道地药材研究模型“丹参毛状根”的评价研究
摘要
作为中医药的精髓,药材的道地性既有来源于历史和文化的属性,又涉及到遗传、环境及生产实践等方方面面。从生物学上说“道地药材”的形成是基因型与环境之间相互作用的产物,可用公式表示:表型=基因型+环境饰变。道地药材是一个复杂系统,它的每一个因素及其相互作用不可能都被同时考察,研究中必须有所取舍,对研究对象降阶降维,才有可能取得突破。环境因子究竟是如何通过修饰道地药材的基因型而发生作用的,生态因子与道地药材次生代谢产物积累的有着怎样的关系,至今缺少直接和系统的实验研究。至此,科学模型因其具有简化复杂研究对象的特点,开始引起道地药材研究者的关注,构建道地药材研究模型(载体),成为道地药材生物学研究的关键。
模型在生物学中的应用相当广泛。通过在基本原型的基础上建立相关的模型,并运用相应的模型分析解释生物学现象、揭示生物本质规律特征,研究某种动态生命系统中各种因子之间相互关系。作为现代生命科学研究不可缺少的重要手段,模型和模型方法很早就已经渗透到生命科学研究的各个环节,例如用于基因工程研究的模式物种,用于医学实验的动物模型等。
毛状根培养是80年代发展起来的基因工程和细胞工程相结合的一项新技术,它是将发根农杆菌的Ri质粒中含有的T-DNA转化到植物细胞的DNA上,诱导植物细胞产生毛状根。毛状根作为一种有效的长期保持物种优良性状的方式,越来越多的被人们作为实验材料,同时获取次生代谢产物。毛状根具有生长速度快,且不受气候、病虫害、地理和季节的限制等特点。据不完全统计,国内外目前已对29科52种药用植物进行了毛状根诱导的研究,建立了长期的毛状根培养系统,获得次生代谢产物。
本论文针对目前道地药材生物学本质研究中需要解决的问题,在本实验室开展工作的基础上,选取丹参毛状根作为研究对象,对其是否具备道地药材研究模型的特性进行了系统的评价研究。即毛状根到底能不能作为模型来研究道地药材?能不能再现原植物药材的特性,是否能作为稳定的可重复的研究工具等。
该模型的评价研究,涉及到植物化学、分子生物学、药理学等许多层面。主要包括三个方面:①再现性,作为研究道地药材的模型要考察毛状根和其原型(即原药材)之间的相似性,包括化学成分,毒性以及药效等,来阐明毛状根是否具有再现原药材的特性。②稳定性,毛状根的核DNA的稳定性、rol基因遗传稳定性、化学成分稳定性等。③实用性,在证明毛状根模型具有稳定性和再现性的基础上,对其作为模型的实用性进行了研究,考察毛状根是否便于取材,是否具备优良的实验物性,作为模型是否易于建立和使用。
1丹参毛状根模型的再现性研究(与原植物的相似性)
1.1化学成分的相似性选取药典中的指标成分丹参酮Ⅱ_A和丹酚酸B,对丹参毛状根和河北丹参药材进行了含量测定,丹参药材中丹参酮Ⅱ_A含量为0.12%,丹酚酸B含量为5.4%;各个批次的毛状根丹参酮Ⅱ_A的含量在0.04%～0.10%之间,平均值与药材相比较低;丹酚酸B的含量在3.21%～7.25%,平均值与药材相比较高。同时,用中药色谱指纹图谱相似度评价系统2004A版软件进行相似度分析,对毛状根与药材的指纹图谱也进行了比较,相似度在0.948以上,说明丹参毛状根的化学成分组成与2个产地(河北灵寿,四川中江)的丹参药材相比差异不显著。由此可见,丹参毛状根与丹参药材具有相似的化学成分。
1.2急性毒性丹参毛状根提取物LD_(50)为63.94g生药/kg,95%可信区间为(58.82～69.51)g生药/kg。按公斤体重计算相当于成人日摄入量(丹参临床人用剂量为9～15g/日,0.25g生药/kg)的255.76倍。结果表明,丹参毛状根最大无毒剂量已超过人临床用药量的100倍。
1.3药效的相似性丹参根为传统的“活血化瘀”中药。其各种制剂应用于冠心病与心血管病治疗收到了良好的效果。本实验从整体、器官以及细胞三个水平上观察丹参毛状根对心肌缺血再灌注损伤的保护作用。①整体实验表明,丹参毛状根组能显著降低缺血再灌注所致VT,VF的发生率,降低血浆LDH,MDA含量,提高SOD活性;与丹参药材相比无显著性差异。②离体实验发现,丹参毛状根能够增加冠脉流量,不但降低心律失常的发生率,而且推迟心律失常的发生时间,缩短持续时间和恢复时间,对LDH,MDA和SOD的释放有明显的改善作用,结果提示丹参毛状根处理对大鼠离体心脏缺血再灌注损伤可能具有保护作用;与丹参药材相比无显著性差异。③细胞实验表明,丹参毛状根可较显著增加H/R心肌细胞的存活率,降低MDA含量,提高SOD活性。各组随剂量增大LDH,MDA含量减少,SOD活性增加,丹参毛状根可拮抗大鼠心肌细胞H/R损伤,对大鼠心肌细胞H/R损伤可能具有保护作用;与丹参药材相比无显著性差异。上述研究表明,丹参毛状根与丹参药材对心肌缺血再灌注损伤的保护作用相当。
