虫草ITS序列分子进化分析及其复合营营养液的体外细胞作用研究
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摘要
药用真菌用于治疗人类疾病有着悠久的历史,由于其具有高蛋白、低脂肪、低热量以及独特的优越性,近年来更被国内外学者关注。虫草、灵芝与猴头三种珍稀的食药用真菌,据研究显示具有抗肿瘤、抗氧化、提高免疫力、调节新陈代谢等多种药用。但野生虫草与猴头资源稀缺,为更好地开发药用真菌资源,使用液体发酵菌丝体己成为替代资源的一条重要的途径。但由于虫草的复杂生活史,以及系统发育地位的混乱,如何正确地鉴定虫草无性型已成为虫草液体发酵并开发其药用功效的首要条件。
本研究采用PCR及DNA测序的方法,对13株不同产地的虫草分离菌ITS区序列进行分析,确认ITS作为条形码的可行性,成功鉴定了供试分离菌。同时采用生物信息学方法对Genbank中收集的虫草属36个物种以及肉座菌目的191个物种的ITS区序列进行多序列比对,构建进化树等分析,探讨虫草的系统发育地位及与相关物种的近缘关系。随后,本研究通过对已鉴定的虫草无性型,猴头,灵芝三种药用真菌进行液体发酵培养,并通过破碎,胶体磨粉碎,超声破壁等方法制备复合营养液,并通过DNS法测定了营养液中的多糖含量。,为了探讨复合营养液的药理作用,本研究采用MTT细胞活力测定,DNA梯状电泳,荧光染色等方法分析了复合营养液对MG63,Hela,HepG-2等体外肿瘤细胞凋亡的诱导作用。另外研究了复合营养液对过氧化氢造成的PC12细胞氧化应激损伤的保护作用,并测定其NO水平和LDH活性的变化。主要研究结果：
1.通过ITS条形码鉴定分析,确认了CM-m5、CM-m7、CM-ml、 CM-m3和CM-m8为蛹虫草的无性型；CS-2和CS-kl为蝙蝠蛾拟青霉。
2.成功制备了虫草复合营养液,采用DNS方法测定还原糖与总糖的方法测定了营养液中含有的多糖含量为22.52mg/ml,并考察了DNS法的稳定性,重复性及加样回收率。
3.复合营养液能够有效抑制MG63、Hela等肿瘤细胞的增殖,并呈剂量依赖性,同时在高剂量营养液的作用下,可抑制HUVEC细胞的增殖,而在低剂量作用时,营养液可促进其生长,但对HepG-2肝癌细胞的抑制作用在48小时内并未体现。通过荧光双染以及DNA Ladder等指标检测凋亡作用,显示复合营养液可通过诱导MG63细胞凋亡从而达到抗肿瘤的效果。
4.使用过氧化氢对PC12细胞进行造模处理,最终确定最佳的H202浓度为400μM,作用时间为24小时。通过MTT实验确认了当加药浓度在5%以下时为复合营养液对PC12细胞的安全浓度,在此基础上考察复合营养液对神经细胞氧化应激的保护作用,结果表明复合营养液能够有效地降低细胞上清LDH活性及NO含量,有效地减轻氧化损伤。
主要结论：
1. ITS序列可以作为虫草条形码对虫草及其相关物种进行鉴定以及系统发育分析,并从分子水平上验证虫草与其无性型之间的对应关系,确认了虫草属被划分为肉座菌目一员的说法,但与虫草与麦角菌科的从属关系相矛盾。
2.复合营养液含有较为丰富的蕈菌多糖,在一定剂量内能够有效抑制肿瘤细胞及与肿瘤生长有关的血管内皮细胞的增殖,并诱导其凋亡,为后续的体内抗肿瘤工作打下基础。
3.复合营养液可通过降低自由基NO水平以及减少LDH渗漏等方式对氧化应激损伤的PC12细胞产生保护作用,也为后续的抗神经退行性疾病作用提供了有效的细胞实验依据。
Medical fungi in the treatment of human disease has a long history. In recent years, more and more scholars pay close attention to them, because of their high protein, low fat, low calorie, and unique advantages. Research shows that they have a variety of pharmacological effects on anti-tumor, anti-oxidation, enhance immunity, and regulation of metabolism. However, wild Cordyceps sinensis and Hericium erinaceus are scarcity resources. In order the better development of the medicinal fungi resources, mycelium of liquid fermentation has become an important way to get alternative resources. However, due to the complex life history and the confused phylogenetic status of the Cordyceps, the correct identification of Cordyceps anamorph has become the first condition for the Cordyceps liquid fermentation and the development of its pharmacological properties.
