耦合Ri质粒介导发根技术的喜树次生代谢物研究
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摘要
喜树为中国特有树种,喜树碱系列衍生物抗癌药物是附加值极高的产品。Ri质粒诱导的发根具有生长迅速、激素自养、生长条件简单、次生代谢产物含量稳定、分化程度高、不易变异等特点,而且可把代谢物分泌至培养基中。本研究用植物转基因工具发根农杆菌,对喜树进行基因干预,实现喜树药用价值次生代谢物的合成；用大孔吸附树脂完成离线分离富集工作,用化学计算指导分子印迹化合物的设计和合成,探讨喜树发根培养次生代谢物的原位捕抓和专一吸附；用细胞模型(黑色素瘤细胞)评价了喜树次生代谢物的药理活性,探讨了次生代谢物的抗氧化能力,进一步阐明了相关物质的抗癌药用机理和抗氧化能力。本研究的具体工作总结如下：
1.建立了Ri质粒介导的喜树发根的培养体系：选择发根农杆菌种ATCC15834作为介导菌株；证实用无菌苗子叶进行发根培养,发根率最高,发根农杆菌经液体培养至OD600为近0.8,稀释5倍用于接种感染喜树外植体；用250mg/L羧苄青霉素+250mg/L替漫汀抗菌；发根在含蔗糖30g/L的HRIM培养基中生长较迅速,液体培养介质有利于喜树发根生长,选8小时光照条件,发根的PH范围以5.5-6.0为优。
2.确定了喜树碱及羟基喜树碱的定量测定方法；毛状根的喜树碱含量0.022mg.g-1(D.W)与正常根含量0.027mg.g-1(D.W)接近；30天培养基中喜树碱含量达根含量的近30%。
3.用大孔吸附树脂离线分离富集培养基中次生代谢物,证实HPD722树脂对喜树碱效果最好；动态参数是：氯仿/乙醇(1：1)作解吸液,解吸液PH值为3,上柱流速1.0ml/min,从培养基中提取了β-谷甾醇、喜树碱、羟基喜树碱；确定了金丝桃苷的存在。
4.以与喜树碱含有相似官能团的茶碱为模板分子,制备了对茶碱有良好吸附能力的单分散或窄分散分子印迹聚合物微球,为喜树碱分子印迹微球的制备筛选了系列参数：GMA和DVB用量配比为4：6得到单分散的聚合物微球,微球的平均粒径为2.65μm；分子印迹聚合物微球的接枝量可以通过控制PGMA-DVB微球表面环氧基含量进行调整；当分子印迹聚合物的接枝量增大到12%时,其吸附量达到14μg/g。
5.运用Gaussian03程序包,采用密度泛函理论(DFT, Density Functional Theory)方法,对喜树碱分子和功能单体分子丙烯酰胺以及二者所形成的复合物的构象进行几何优化,在此基础上结合自然键轨道(NBO)进行计算并分析喜树碱E环C-0键的成键性质,解释其易分解的原因。对化合物数据库进行搜索,寻找与喜树碱结构近似的分子：H(8-ethyl-8-hydroxybenzo[6,7]indolizino[2,3-b]quinoline-9,12(6H,8H)-d ione)
6.证实喜树碱、羟基喜树碱、β-谷甾醇对人A375细胞及鼠B16-F10细胞生长有显著抑制作用,不同浓度处理后与对照组比较差别有显著性(P<0.05),呈浓度时间依赖性;而金丝桃苷、白桦酯酸对人A375细胞及鼠B16-F10细胞生长没有显著抑制作用；喜树碱的抑制浓度(IC50)测得是35μmol/L,羟基喜树碱的抑制浓度(IC50)测得是32μmol/L,β-谷甾醇的抑制浓度(IC50)测得是0.75mmol/L；喜树碱对B16-F10细胞周期的影响是：改变细胞的周期分布,使细胞周期出现S期延长,G0-G1期缩短。
7.通过邻苯三酚自氧化法、清除DPPH自由基法和清除ABTS自由基三种方法得到金丝桃苷的抗氧化能力接近VC,β-谷甾醇的抗氧化能力与BTH接近的结果,金丝桃苷的IC50为0.043g/L。β-谷甾醇的IC50为0.73g/L
Camptotheca acuminate is a special plant in China. Camptothecin and its analogues are highly added value products. The hairy roots of Camptotheca acuminate induced by Ri plasmid, have the advantages of rapid growth, hormone autotrophy, requiring simple growth conditions, producing high and stable content of secondary metabolites, containing high differentiation degree and low variation rate, etc.. Furthermore, they are capable to excrete the secondary metabolites in medium. Because the concentration of secondary metabolites that Camptothecin excretes in medium is considered to be low, so the condition of enrichment and extraction for Camptothecin and analogue into medium with macroporous resins need be investigated. The effect of Camptothecin and analogue on B16-F10mouse melanoma cells and A375human melanoma cells was examined. The results followed:
1.Camptothecin is an anticancer and antiviral alkaloid produced by the Camptotheca acuminata (Nyssaceae). Hairy root cultures of Camptotheca acuminata were worked out where tissues took out from Agrobacterium rhizogenes strains ATCC15834. The hairy roots grew rapidly in HRIMI medium which contains30g/L sugar. The electrophoretic analysis indicated that the gene in Ri plasmid of Agrobacterium rhizogenes had been integrated into the genome of the hairy root and had been expressed. The suitable PH value for cultivation system were5.5-6.0.
