【作者】 徐志敏；

【导师】 瞿伟菁；

【作者基本信息】 华东师范大学 ， 植物学， 2007， 硕士

【摘要】 本文基于建立香叶天竺葵（Pelargonium graveolens L’Hér）生物反应器体系，首先研究了内生菌污染的控制，悬浮细胞和毛状根培养系统的建立，并对组培材料和大田材料精油提取方法和影响因素进行了比较。在此基础上，分析了组培苗精油的化学成分，进行体外鼻咽癌细胞生长抑制试验，以研究组培香叶油的药效物质基础。内生菌污染是香叶天竺葵在组织培养增殖过程中影响实验体系建立的技术障碍，为此研究首先就不同抑菌方法进行了试验，结果显示：Amp150 mg／L和Amp100mg／L+Km50mg／L处理的植株生长旺盛，生根率均高于90％，植株的精油成份含量略低于阴性对照，表明抑菌剂对精油成分含量的影响较小，但同时这两种组合仍存在一定的污染率。在建立细胞悬浮培养体系的过程中，筛选了愈伤组织28种固体培养基、5种蔗糖浓度；悬浮细胞4种液体培养基。结果表明，1％蔗糖浓度的MS+BAP1.0mg／L+2，4-D0.7mg／L和MS+BAP1.0mg／L+2，4-D1.0mg／L培养基适合产生松散且未分化的愈伤组织。31号培养悬浮细胞效果最好，细胞增殖较快，分散度好。对悬浮细胞培养液进行光镜观察，发现悬浮液中确实有单个细胞及小细胞团存在。扫描电镜观察发现，单个细胞具有球形、长梭形等各种形态，球形直径在60.100μm不等，长梭形大小在（25-80）×（25-80）×（200-300）gm³不等，基本没有细胞分化。对悬浮细胞培养条件的筛选结果表明，50mL装液量、4％蔗糖浓度、120rpm暗培养适宜悬浮细胞培养。为建立香叶天竺葵毛状根生物反应器系统，以发根农杆菌A4和C58C1转染植物，发根诱导率结果显示：以叶片为外植体，于C58C1菌原液中浸泡5min，光照16h／d条件下，在不添加甘露醇、无激素的MS₀培养基中共培养2d是较理想的转化方式。PCR检测结果表明，发根基因ro1B、ro1C基因已整合进香叶天竺葵毛状根基因组中。毛状根生长周期测定显示第40天时生物量达到最大值，湿重增长10.2倍，干重增长11.8倍；葡萄糖6-磷酸脱氢酶（G-6-PDH）活性变化的统计分析结果显示，G-6-PDH活性在第36天时达到最大值40.50±1.926，与接种时相比极显著增加（P＜0.01）。将毛状根用正己烷萃取后进行GC-MS组分分析，结果表明，接种时鉴定出14种精油组分，含量仅占总萃取物的0.95％；第44天时鉴定的组分最多，达26种，占总含量的6.93％；香茅醇、甲酸香茅酯等主要成份含量于48天时达到最高值，占萃取物的1.53％。本文进一步对不同培养基进行筛选，确定了毛状根培养条件。结果显示，与基础培养基培养48天的毛状根相比，1／2MS₀添加NAA0.1mg／L培养的毛状根仅生物量显著降低（P＜0.05）、葡萄糖-6-磷酸脱氢酶活性（P＜0.01）及薄荷醇、香茅醇、甲酸香茅酯含量均较高。毛状根诱导的再生苗香叶油组分分析表明，MS₀和MS₂培养基上诱导的再生苗与对照组培普通生根苗的精油成份相似，含量稍有差别。MS₀培养基诱导的再生苗香茅醇、甲酸香茅酯含量最高，占总量的39.33％，表明MS₀适合毛状根再生苗的诱导和生长。由于组织培养材料的精油含量较少，本文进一步对大田、组培材料的培养时间、质量、水蒸气蒸馏时间或正己烷萃取等影响精油成份含量的因素进行比较，结果表明，30g茎叶蒸馏12min是50g以下大田材料蒸馏精油的理想条件；7月份较适合蒸馏和萃取大田茎叶的香叶油；培养50天的组培无根叶和40天的有根叶较分别适用于水蒸气蒸馏和正己烷萃取；7月份的大田根和液体培养60天的组培根适用于正己烷萃取。为分析香叶天竺葵组培苗精油的化学成分，研究其药效物质基础，本文进行了组培香叶油等3种香叶油及其主要单体组分对体外人鼻咽癌细胞的生长抑制试验。结果显示以香叶油及单体处理鼻咽癌细胞24h后，产生了不同程度的增殖抑制作用，随药物浓度上升而增强，表现出剂量效应关系。组培香叶油和乙酸香茅酯抑癌作用较强，IC₅₀值分别达到94.25 nL／mL和43.98 nL／mL。3种香叶油及松油醇和乙酸香茅酯处理后细胞形态均发生较大改变，大部分细胞变圆缩小，出芽并伴有凋亡小体形成；用PI荧光探针结合流式细胞术检测细胞周期变化发现，香叶油及松油醇可能是通过提高鼻咽癌细胞的凋亡率来抑制肿瘤细胞生长。更多还原

