物质喜树碱将是一个很好的选择。本文以喜树(Camptotheca acuminata Decsne.CPA)愈伤组织为材料,比较系统的研究了悬浮培养条件下光照、激素以及多种理化因子喜树细胞悬浮培养生长、生理生化特性的影响,使喜树细胞培养生长速度有明显提高,为进一步研究积累了资料。
1.植物生长调节物质的影响
不同种类、不同浓度的激素及其组合对细胞生长效应不相同。NAA浓度为0.5mg/1时最有利于细胞生长:在NAA与KT组合中,以NAA0.5mg/1+KT0.1mg/1的效果最佳,细胞生长速度最快。
2.生理生化特性的研究
在光照条件下,细胞生长周期为30天;而在黑暗条件下,生长周期为27天,细胞增殖曲线均呈“S”型,可大致分为延迟期、指数生长期和静止期:在两种光照条件下,其培养液的pH先下降后都呈上升趋势:培养细胞中可溶性蛋白质含量变化曲线相似,在12天和21天出现两个高峰,黑暗条件下的含量低于光照条件下的含量;培养细胞的过氧化物酶活性变化曲线相似,均出现两个高峰,第一个高峰均出现在第3天,光照条件下的第二个高峰出现在15天,而黑暗条件下出现在18天;培养细胞超氧化物歧化酶活性变化曲线相似,均出现两个峰值,光照条件下的第一个高峰出现在18天,而黑暗条件下出现在21天,两种情况下的第二个高峰均出现在第27天。
d\硕士学位论文
啥啼动妒M从几rsul0JS
3.不同理化因子的影响
在喜树细胞悬浮培养中,BS培养基和仆h:培养基较有利于细胞的生长:
适宜的接种量为3-蛇鲜重八00ml培养基:随着蔗糖和葡萄糖浓度的升高,细
胞生长速度呈上升趋势,且在浓度为3%~4池八时最有利于细胞生长;添
加适宜的琼脂糖能促进细胞生长:NO.;和N比“的比例不同,细胞生长效应也不同。
最后,对作为药用资源的植物细胞悬浮培养进行了总结,对细胞悬浮培养
的开发,出现的问题以及对这些问题的探索进行了讨论。同时对今后的研究方
向也作了展望。
Camptothecin is a kind of anticancer medicine having good prospect of exploitation, CPT and analogue are get from CPA. at the moment, output of CPT was effected by the growth and environment of CPA. so production of CPT by cell culture may be a promising choice .In the paper ,based on callus of Camptotheca acuminata Decsne., the influences of light, hormones and several factors on the growth, physiological, biochemical characters of suspension culture of Camptotheca acuminata Decsne. Were systematically studied, cell growth speed was obviously improved, it would give references for future studies.
1.Effects of hormones
Different kinds, different concentration and combinations of hormones had different effects on cell growth. 0.5mg/l NAA was optimal for the cell growth ; Among the combination of NAA and KT ,0.5mg/lNAA+0.1mg/lKT was optimal for the cell growth.
2.Study of physiological and biochemical characters
When CPA was culture in the light and in the dark ,both of their cell growth curves were like "S", but their growth circles were about 30 and 27 days respectively ,It could be divided into lag phase, exponential growth phase and stationary phase .The cell grew more rapidly in the white light than in the dark ;The PH values of culture mediums lowered and then showed up gradually ;Two peaks of soluble protein contents and peroxides and superoxide dismutase activities of culture cell were showed up ,but they were on the different days ;the soluble protein content was higher in the white light.
3.Effects of different physical and chemical factors
In the suspension culture ,B5medium and White medium were optimal for cell growth ;The inoculums quantity of 3-4g @
FW/1001 was better for the cell
growth ;The growth rate increased with the increasing concentrations of Sucrose and Glucose ;The medium with certain concentration of Agarose could increase the growth rate ; Effect of cultural cell growth was different with different proportion of NO3-and NH4+.
Finally, the conditions in cell suspension culture of some medicine plants were analyzed ;research and development -, problems and resolutions were discussed; prospects of future researches were also given.
引文
1. Wall M E Wani,et al.The isolation and structure of Camptothecin a novel alkaloidal leukemia and tumor inhibitor from Camptothecin acuminata J.AM.chem..Soc.,1966, 88:3888-3890.