2丹参毛状根模型的稳定性研究
2.1核DNA的稳定性通过流式细胞仪对丹参毛状根核DNA浓度的分析,选取大豆作为内参物,结果表明所选取的5批丹参毛状根样品,核DNA含量平均为0.18pg/2C,基因组大小平均为87.8 Mbp,RSD为6.6%,核DNA的浓度比较稳定。
2.2 rol基因的稳定性从随机培养的丹参毛状根中任选10批,提取新鲜组织的DNA,并进行PCR扩增,结果表明,外源基因rolA在10批样品的基因组DNA中均可以检测到,并将rolA基因作为目的基因进行了回收测序与GeneBank登录序列(X12579)的同源性为100%。由此可以说明10个批次的丹参毛状根样品中rol基因是稳定的。
2.3化学成分的稳定性通过对丹参毛状根的指纹图谱研究,对其化学成分的稳定性进行了分析。利用中药色谱指纹图谱相似度评价系统2004A版软件进行相似度分析,从计算结果可以看出,10批毛状根的相似度均在0.970以上,说明不同批次丹参毛状根样品间化学成分组成的差异不显著。同时,通过对不同批次的丹参毛状根的生长情况的分析表明,不同批次生长率无显著性差异,比较稳定。
3丹参毛状根模型的实用性研究
3.1丹参毛状根的生长周期研究6,7-V培养基上培养丹参毛状根,分别在第2,4,6,8,10周收获,测定其生长量。高效液相色谱法测定丹参毛状根中丹参酮Ⅱ_A和丹酚酸B的含量。在固体培养基中培养的毛状根,收获时应选择6周比较合适,此时毛状根的增长倍数大,有效成分相对来说也比较高。由此可见,丹参毛状根培养周期短,6-8周即可收获,具有快速繁殖的特性,并能迅速积累次生代谢产物。
3.2矿质营养元素对丹参毛状根生长和有效成分积累的影响本实验通过在6,7-V培养基的矿质营养元素配方基础上进行加减,深入研究了培养基中氮、钾、铁、镁4种单一矿质营养元素的浓度,对丹参毛状根生长和有效成分积累的影响。结果表明,氮元素对毛状根的生长影响最大,其次是钾元素。铁元素和镁元素对丹参毛状根的生长量影响不大。从有效成分含量的积累来看,低氮以及完全处理组的铁和镁元素有利于丹参酮Ⅱ_A的累积。高氮,高钾有利于丹酚酸B的积累。培养基中这4种元素浓度的改变都会对毛状根产生不同程度的刺激,进而影响其生长和有效成分的积累,由此证明丹参毛状根对矿质营养元素的刺激具有灵敏性,因此,可以用毛状根作为模型进行更深一步的道地产区环境因子变化的分析研究。
结论
本论文的研究结果表明:丹参毛状根具有良好的再现原药材的特性,可以替代原药材作为研究模型,各个批次遗传、化学比较稳定,取材方便,生长繁殖快,具有优良的实验物性,作为模型具有一定的实用性。
通过对丹参毛状根的评价研究,认为毛状根可以作为研究模型。用毛状根为模型,可以以道地药材表型与生境及基因型的相互关系为切入点,深入研究道地药材对环境的响应,基因的功能、表达的调控机制等。
本论文首次提出道地药材研究模型,并系统的对毛状根作为模型进行评价,为道地药材的研究提供实验平台,是本论文的创新点。
As the soul of traditional Chinese medicine,the geo-herblism of Chinese material medica not only has historical and cultural nature,but also concerns heredity, environment and production,etc.The forming of geoherb is resulted from the interaction of genotype and environment.It can be expressed by this formula: phenotype = genotype + environmental modification.Geoherbs is a complex system, and we can not study the factors and interaction among them simultaneously,but we should grasp the key point and simplify the object to find a breakthrough.And the model for scientific study is used by the researchers of geoherbs,which has the characteristics of simplified objects.It is very important to construct the geoherb's model in studying the biology of it.