【Abstract】 To establish cell suspension and hairy root culture system, inhibition of bacterial contamination, essential oil extraction methods and affecting factors, components of Pelargonium graveolens oil from tissue-cultured plantlet and inhibition on cell growth of human nasopharyngeal carcinoma were studied in this paper.Bacterial contamination is observed in tissue subculture period of Pelargonium graveolens micropropagation system. Ampicillin 150mg/L and ampicillin 100mg/L combining with kanamycin 50mg/L were efficient in bacterial contamination controlling not only because they made the rate of root formation higher than 90%, but also because through the GC-MS analysis, these medium made the contents of the plantlets almost be the same as control. They are fit for controlling the bacterial contamination.In the establishment of in vitro cell suspension culture, 28 solid culture mediums, 5 sucrose concentrations and 4 liquid culture mediums were observed. It indicated that addition of BAP1.0mg/L combining with 2,4-D0.7mg/L, BAP1.0mg/L combining with 2,4-D1.0mg/L and 1% sucrose in MS solid medium were fit for forming incompact and indifferentiationed callus. Addition of BAP1.0mg/L combining with 2,4-D0.7mg/L in MS liquid medium was fit for cell suspension. Single cell and small group of cells in liquid medium were observed with microscope and scaning electron microscope. There were various shapes like ball or long shuttle of single cells. The diameter of ball cells was about 60-100μm and the size of long shuttle cells was about （25-80）×（25-80）×（200-300）μm³ .The cells had not differentiationed yet. Then we observed the factors for cell suspension culture, it came to the results that the cells were fit for culturing in 50mL medium, 4% sucrose, 120rpm rotation in dark condition.In order to establish the in vitro system of hairy root culture, the leaf explants of Pelargonium graveolens were infected with Agrobacterium rhizogenes strains A4 and C58C1. To obtain hairy roots efficiently, dipping in C58C1 solution for 5 minutes, co-culturing for 2 days by illumination for 16h/d in MS medium without mannitol and phytohormones were necessary. The PCR result confirmed the transformation of rolB and rolC genes from tDNA of Ri plasmid to the hairy root of Pelargonium graveolens genome. The wet weight of hairy root showed a 10.2-fold and the dry weight a 11.8-fold enhancement in the 40^th day in growth cycle. The G-6-PDH activity in the growth cycle came to the highest value of 40.50±1.926 in the 36^th day, markedly higher than that of inoculation （P<0.01） .Through the GC-MS analysis of the hairy root in growth cycle, it could be identified 14 compounds with the content of 0.95% when we inoculated the hairy root, then it enhanced to 26 compounds which counts for 6.93%in the 44^th day. The content of citrinellol and citronellyl formate（total 40.70%） showed as the highest in the 48^th day.To select the suitable liquid medium for culturing hairy root, 19 mediums were valued. The biomass, G-6-PDH activity and the content of menthol, citrinellol and citronellyl formate of the hairy roots which were cultured in l/2MSo added with NAA0.1mg/L all kept in a high level,so this medium was suitable for hairy root culture.Then we characterized the components of plantlets induced by hairy root which were cultured in 3 solid mediums. The essential oils from plantlets in MS₀ （MS₀O）and MS₂ （MS₂O）mediums were similar to the one from 30d-cultured plantlets which were induced by normal callus of Pelargonium graveolens. The content of citrinellol and citronellyl formate（total 39.33%） of plantlet from MS₀ medium was the highest. MS₀ medium was fit for culturing plantlets induced by hairy root.Then we studied and compared factors such as culturing time and weight of cultured and field materials, vapor distillation time or n-hexane extraction affecting the components and contents of essential oil. The results indicated that it is efficient to get essential oil in vapor distillation by 30g leaves of Pelargonium graveolens from field for 12min. The leaves from field could be vapor distillated and extracted in July. Materials which were just adventitious buds from cultured conditions were good for vapor distillation by culturing 50 days.The plantlets which were rooted were better for extraction by culturing 40 days.To analysis the components of Pelargonium graveolen L’Hér oil （PLO） from tissue-culture plantlet and study its pharmacological effect of inhibition on cell growth of human nasopharyngeal carcinoma（CNE）, GC-MS analysis was used to identify the components of PLOs from different sources. MTT assay, Wright dyeing were carried out to measure the antitumor activity of the oils and monoments.The results showed that after treating with PLOs and monoments for 24h, the antitumor effects were different and toned up along with the rising of drug concentrations.They represented as dosage effect relationship. The antitumor effect of PLO from tissue-culture plantlet and citronellyl acetate were better than other oils or monomers, their IC₅₀ values were 94.25 nL/mL and 43.98 nL/mL. After treated with PLOs and 2 monomers, the CNE cell had a markedly morphological change. The cell line exhibited typical morphological alterations of apoptosis, such as rounding of cells, apoptotic body around the cells. Through the acridine orange staining, the cell cycle were observed by flow cytometer. We concluded that PLOs and terpineol inhibited the growth of CNE cell lines by increasing the rate of apoptosis.更多还原