2.聂纯。天然药物抗癌有效成分研究进展。中草药。1999,30:65-69。
3. C.G.Moertel.A.J.Schutt,R,J,Reitemeir. R.G.Hahn,cancer chemither. Reptl,Pt. 1,56-95.
4. J.A.Gottlieb,J.K.Luce. Cancer Chemother. Rept. 1.Pt. 1.56.103.
5. A.G.Schultz,Chem Rev. 1973,73:385.
6. M.Shamma. V. StGeorgiev, J.Pharm. Sci.6974,63:163.
7. Hsiang Y.H.R Hertzberg,et al.Camptothecin induces protein-linked DNA Topoismerase,J. Biol.Chem., 1985,260:14873-14878.
8. Kuniaki Sakato. Masanaru Misawa. Effects of chemical and physical on growth of Camptotheca acuminata cell culture Agr. Bio. Chem., 1974,38(3):491-497.
9. Perdue J.RE.Smith,et al.Camptotheca acuminata Decsne(Nyssaceae),Source of Camptothecin,an antileukemic allcaloid US,Tech,Bull,NO. 1415.
10. Sakato K,Tanaka H.,et al. Isolation and identification of Camptothecin from cell of Camptotheca acuminata suspention culture,Agr. Biol.Chem.,38:217-218.
11. Arjon J.Van Hengel,M P Harkes,et al.,Characterzation of callus formation and Camptothecin prodution by cell lines of CPT.plant cell,Tissure and Organ Culture, 1992,28:11-18.
12. A.K Jain,CL Nessler clonal propagtiom of CPT through shoot bud culture. Plant Cell.T issure and Organ Culture, 1996,44:229-233.
13.董锡裕等.喜树碱类抗癌药——又一世界性热门课题中草药,1996,27(4):243。
14.聂纯.天然药物抗癌有效成分研究进展。中草药。1999,30(1):65-67。
15.郭新娜,赵彼得等.羟基喜树碱膀胱灌注合并高频透热治疗膀胱癌近期疗效观察。军医进修2学院院报,1994,15(3):182.
16.林国础,陈延昌等。生长转化羟基喜树碱治疗口腔颌面涎腺腺源上皮癌的临床观察。华医口腔医学杂志。1994,12(1):51-52。
17. Mc Donald C. Camptotheca acuminata:China's 'Tree of joy'Offers Hope in the U.S.(Draft report), 1997.
18. Priel E,Yosef O et al. Detection of a novel DNA topoisomerase Ⅰ activity associated with human immunodeficiency virus (HIV) and other retrovirus particles International Conference on Aids, 1989,5,586.
19. James J S. Topotecan,CPT-11 (irinotecan), Camptothecin, and other topoisomerase Ⅰ inhibitors,AIDS Treatment.
20.焦敬荣,李习舜,喜树碱二甲基砜溶液治疗银屑病的疗效观察。中华医学杂志。11974,54(4):208-210。
21.郭茜茹,李舒茵等。喜树碱抑制滤过光泡瘢痕化的临床研究,眼科研究。1995,13(4):262-264。
22. Buta J C,Joseph FW. Camptothecin,aselective Plant growth regulator. J, Agric. Food chem..1976,24(5): 1085-1086.
23. Wall M E et al. Structure activity relationship of plant antiumor agents related to camptothecin and quassinoids 1980:Intrnational Research congress on Medicinal Plant Research.
24. Wani M C, Ronman P E et al. Sythensis and biological activity of camptothecin analogues. J.Med.Chem. 1980,23(5):554-560.
25.郭舜民。喜树碱类似物的研究进展。国外医学,药学分册,1994,21(5):270-273。
26.上海药物研究所药理研究室。喜树抗癌作用的实验研究。中华医学杂志,1975,55,274。
27. Lopez-Meyer M,Nessler CL,et al. Spite of accumlation of the antiturmor alkzliod CPT in CPA.Plant Med, 1994,60:558-560.
28. Vineent R.Lopez-Meyer M,et al.Sustained havest of CPT from the leaves of CPA J.Nat,Pro. 1997,60:618-619.
29. Gamborg OL Miller RA&Ojima V. Natrient requairements of suspention Culture of soybean root cels.EPT. Cell Res, 1968,50:151-158
30. Mura shige T&Skoog F.A revised medium for rapid groom and bioassays with tobacco tissue culture.Physiol.Plant, 1962,15:473-497.
31. Georage L.Bapat VA al Rao PS in vitro multiplieation of sesame through tissue culture. Annals of Bot.60:17-21.
32.赵洁。光质和温度与2、4-D组合对CPT叶片脱分化的效应。华中师范大学学报(自然科学版),1992,28(4):482-486。
33.陕西医药总公司情报站。医药动态,1997,(6):23。
34. Erdelmeier CAJ.Erdelemier,Kinghorn AP&Farnsworth NR Use of over Pressure layre chromatography(OPLC) for the seperation of Nature produets with antineoplastic activity,.J.Natural Prod,49:1133-1137.