The model,constructed from archetype,is widely used in biology,such as to analyze the phenomena of biology,to post the rules and characters of life essence and to study the interaction of the factors in dynamic life systems etc.Model and model methods to study the object become very important in life sciences,for example the bio-model in genome research and medicinal experiment.
The culture of hairy root is a new technology combined with gene engineering and cell engineering developed from 1980's.It transforms the T-DNA in Ri plasmid to the DNA of plant cell,and induced the hairy root.Hairy root culture is an effective way for long-term preservation of valuable genetic materials,and to obtain active compounds of some medical plants,especially for those plants,the interest secondary metabolites of which are accumulated in roots.The growth of hairy root is rapid and it is not restricted by climate,plant disease and insect pests,geography and seasons.
Genetic instability would be induced during continuous terms in vitro maintenance.Structural chromosomal rearrangements,polyploidy,and mutations also contribute to the culture-induced variations.So whether the hairy root can be used as model to study the geoherb,whether it is reappearance to the original plant,whether it is stable to repeat the results as a tool,all need our evaluating systemically.
According to the problems which needs resolving in geoherbs study,this dissertation uses hairy root of Salvia miltiorrhiza as a model based on the job of out laboratory,to study whether the hairy root has the attribute of model in geoherb research.The evaluation of the model includes many technologies such as phytochemistry,molecular biology,pharmacology and so on.It is mainly include 3 aspects:①Reappearance study.Evaluating the comparability of hairy root and its original plant include chemical components,toxity and pharmacokinetic study to clarify whether the hairy root can replace the original plant S.miltiorrhiza.②Stability study.Evaluating the stability of rol gene,nucleus DNA and chemical contents.③Practicability study:Evaluating the practicability based on the above two phases.The aim is to study the phenotype variation such as growth rate and the content of chemical components of hairy root by changing the culture condition.
1 Reappearance study on hairy root of S.miltiorrhiza(comparability with original plant)
1.1 Comparability of chemical components Determinate the content of tanshinoneⅡ_A and tashinoate B which index in Chinese pharmacopoeia by HPLC method of hairy root and S.miltiorrhiza from Hebei province.The content of tanshinoneⅡ_A and tashinoate B in S.miltiorrhiza from Hebei province are 0.12%, 5.4%.In hairy roots,the content of tanshinoneⅡ_A is 0.04%～0.10%,the average value is lower than plant;the content of tashinoate B is 3.21%～7.25%,the average value is higher than plant.Meanwhile,the fingerprints of hairy root and S.miltiorrhiza from Hebei and Sichuan province were compared.The similarity of them is higher than 0.948 after detected by software "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" published by Chinese pharmacopoeia commission(Version 2004A).It's no significant different of them.So the chemical components of hairy root and its original plant S.miltiorrhiza are similar.
1.2 Acute toxicity The LD_(50) of abstract of hairy root is 63.94g crude drug/kg,95% confidence interval is(58.82～69.51)g crude drug/kg,which is 255.76 times of an adult a day's quantity in clinical.It showed that the hairy root is lower toxicity.
