| 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 tanshinoneⅡA and tashinoate B which index in Chinese pharmacopoeia by HPLC method of hairy root and S.miltiorrhiza from Hebei province.The content of tanshinoneⅡA and tashinoate B in S.miltiorrhiza from Hebei province are 0.12%, 5.4%.In hairy roots,the content of tanshinoneⅡA 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 LD50 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.miltiorrhiza2.1 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.miltiorrhiza3.1 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 tanshinoneⅡA 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 tanshinoneⅡA.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.ConclusionThe 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. |
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