35.林启寿。中草药成分化学。北京:科学出版社,1977。719-720。
36.上海药物研究所植化室喜树组。喜树果实中抗癌有效成分CPT与10羟基喜树碱。中草药通讯,1975,(5):17-19。
37.张红,王永耀等,大孔吸附树脂提取CPT的眼。离子交换与吸附。1995,12(2):145-150。
38.张正秀。抗癌药物CPT生产工艺研究。中国野生植物资源。1993,(1):1-4。
39.上海市卫生局。上海市药品标准(下册)。上海科学技术出版社,1980,274。
40.姚培潢。HPLC测定CPT的含量。中国药学杂志。1994,29(3):165-166。
41.张清明,王鹏等。三波长分光光度计法测量喜树碱多相脂质体中CPT的含量。沈阳药学院学报,1991,8(1):48-50。
42.冯建灿。CPA与CPT开发利用进展。林业科学,2000,9(5):36。
43.刘春朝,王玉春,欧阳藩。植物组织培养生产有用次生代谢物的研究进展,生物技术通报。1997,5:62。
44.徐礼,沙世炎,中草药成分分析法(下册),1984,人民卫生出版社[M],北京。
45.张志良,植物生理学实验指导,高等教育出版社[M],北京。1990
46. Shubal K.,AR.Menta,Changes in enzyme activity and isoperexidases in haploid Tobacco callus during organnogenesis, 1982,Plant Sci Lett[J], 24(1):67-77
47.郑光植,梁峥,药用植物组织培养的研究,植物学报[J],1976,18(2):163-169
48.候嵩,李新明,尚延兰等,九连小蘖悬浮细胞培养生理生化特性的研究,武汉植物学研究[J],1988,6(4):391-194
49.周立刚,郑光植,人参细胞大量培养的研究,天然产物研究与开发[J],1991,3(1):
50.杨和平,程井辰,马唐胚性与非胚性愈伤组织生理差异的初步研究,植物生理学通讯[J],1991,27(5):337-340
51.王亚馥,王仑山,陆卫等,枸杞组织培养过程中过氧化物酶和可溶性蛋白质的变化,实验生物学报[J],1989,22(1):1-4
52.张静兰,1982,放线菌D和环己亚胺对激素诱导绿豆叶脱分化及其核酸、蛋白质代谢的作用,植物学报[J],24(5):433-439
53. De Capite. Action of light and Temperature on Growth of plant Tissue Culture in Vitro, American Journal of Botany [J], 1895,4:869-87
54. Lee, E.H.and L,H.Bennett, superoxide dismutase A possible protective enzyme against ozone injury in snap beans (phasealus vulgarisL).Plant Physiol., 1982,69:1444-1449.
55.曹孜义,刘国民.实用植物组织培养技术教程[M].第一版.甘肃科学技术出版社,
1996,21-31。
56.高向阳,叶国洪,等.几种营养物质对烟草细胞生长和CoQl0含量的影响.生物工程学报,2001,1(16):78-81.
57.甘焕远,郑光植,等.云南红豆杉细胞的悬浮培养.植物生理学报,1997,23(1):43-46.
58. Mizukami H.Konoshima M,Tabata M.Effect of nutritional factors on Shikonin derivative formation in Lithosperman callus culture. Photochemistry, 1977,18:1183-1186.
59.郑光植,何静波,王世林。三分三愈伤组织的悬浮培养。云南植物研究。1982,4(2):203-206。
60.甘焕远,郑光植,彭丽萍。云南红豆杉细胞的悬浮培养。植物生理学报,1997,23(1):43-46。
61. Kurz WGM,The several factors which influence secondary metabolites biosynthesis and biotransformation on plant cell cultures. Critical Rev Biotech, 1985,2:105-121.
62.周立刚,郑光植。影响植物细胞发酵培养的诸因素。国外医学。植物学分册。1991,6 (3):112-114。
63. Keller. F.Transport of stachyose and sucrose by vacuoles of Japanese artichoke tubers. Plant Physics. 1992,98:442-445.
64.许建峰,刘传斌,方晓舟。高山红景天细胞悬浮培养中pH值对红景天甙胞外释放及细胞活性的影响,植物学报。1997,39(1):1022-1029。