1.3 Comparability of pharmacodynamics Studying on protection by S.miltiorrhiza hairy root on myocardial ischemia and reperfusion injury through the three levels of intact rats,isolated rat hearts and cultured neonatal rat cardiomyocytes.①Protection by S.miltiorrhiza hairy root on myocardial ischemia and reperfusion injury in the anesthetized rat.The result showed that the protection induced by S.miltiorrhiza hairy root reduced the incidence of ventricular tachycardia(VT) and ventricular fibrillation (VF) during the period of ischemia and reperfusion,the rise of plasma level of LDH, MDA decreased and the activity of SOD increased.It's no significant different between the hairy root and S.miltiorrhiza.②Protection by S.miltiorrhiza hairy root on myocardial ischemia and reperfusion injury in isolated Langendorff rat hearts.Our results showed that S.miltiorrhiza hairy root preconditioning can pervent postischmic abnormalities in reperfusion arrhythmias,aortic flow and myocardium metabolism. It's no significant different between the hairy root and S.miltiorrhiza.③Protection by S.miltiorrhiza hairy root on hypoxia and reoxygenation injury in cultured neonatal rat cardiomyocytes.The results demonstrated that S.miltiorrhiza hairy root increased the survival rate during hypoxia and reoxygenation.The rise of LDH and MDA significantly decreased and the activity of SOD significantly increased.The results above can show that preconditioning by S.miltiorrhiza hairy root demonstrated protection on hypoxia and reoxygenation injury in cultured neonatal rat cardiomyocytes.It's no significant difference between the hairy root and S. miltiorrhiza.
2 Stability study on hairy root of S.miltiorrhiza
2.1 Stability of nucleus DNA Flow cytometry analyses were carried out with S. miltiorrhiza hairy roots.The nucleus from leaf tissues of 2-week old seedling Glycine max was used as internal reference standard.The long-term in vitro maintained hairy root lines of five passages had nucleus DNA contents average 0.18 pg/2C.RSD is 6.6%.The variations in the nucleus DNA contents of the hairy root lines have no significantly difference.
2.2 Stability of rol genes The rol gene was detected by PCR in DNA from cultures in 10 passages.We found the rolA gene in all the 10 passages after detected by PCR in DNA.We recovered the rolA gene,sequencing and alignment results showed that it is homogeneous with Genbank(X12579).It showed that the rol gene in hairy root lines of 10 passages is stable.
2.3 Stability of chemical components The fingerprints of hairy root lines of 10 passages were compared.The similarity of them is higher than 0.970 after detected by software "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" published by Chinese pharmacopoeia commission (Version 2004A).It's no significant different of them.Meanwhile,determinate the growth rate of hairy root of 10 passages,it's no significant difference of them too.
3 Practicability study on hairy root of S.miltiorrhiza
3.1 Study on the effective component of S.miltirrhiza hairy root in different cultivating time.Cultivate the hairy root in 6,7-V culture medium,harvest in the 2th, 4th,6th,8th,10th weeks.Determinate the growth rate and the content of tanshinoneⅡ_A and tashinoate B by HPLC method.The 6th week is the proper time to harvest the hairy roots which the growth rate and the content were higher.So the period of Salvia miltirrhiza hairy root is short,6-8 weeks can be harvest,.The growth of S.miltirrhiza hairy root and obtain active compounds are rapidly.
3.2 Study on the effects of mineral nutrition in the growth and secondary metabolism of S.miltiorrhiza hairy root This experiment studied on the effects of mineral nutrition nitrogen(N),potassium(K),ferrum(Fe),magnesium(Mg),single element,in the growth and secondary metabolism of S.miltiorrhiza hairy root base on the 6,7-V medium.It is showed that N has significant affect to growth,secondly is K, Fe and Mg have no significant affect to growth.The lower concentration of N and CK of Fe and Mg are good for accumulating of tanshinoneⅡ_A.Higher concentration of N and K are good for accumulating of tashinoate B.The hairy root would be stimulated differently by the change of this 4 elements' concentration.It proved that the hairy root is sensitive to mineral nutrition and can be used as model to study fatherly.
Conclusion
The results of this dissertation indicate that the S.miltirrhiza hairy root has good reappearance character to S.miltirrhiza.It can be used as a model to study replacing original plants because of its stability in genetic,chemical character and because it is easy to harvest from rapid growth and is practicable.