2. The hairy roots which induced by Ri plasmid, was ascertained by LC-ESI-MS detection that it could release camptothecin in condition of medium. It is be worked out, the method of determination for camptothecin content from the hairy root culture medium with HPLC. The cultures were able to synthesize the alkaloids at the levels of0.022mg.g-1(DW), and CPT from the hairy root in plant was tested as0.027mg/g (DW).
3. The camptothecin from the medium was effectively enriched and recovered by selecting non-polar adsorptive resin HPD-722,The camptothecin. Hydroxycamptothecin、P-sitosterol in the hairy root culture medium were effectively enriched and recovered. It was also be confirmed the hyperin in hairy root culture medium by the HPLC method of determination.
4. With theophylline containing similar functional groups as camptothecin for the molecular template.the monodisperse molecularly imprinted polymer microspheres were Syntheszised. The condition for the Synthesis of surface molecularly imprinted microsphere werer optimizated.The proportioning of GMA and DVB is4:6.the size particle of microspheres was tested as2.65μm.The grafting of Polymicrospheres microsphere was controlled by the adjusted the content of cyclooxy group on the surface Poly9(PGMA-DVB)microspheres microsphere. As the grafting of Polymicrospheres microsphere increased to12%,the adsorption of molecularly imprinted polymer microspheres was14μg/g.
5. The geometry structures of Complex synthesized with CPT and acrylamide as a functional monomer are optimized using density functional theory(DFT) as implemented in GAUSSIAN03program. The bonding property of the C-O Bond in E Ring from CPT was calculated with natural bond orbital analyses. With search through the compound databases, the approximate molecule of CPT was found. It is H (8-ethyl-8-hydroxybenzo[6,7]indolizino[2,3-b]quinoline-9,12(6H,8H)-dione)
6. Both CPT and HCPT have a stronger inhibitory effect to B16-F10malignant melanoma cells. The inhibitory effect of CPT and HCPT is dose dependent, and the IC50of CPT is35μmol/L, the IC50of HCPT is32μmol/L. β-sitosterol has a significant inhibitory effect to B16-F10malignant melanoma cells. The inhibitory effect of β-sitosterol is dose dependent, and the IC50is0.75mmol/L
7. With the ways of the autoxidation of pyrogallol, eliminating DPPH·and ABTS eliminating free radicals, the antioxidant abilities of β-sitosterol and hyperin were be determinated. The results showed:The antioxidant effect of β-sitosterol and hyperin are dose dependent, and the IC50ofβ-sitosterol is0.73g/L The IC50of hyperin is0.043g/L.It showed the antioxidant ability of hyperin is similar the antioxidant ability of Vc.
1.建立了Ri质粒介导的喜树发根的培养体系：选择发根农杆菌种ATCC15834作为介导菌株；证实用无菌苗子叶进行发根培养,发根率最高,发根农杆菌经液体培养至OD600为近0.8,稀释5倍用于接种感染喜树外植体；用250mg/L羧苄青霉素+250mg/L替漫汀抗菌；发根在含蔗糖30g/L的HRIM培养基中生长较迅速,液体培养介质有利于喜树发根生长,选8小时光照条件,发根的PH范围以5.5-6.0为优。
2.确定了喜树碱及羟基喜树碱的定量测定方法；毛状根的喜树碱含量0.022mg.g-1(D.W)与正常根含量0.027mg.g-1(D.W)接近；30天培养基中喜树碱含量达根含量的近30%。
3.用大孔吸附树脂离线分离富集培养基中次生代谢物,证实HPD722树脂对喜树碱效果最好；动态参数是：氯仿/乙醇(1：1)作解吸液,解吸液PH值为3,上柱流速1.0ml/min,从培养基中提取了β-谷甾醇、喜树碱、羟基喜树碱；确定了金丝桃苷的存在。
4.以与喜树碱含有相似官能团的茶碱为模板分子,制备了对茶碱有良好吸附能力的单分散或窄分散分子印迹聚合物微球,为喜树碱分子印迹微球的制备筛选了系列参数：GMA和DVB用量配比为4：6得到单分散的聚合物微球,微球的平均粒径为2.65μm；分子印迹聚合物微球的接枝量可以通过控制PGMA-DVB微球表面环氧基含量进行调整；当分子印迹聚合物的接枝量增大到12%时,其吸附量达到14μg/g。
5.运用Gaussian03程序包,采用密度泛函理论(DFT, Density Functional Theory)方法,对喜树碱分子和功能单体分子丙烯酰胺以及二者所形成的复合物的构象进行几何优化,在此基础上结合自然键轨道(NBO)进行计算并分析喜树碱E环C-0键的成键性质,解释其易分解的原因。对化合物数据库进行搜索,寻找与喜树碱结构近似的分子：H(8-ethyl-8-hydroxybenzo[6,7]indolizino[2,3-b]quinoline-9,12(6H,8H)-d ione)
6.证实喜树碱、羟基喜树碱、β-谷甾醇对人A375细胞及鼠B16-F10细胞生长有显著抑制作用,不同浓度处理后与对照组比较差别有显著性(P<0.05),呈浓度时间依赖性;而金丝桃苷、白桦酯酸对人A375细胞及鼠B16-F10细胞生长没有显著抑制作用；喜树碱的抑制浓度(IC50)测得是35μmol/L,羟基喜树碱的抑制浓度(IC50)测得是32μmol/L,β-谷甾醇的抑制浓度(IC50)测得是0.75mmol/L；喜树碱对B16-F10细胞周期的影响是：改变细胞的周期分布,使细胞周期出现S期延长,G0-G1期缩短。
7.通过邻苯三酚自氧化法、清除DPPH自由基法和清除ABTS自由基三种方法得到金丝桃苷的抗氧化能力接近VC,β-谷甾醇的抗氧化能力与BTH接近的结果,金丝桃苷的IC50为0.043g/L。β-谷甾醇的IC50为0.73g/L