In this study, PCR and DNA sequencing methods were used to analyze the isolated ITS sequences, confirm the feasibility of ITS as a barcode and identified13isolated strain from different origin. Multiple sequences alignment of ITS region sequences for36species of Cordyceps genus and191species of hypocreales from Genbank, phylogenetic tree analysis were used to investigate the phylogenetic position of Cordyceps and related species by bioinformatics. In this study, Cordyceps militaris, Hericium erinaceus, and Ganoderma lucidum were carried out liquid fermentation culture. The complex nutrient liquid was prepared by the method of the broken, colloid mill grinding, ultrasonic broken. Polysaccharide content of the nutrient liquid was determined by the DNS method.
In order to investigate the pharmacological effects of the complex nutrient liquid, the MTT cell viability determination, DNA ladder electrophoresis, staining were used to analysis the induction of apoptosis to the MG63, Hela, HepG-2cells in vitro. In addition, protective effect of the complex nutrient liquid in PC12cells caused by oxidative stress injury of hydrogen peroxide was researched, The changes its level of NO and LDH activity were determined.
Major results:
1. Through the ITS barcode identification, CM-m5, CM-m7CM-ml, CM-m3, CM-m8were confirmed for the anamorph of Cordyceps militaris and CS-2and CS-kl were Hepialidae Paecilomyces.
2. The complex nutrient liquid was successfully prepared. Polysaccharide content for22.52mg/ml and the stability, reproducibility and sample recovery of the DNS method were studied.
3. The complex nutrient liquid can effectively inhibit the proliferation of MG63and Hela celsl, showing a dose-dependent. High levels of complex nutrient liquid could inhibit the proliferation of HUVEC, but the low-dose one can promote the growth of HUVEC. The complex nutrient liquid does not reflect the inhibition of HepG-2hepatoma cells within48hours. Fluorescent double staining and DNA ladder showed that complex nutrient liquid induced apoptosis in MG63cells to achieve anti-tumor effect.
4. Hydrogen peroxide was used in PC12cells modeling, the optimal concentration of H2O2was400μM, for24h. The safe concentration of the composite nutrient solution in PC12cells was dosing concentration below5%, confirmed by MTT assay. On this basis, the protective effect of The complex nutrient liquid on the nerve cells to oxidative stress was examined. The results showed that the complex nutrient liquid can effectively reduce the cell supernatant LDH activity and NO content, and reduce the oxidative damage.
Main conclusions:
1. ITS sequence could be used as a barcode to identification and phylogenetic analysis in Cordyceps and its related species. Verify the correspondence between Cordyceps and its anamorphs in the molecular level and confirm that Cordyceps is divided into Hypocreales.
2. The complex nutrient liquid has rich mushroom polysaccharide, which inhibit the proliferation of tumor cells and endothelial cells in tumor growth and induce apoptosis with a certain dose. That lay the foundation for the subsequent study in vivo.
3. The complex nutrient liquid can produce a protective effect on PC12cells to oxidative stress injury by reducing the free radical NO level and the LDH leakage. Also provide an effective experimental basis for the next study, which can be used in anti-neurodegenerative diseases.