It provides a good evidence to using S.miltirrhiza hairy root fatherly based on the evaluation of S.miltirrhiza hairy root as a model to study on geoherbs biology characteristics more deeply.For example,we can study the phenotype variation of hairy root by simulating one or more factors and making a set of grads concentration which affects the lives and quality of geoherbs.We can also study the function gene's express and regulation in geoherbs by transforming exogenous genes to hairy root.
Evaluating hairy root as model and putting forward that the hairy root can be used in studying biological essence of geoherbs,are the innovations of this dissertation.
模型在生物学中的应用相当广泛。通过在基本原型的基础上建立相关的模型,并运用相应的模型分析解释生物学现象、揭示生物本质规律特征,研究某种动态生命系统中各种因子之间相互关系。作为现代生命科学研究不可缺少的重要手段,模型和模型方法很早就已经渗透到生命科学研究的各个环节,例如用于基因工程研究的模式物种,用于医学实验的动物模型等。
毛状根培养是80年代发展起来的基因工程和细胞工程相结合的一项新技术,它是将发根农杆菌的Ri质粒中含有的T-DNA转化到植物细胞的DNA上,诱导植物细胞产生毛状根。毛状根作为一种有效的长期保持物种优良性状的方式,越来越多的被人们作为实验材料,同时获取次生代谢产物。毛状根具有生长速度快,且不受气候、病虫害、地理和季节的限制等特点。据不完全统计,国内外目前已对29科52种药用植物进行了毛状根诱导的研究,建立了长期的毛状根培养系统,获得次生代谢产物。
本论文针对目前道地药材生物学本质研究中需要解决的问题,在本实验室开展工作的基础上,选取丹参毛状根作为研究对象,对其是否具备道地药材研究模型的特性进行了系统的评价研究。即毛状根到底能不能作为模型来研究道地药材?能不能再现原植物药材的特性,是否能作为稳定的可重复的研究工具等。
该模型的评价研究,涉及到植物化学、分子生物学、药理学等许多层面。主要包括三个方面:①再现性,作为研究道地药材的模型要考察毛状根和其原型(即原药材)之间的相似性,包括化学成分,毒性以及药效等,来阐明毛状根是否具有再现原药材的特性。②稳定性,毛状根的核DNA的稳定性、rol基因遗传稳定性、化学成分稳定性等。③实用性,在证明毛状根模型具有稳定性和再现性的基础上,对其作为模型的实用性进行了研究,考察毛状根是否便于取材,是否具备优良的实验物性,作为模型是否易于建立和使用。
1丹参毛状根模型的再现性研究(与原植物的相似性)
1.1化学成分的相似性选取药典中的指标成分丹参酮Ⅱ_A和丹酚酸B,对丹参毛状根和河北丹参药材进行了含量测定,丹参药材中丹参酮Ⅱ_A含量为0.12%,丹酚酸B含量为5.4%;各个批次的毛状根丹参酮Ⅱ_A的含量在0.04%～0.10%之间,平均值与药材相比较低;丹酚酸B的含量在3.21%～7.25%,平均值与药材相比较高。同时,用中药色谱指纹图谱相似度评价系统2004A版软件进行相似度分析,对毛状根与药材的指纹图谱也进行了比较,相似度在0.948以上,说明丹参毛状根的化学成分组成与2个产地(河北灵寿,四川中江)的丹参药材相比差异不显著。由此可见,丹参毛状根与丹参药材具有相似的化学成分。
1.2急性毒性丹参毛状根提取物LD_(50)为63.94g生药/kg,95%可信区间为(58.82～69.51)g生药/kg。按公斤体重计算相当于成人日摄入量(丹参临床人用剂量为9～15g/日,0.25g生药/kg)的255.76倍。结果表明,丹参毛状根最大无毒剂量已超过人临床用药量的100倍。
1.3药效的相似性丹参根为传统的“活血化瘀”中药。其各种制剂应用于冠心病与心血管病治疗收到了良好的效果。本实验从整体、器官以及细胞三个水平上观察丹参毛状根对心肌缺血再灌注损伤的保护作用。①整体实验表明,丹参毛状根组能显著降低缺血再灌注所致VT,VF的发生率,降低血浆LDH,MDA含量,提高SOD活性;与丹参药材相比无显著性差异。②离体实验发现,丹参毛状根能够增加冠脉流量,不但降低心律失常的发生率,而且推迟心律失常的发生时间,缩短持续时间和恢复时间,对LDH,MDA和SOD的释放有明显的改善作用,结果提示丹参毛状根处理对大鼠离体心脏缺血再灌注损伤可能具有保护作用;与丹参药材相比无显著性差异。③细胞实验表明,丹参毛状根可较显著增加H/R心肌细胞的存活率,降低MDA含量,提高SOD活性。各组随剂量增大LDH,MDA含量减少,SOD活性增加,丹参毛状根可拮抗大鼠心肌细胞H/R损伤,对大鼠心肌细胞H/R损伤可能具有保护作用;与丹参药材相比无显著性差异。上述研究表明,丹参毛状根与丹参药材对心肌缺血再灌注损伤的保护作用相当。
