【作者】 滕建北；

【导师】 万德光；

【作者基本信息】 成都中医药大学 ， 中药学， 2009， 博士

【摘要】 中药石斛(Caulis Dendrobii)为来源于兰科石斛属多种植物,长期以来供需矛盾较为突出。石斛的来源植物生长缓慢,尤以细茎类的石斛如铁皮石斛(Dendrobium candidum Wall Ex Lindl)、美花石斛(Dendrobium loddigesii Rolfe)等为甚；由于其生长缓慢、产量低,导致野生资源日渐稀少、人工栽培效益低而推广困难。进行石斛的生长机理研究、促生长技术研究和药材质量形成研究是解决上述问题的根本途径。课题首先进行了本草考证,阐述了石斛药材的药用历史,明确了古今药名的一致性；品种考证探讨了古今对应的石斛品种；阐述了石斛功效的历史沿革；考证了本草记载并结合现代文献明确了石斛的道地产区的多样性。课题对石斛道地.产区之一的广西进行了石斛资源和生境调查。通过铁皮石斛、美花石斛茎尖细胞分裂节律研究,首次发现其昼夜均可进行细胞分裂,并发现了细胞分裂高峰期；其茎尖细胞分裂指数极低,是导致石斛生长缓慢的主要原因。实验对不同实验组(喷施6-苄基氨基嘌呤(6-BA)；特殊光照处理；对照组)铁皮石斛、美花石斛茎尖分生组织分区研究。结果揭示了石斛茎尖分生组织细胞的分层和分区情况；探讨了人工干预措施(喷施6-BA；特殊光照处理)对其的影响。进行铁皮石斛和美花石斛茎尖细胞死亡性质的研究。通过显微、超微的细胞形态观察,末端脱氧核苷酸转移酶介导的生物素化的dUTP切口末端标记(TUNEL法)以及吖啶橙荧光法研究发现,其茎尖细胞死亡性质属于细胞坏死。认为TUNEL法较适用于石斛茎尖细胞死亡性质的研究。进行了铁皮石斛和美花石斛茎尖细胞ATP酶的显微定位研究。实验明确了ATP酶活性显著的部位,揭示了不同阶段、不同实验处理的茎尖细胞ATP酶的活性差异。进行了铁皮石斛和美花石斛的抗逆性研究。通过测定不同时期、不同实验处理的石斛过氧化物酶(POD)、过氧化氢酶(CAT)的活性和丙二醛(MDA)的含量,研究其抗逆性及人工干预措施对其抗逆性的影响。实验发现POD、CAT活性,铁皮石斛和美花石斛均以对照组为最低；MDA则以对照组为最高。铁皮石斛激素组、物理组的POD、CAT活性,以及对照组CAT活性,均以5月最高,10月最低。美花石斛激素组、物理组的POD活性无月份差异,对照组则由5月、10、次年1月递增。首次研究了不同时期、不同实验处理的铁皮石斛与美花石斛叶片蔗糖合成酶活性,探讨石斛生长过程的生理变化以及人工干预措施对其的影响。实验发现5月份的蔗糖合成酶活性,两种石斛均为对照组高于激素组或物理组,10月、次年1月则相反,对照组低于激素组或物理组。以多糖含量为指标,研究了不同时期、不同实验处理的铁皮石斛与美花石斛药材的质量,探讨人工干预措施对其质量的影响；以浓硫酸蒽酮法首次用于石斛多糖含量的测定。生长时间为3、6、12个月的多糖含量比较表明,铁皮石斛和美花石斛各组的多糖含量均呈递增趋势；生长3-12个月时,铁皮石斛和美花石斛均为激素组的多糖含量最低；美花石斛生长18个月,激素组>物理组>对照组。更多还原

【Abstract】 Caulis Dendrobii, traditional Chinese medicinal material, which origin is plants of Dendrobium, has been in a condition of much demand but poor supply for long-term. Lack of Caulis Dendrobii is principally attributed to retarded growth of the plants, especially of the tiny stem species, such as Dendrobium candidum Wall Ex Lindl and Dendrobium loddigesii Rolfe. To study on the growing mechanism and growth-promoting technique applying is considered optimum key to the problem.History used as medicine of Caulis Dendrobii was expatiated and the consistency of medicine name "Shihu" all through the ages was shown by herbal textual. Varieties applied both ancient and modern were collated, evolution of efficacy was also explained, and the diversity of trueborn producing areas was confirmed by both herbal textual and modern literature research. Resource and habitat of the plants in Guangxi province were studied on the spot.Cell divisional rhythm of stem apex of D. candidum and D. loddigesii was discovered. Fastigiumof divisional index was found to be at 9:00AM, which reached 1.5×1O^-5 in D. candidum and in 3.3×1O^-4 in D. loddigesii; Cell division generated around the clock. May was found the most active month while monthly rhythm was reviewed. The low divisional index of stem apex cells was considered the reason for retarded growth of D. candidum and D. loddigesii.Structure differentiation of stem growing tips of D. candidum and D. loddigesii was found and effects of intervention by 6-BA spraying and specific lighting were found.Kind of cell death occurred in D. candidum stem apex and D. loddigesii stem apex was ascertained to be necrosis,which opposite kind is programmed cell death, by applying microscope, transmission electron microscope, terminal deoxynucleotibyl transferase mediated dUTP-biotin nick-end labeling and acridine orange staining.Location of adenosine triphosphatase in cell was shown by cytochemical reaction and observed by using transmission electron microscope. Difference of ATPase activity among experimental groups were revealed. Resistance to environmental stress in May, October and January of the year ensuing, shown by activity of peroxidase, catalase, and the content of malomdiadehyde in D. candidum leaves and D. loddigesii leaves,was studied. Difference of the three indexs among experimental groups were revealed. In comparison among groups, activity of POD and CAT in control group was found to be the lowest both in candidum and D. loddigesii, while content of MDA to be the highest.In monthly comparison, activity of POD and CAT, in hormone group and lighting group of D. candidum, was found to be the highest in May, while to be the lowest in October. No significant difference was found in the activity of POD in hormone group and lighting group of D. loddigesii, while step-up trend shown in controlgroup. Same monthly change of CAT activity and MDA content was revealed in D. loddigesii as in D. candidum. So was of MDA content. Sucrose synthase activity in leaves of was also assayed. Activity of sucrose synthase was found to be the highest in May in control group of both plants, while found to be the lowest in Oct. and Jan.the the year ensuing, on the contrary.Polysaccharide content of D. candidum stems and D. loddigesii stems of all groups, grown for different time, was determined and compared. Quanlity of the Caulis Dendrobii was valued by the polysaccharide content and the effects of intervention,6-BA spraying and specific lighting, were discovered. Step-up trend of polysaccharide content, assayed in seriate growth periods before 18th month, was discovered both in D. candidum stems and D. loddigesii. In particular, polysaccharide content in hormone group of D. loddigesii was detected to be the lowest before 12th month’s growth, but to be the highest in 18^th month.更多还原