Camptotheca acuminate is a special plant in China. Camptothecin and its analogues are highly added value products. The hairy roots of Camptotheca acuminate induced by Ri plasmid, have the advantages of rapid growth, hormone autotrophy, requiring simple growth conditions, producing high and stable content of secondary metabolites, containing high differentiation degree and low variation rate, etc.. Furthermore, they are capable to excrete the secondary metabolites in medium. Because the concentration of secondary metabolites that Camptothecin excretes in medium is considered to be low, so the condition of enrichment and extraction for Camptothecin and analogue into medium with macroporous resins need be investigated. The effect of Camptothecin and analogue on B16-F10mouse melanoma cells and A375human melanoma cells was examined. The results followed:
1.Camptothecin is an anticancer and antiviral alkaloid produced by the Camptotheca acuminata (Nyssaceae). Hairy root cultures of Camptotheca acuminata were worked out where tissues took out from Agrobacterium rhizogenes strains ATCC15834. The hairy roots grew rapidly in HRIMI medium which contains30g/L sugar. The electrophoretic analysis indicated that the gene in Ri plasmid of Agrobacterium rhizogenes had been integrated into the genome of the hairy root and had been expressed. The suitable PH value for cultivation system were5.5-6.0.
2. The hairy roots which induced by Ri plasmid, was ascertained by LC-ESI-MS detection that it could release camptothecin in condition of medium. It is be worked out, the method of determination for camptothecin content from the hairy root culture medium with HPLC. The cultures were able to synthesize the alkaloids at the levels of0.022mg.g-1(DW), and CPT from the hairy root in plant was tested as0.027mg/g (DW).
3. The camptothecin from the medium was effectively enriched and recovered by selecting non-polar adsorptive resin HPD-722,The camptothecin. Hydroxycamptothecin、P-sitosterol in the hairy root culture medium were effectively enriched and recovered. It was also be confirmed the hyperin in hairy root culture medium by the HPLC method of determination.
4. With theophylline containing similar functional groups as camptothecin for the molecular template.the monodisperse molecularly imprinted polymer microspheres were Syntheszised. The condition for the Synthesis of surface molecularly imprinted microsphere werer optimizated.The proportioning of GMA and DVB is4:6.the size particle of microspheres was tested as2.65μm.The grafting of Polymicrospheres microsphere was controlled by the adjusted the content of cyclooxy group on the surface Poly9(PGMA-DVB)microspheres microsphere. As the grafting of Polymicrospheres microsphere increased to12%,the adsorption of molecularly imprinted polymer microspheres was14μg/g.
5. The geometry structures of Complex synthesized with CPT and acrylamide as a functional monomer are optimized using density functional theory(DFT) as implemented in GAUSSIAN03program. The bonding property of the C-O Bond in E Ring from CPT was calculated with natural bond orbital analyses. With search through the compound databases, the approximate molecule of CPT was found. It is H (8-ethyl-8-hydroxybenzo[6,7]indolizino[2,3-b]quinoline-9,12(6H,8H)-dione)
6. Both CPT and HCPT have a stronger inhibitory effect to B16-F10malignant melanoma cells. The inhibitory effect of CPT and HCPT is dose dependent, and the IC50of CPT is35μmol/L, the IC50of HCPT is32μmol/L. β-sitosterol has a significant inhibitory effect to B16-F10malignant melanoma cells. The inhibitory effect of β-sitosterol is dose dependent, and the IC50is0.75mmol/L
7. With the ways of the autoxidation of pyrogallol, eliminating DPPH·and ABTS eliminating free radicals, the antioxidant abilities of β-sitosterol and hyperin were be determinated. The results showed:The antioxidant effect of β-sitosterol and hyperin are dose dependent, and the IC50ofβ-sitosterol is0.73g/L The IC50of hyperin is0.043g/L.It showed the antioxidant ability of hyperin is similar the antioxidant ability of Vc.
引文
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