2丹参毛状根模型的稳定性研究
2.1核DNA的稳定性通过流式细胞仪对丹参毛状根核DNA浓度的分析,选取大豆作为内参物,结果表明所选取的5批丹参毛状根样品,核DNA含量平均为0.18pg/2C,基因组大小平均为87.8 Mbp,RSD为6.6%,核DNA的浓度比较稳定。
2.2 rol基因的稳定性从随机培养的丹参毛状根中任选10批,提取新鲜组织的DNA,并进行PCR扩增,结果表明,外源基因rolA在10批样品的基因组DNA中均可以检测到,并将rolA基因作为目的基因进行了回收测序与GeneBank登录序列(X12579)的同源性为100%。由此可以说明10个批次的丹参毛状根样品中rol基因是稳定的。
2.3化学成分的稳定性通过对丹参毛状根的指纹图谱研究,对其化学成分的稳定性进行了分析。利用中药色谱指纹图谱相似度评价系统2004A版软件进行相似度分析,从计算结果可以看出,10批毛状根的相似度均在0.970以上,说明不同批次丹参毛状根样品间化学成分组成的差异不显著。同时,通过对不同批次的丹参毛状根的生长情况的分析表明,不同批次生长率无显著性差异,比较稳定。
3丹参毛状根模型的实用性研究
3.1丹参毛状根的生长周期研究6,7-V培养基上培养丹参毛状根,分别在第2,4,6,8,10周收获,测定其生长量。高效液相色谱法测定丹参毛状根中丹参酮Ⅱ_A和丹酚酸B的含量。在固体培养基中培养的毛状根,收获时应选择6周比较合适,此时毛状根的增长倍数大,有效成分相对来说也比较高。由此可见,丹参毛状根培养周期短,6-8周即可收获,具有快速繁殖的特性,并能迅速积累次生代谢产物。
3.2矿质营养元素对丹参毛状根生长和有效成分积累的影响本实验通过在6,7-V培养基的矿质营养元素配方基础上进行加减,深入研究了培养基中氮、钾、铁、镁4种单一矿质营养元素的浓度,对丹参毛状根生长和有效成分积累的影响。结果表明,氮元素对毛状根的生长影响最大,其次是钾元素。铁元素和镁元素对丹参毛状根的生长量影响不大。从有效成分含量的积累来看,低氮以及完全处理组的铁和镁元素有利于丹参酮Ⅱ_A的累积。高氮,高钾有利于丹酚酸B的积累。培养基中这4种元素浓度的改变都会对毛状根产生不同程度的刺激,进而影响其生长和有效成分的积累,由此证明丹参毛状根对矿质营养元素的刺激具有灵敏性,因此,可以用毛状根作为模型进行更深一步的道地产区环境因子变化的分析研究。
结论
本论文的研究结果表明:丹参毛状根具有良好的再现原药材的特性,可以替代原药材作为研究模型,各个批次遗传、化学比较稳定,取材方便,生长繁殖快,具有优良的实验物性,作为模型具有一定的实用性。
通过对丹参毛状根的评价研究,认为毛状根可以作为研究模型。用毛状根为模型,可以以道地药材表型与生境及基因型的相互关系为切入点,深入研究道地药材对环境的响应,基因的功能、表达的调控机制等。
本论文首次提出道地药材研究模型,并系统的对毛状根作为模型进行评价,为道地药材的研究提供实验平台,是本论文的创新点。
As the soul of traditional Chinese medicine,the geo-herblism of Chinese material medica not only has historical and cultural nature,but also concerns heredity, environment and production,etc.The forming of geoherb is resulted from the interaction of genotype and environment.It can be expressed by this formula: phenotype = genotype + environmental modification.Geoherbs is a complex system, and we can not study the factors and interaction among them simultaneously,but we should grasp the key point and simplify the object to find a breakthrough.And the model for scientific study is used by the researchers of geoherbs,which has the characteristics of simplified objects.It is very important to construct the geoherb's model in studying the biology of it.
The model,constructed from archetype,is widely used in biology,such as to analyze the phenomena of biology,to post the rules and characters of life essence and to study the interaction of the factors in dynamic life systems etc.Model and model methods to study the object become very important in life sciences,for example the bio-model in genome research and medicinal experiment.
The culture of hairy root is a new technology combined with gene engineering and cell engineering developed from 1980's.It transforms the T-DNA in Ri plasmid to the DNA of plant cell,and induced the hairy root.Hairy root culture is an effective way for long-term preservation of valuable genetic materials,and to obtain active compounds of some medical plants,especially for those plants,the interest secondary metabolites of which are accumulated in roots.The growth of hairy root is rapid and it is not restricted by climate,plant disease and insect pests,geography and seasons.
Genetic instability would be induced during continuous terms in vitro maintenance.Structural chromosomal rearrangements,polyploidy,and mutations also contribute to the culture-induced variations.So whether the hairy root can be used as model to study the geoherb,whether it is reappearance to the original plant,whether it is stable to repeat the results as a tool,all need our evaluating systemically.
According to the problems which needs resolving in geoherbs study,this dissertation uses hairy root of Salvia miltiorrhiza as a model based on the job of out laboratory,to study whether the hairy root has the attribute of model in geoherb research.The evaluation of the model includes many technologies such as phytochemistry,molecular biology,pharmacology and so on.It is mainly include 3 aspects:①Reappearance study.Evaluating the comparability of hairy root and its original plant include chemical components,toxity and pharmacokinetic study to clarify whether the hairy root can replace the original plant S.miltiorrhiza.②Stability study.Evaluating the stability of rol gene,nucleus DNA and chemical contents.③Practicability study:Evaluating the practicability based on the above two phases.The aim is to study the phenotype variation such as growth rate and the content of chemical components of hairy root by changing the culture condition.
1 Reappearance study on hairy root of S.miltiorrhiza(comparability with original plant)
1.1 Comparability of chemical components Determinate the content of tanshinoneⅡ_A and tashinoate B which index in Chinese pharmacopoeia by HPLC method of hairy root and S.miltiorrhiza from Hebei province.The content of tanshinoneⅡ_A and tashinoate B in S.miltiorrhiza from Hebei province are 0.12%, 5.4%.In hairy roots,the content of tanshinoneⅡ_A is 0.04%～0.10%,the average value is lower than plant;the content of tashinoate B is 3.21%～7.25%,the average value is higher than plant.Meanwhile,the fingerprints of hairy root and S.miltiorrhiza from Hebei and Sichuan province were compared.The similarity of them is higher than 0.948 after detected by software "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" published by Chinese pharmacopoeia commission(Version 2004A).It's no significant different of them.So the chemical components of hairy root and its original plant S.miltiorrhiza are similar.
1.2 Acute toxicity The LD_(50) of abstract of hairy root is 63.94g crude drug/kg,95% confidence interval is(58.82～69.51)g crude drug/kg,which is 255.76 times of an adult a day's quantity in clinical.It showed that the hairy root is lower toxicity.
1.3 Comparability of pharmacodynamics Studying on protection by S.miltiorrhiza hairy root on myocardial ischemia and reperfusion injury through the three levels of intact rats,isolated rat hearts and cultured neonatal rat cardiomyocytes.①Protection by S.miltiorrhiza hairy root on myocardial ischemia and reperfusion injury in the anesthetized rat.The result showed that the protection induced by S.miltiorrhiza hairy root reduced the incidence of ventricular tachycardia(VT) and ventricular fibrillation (VF) during the period of ischemia and reperfusion,the rise of plasma level of LDH, MDA decreased and the activity of SOD increased.It's no significant different between the hairy root and S.miltiorrhiza.②Protection by S.miltiorrhiza hairy root on myocardial ischemia and reperfusion injury in isolated Langendorff rat hearts.Our results showed that S.miltiorrhiza hairy root preconditioning can pervent postischmic abnormalities in reperfusion arrhythmias,aortic flow and myocardium metabolism. It's no significant different between the hairy root and S.miltiorrhiza.③Protection by S.miltiorrhiza hairy root on hypoxia and reoxygenation injury in cultured neonatal rat cardiomyocytes.The results demonstrated that S.miltiorrhiza hairy root increased the survival rate during hypoxia and reoxygenation.The rise of LDH and MDA significantly decreased and the activity of SOD significantly increased.The results above can show that preconditioning by S.miltiorrhiza hairy root demonstrated protection on hypoxia and reoxygenation injury in cultured neonatal rat cardiomyocytes.It's no significant difference between the hairy root and S. miltiorrhiza.
2 Stability study on hairy root of S.miltiorrhiza
2.1 Stability of nucleus DNA Flow cytometry analyses were carried out with S. miltiorrhiza hairy roots.The nucleus from leaf tissues of 2-week old seedling Glycine max was used as internal reference standard.The long-term in vitro maintained hairy root lines of five passages had nucleus DNA contents average 0.18 pg/2C.RSD is 6.6%.The variations in the nucleus DNA contents of the hairy root lines have no significantly difference.
2.2 Stability of rol genes The rol gene was detected by PCR in DNA from cultures in 10 passages.We found the rolA gene in all the 10 passages after detected by PCR in DNA.We recovered the rolA gene,sequencing and alignment results showed that it is homogeneous with Genbank(X12579).It showed that the rol gene in hairy root lines of 10 passages is stable.
2.3 Stability of chemical components The fingerprints of hairy root lines of 10 passages were compared.The similarity of them is higher than 0.970 after detected by software "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" published by Chinese pharmacopoeia commission (Version 2004A).It's no significant different of them.Meanwhile,determinate the growth rate of hairy root of 10 passages,it's no significant difference of them too.
3 Practicability study on hairy root of S.miltiorrhiza
3.1 Study on the effective component of S.miltirrhiza hairy root in different cultivating time.Cultivate the hairy root in 6,7-V culture medium,harvest in the 2th, 4th,6th,8th,10th weeks.Determinate the growth rate and the content of tanshinoneⅡ_A and tashinoate B by HPLC method.The 6th week is the proper time to harvest the hairy roots which the growth rate and the content were higher.So the period of Salvia miltirrhiza hairy root is short,6-8 weeks can be harvest,.The growth of S.miltirrhiza hairy root and obtain active compounds are rapidly.
3.2 Study on the effects of mineral nutrition in the growth and secondary metabolism of S.miltiorrhiza hairy root This experiment studied on the effects of mineral nutrition nitrogen(N),potassium(K),ferrum(Fe),magnesium(Mg),single element,in the growth and secondary metabolism of S.miltiorrhiza hairy root base on the 6,7-V medium.It is showed that N has significant affect to growth,secondly is K, Fe and Mg have no significant affect to growth.The lower concentration of N and CK of Fe and Mg are good for accumulating of tanshinoneⅡ_A.Higher concentration of N and K are good for accumulating of tashinoate B.The hairy root would be stimulated differently by the change of this 4 elements' concentration.It proved that the hairy root is sensitive to mineral nutrition and can be used as model to study fatherly.
Conclusion
The results of this dissertation indicate that the S.miltirrhiza hairy root has good reappearance character to S.miltirrhiza.It can be used as a model to study replacing original plants because of its stability in genetic,chemical character and because it is easy to harvest from rapid growth and is practicable.
It provides a good evidence to using S.miltirrhiza hairy root fatherly based on the evaluation of S.miltirrhiza hairy root as a model to study on geoherbs biology characteristics more deeply.For example,we can study the phenotype variation of hairy root by simulating one or more factors and making a set of grads concentration which affects the lives and quality of geoherbs.We can also study the function gene's express and regulation in geoherbs by transforming exogenous genes to hairy root.
Evaluating hairy root as model and putting forward that the hairy root can be used in studying biological essence of geoherbs,are the innovations of this dissertation.
引文
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