本研究采用PCR及DNA测序的方法,对13株不同产地的虫草分离菌ITS区序列进行分析,确认ITS作为条形码的可行性,成功鉴定了供试分离菌。同时采用生物信息学方法对Genbank中收集的虫草属36个物种以及肉座菌目的191个物种的ITS区序列进行多序列比对,构建进化树等分析,探讨虫草的系统发育地位及与相关物种的近缘关系。随后,本研究通过对已鉴定的虫草无性型,猴头,灵芝三种药用真菌进行液体发酵培养,并通过破碎,胶体磨粉碎,超声破壁等方法制备复合营养液,并通过DNS法测定了营养液中的多糖含量。,为了探讨复合营养液的药理作用,本研究采用MTT细胞活力测定,DNA梯状电泳,荧光染色等方法分析了复合营养液对MG63,Hela,HepG-2等体外肿瘤细胞凋亡的诱导作用。另外研究了复合营养液对过氧化氢造成的PC12细胞氧化应激损伤的保护作用,并测定其NO水平和LDH活性的变化。主要研究结果：
1.通过ITS条形码鉴定分析,确认了CM-m5、CM-m7、CM-ml、 CM-m3和CM-m8为蛹虫草的无性型；CS-2和CS-kl为蝙蝠蛾拟青霉。
2.成功制备了虫草复合营养液,采用DNS方法测定还原糖与总糖的方法测定了营养液中含有的多糖含量为22.52mg/ml,并考察了DNS法的稳定性,重复性及加样回收率。
3.复合营养液能够有效抑制MG63、Hela等肿瘤细胞的增殖,并呈剂量依赖性,同时在高剂量营养液的作用下,可抑制HUVEC细胞的增殖,而在低剂量作用时,营养液可促进其生长,但对HepG-2肝癌细胞的抑制作用在48小时内并未体现。通过荧光双染以及DNA Ladder等指标检测凋亡作用,显示复合营养液可通过诱导MG63细胞凋亡从而达到抗肿瘤的效果。
4.使用过氧化氢对PC12细胞进行造模处理,最终确定最佳的H202浓度为400μM,作用时间为24小时。通过MTT实验确认了当加药浓度在5%以下时为复合营养液对PC12细胞的安全浓度,在此基础上考察复合营养液对神经细胞氧化应激的保护作用,结果表明复合营养液能够有效地降低细胞上清LDH活性及NO含量,有效地减轻氧化损伤。
主要结论：
1. ITS序列可以作为虫草条形码对虫草及其相关物种进行鉴定以及系统发育分析,并从分子水平上验证虫草与其无性型之间的对应关系,确认了虫草属被划分为肉座菌目一员的说法,但与虫草与麦角菌科的从属关系相矛盾。
2.复合营养液含有较为丰富的蕈菌多糖,在一定剂量内能够有效抑制肿瘤细胞及与肿瘤生长有关的血管内皮细胞的增殖,并诱导其凋亡,为后续的体内抗肿瘤工作打下基础。
3.复合营养液可通过降低自由基NO水平以及减少LDH渗漏等方式对氧化应激损伤的PC12细胞产生保护作用,也为后续的抗神经退行性疾病作用提供了有效的细胞实验依据。
Medical fungi in the treatment of human disease has a long history. In recent years, more and more scholars pay close attention to them, because of their high protein, low fat, low calorie, and unique advantages. Research shows that they have a variety of pharmacological effects on anti-tumor, anti-oxidation, enhance immunity, and regulation of metabolism. However, wild Cordyceps sinensis and Hericium erinaceus are scarcity resources. In order the better development of the medicinal fungi resources, mycelium of liquid fermentation has become an important way to get alternative resources. However, due to the complex life history and the confused phylogenetic status of the Cordyceps, the correct identification of Cordyceps anamorph has become the first condition for the Cordyceps liquid fermentation and the development of its pharmacological properties.
In this study, PCR and DNA sequencing methods were used to analyze the isolated ITS sequences, confirm the feasibility of ITS as a barcode and identified13isolated strain from different origin. Multiple sequences alignment of ITS region sequences for36species of Cordyceps genus and191species of hypocreales from Genbank, phylogenetic tree analysis were used to investigate the phylogenetic position of Cordyceps and related species by bioinformatics. In this study, Cordyceps militaris, Hericium erinaceus, and Ganoderma lucidum were carried out liquid fermentation culture. The complex nutrient liquid was prepared by the method of the broken, colloid mill grinding, ultrasonic broken. Polysaccharide content of the nutrient liquid was determined by the DNS method.
In order to investigate the pharmacological effects of the complex nutrient liquid, the MTT cell viability determination, DNA ladder electrophoresis, staining were used to analysis the induction of apoptosis to the MG63, Hela, HepG-2cells in vitro. In addition, protective effect of the complex nutrient liquid in PC12cells caused by oxidative stress injury of hydrogen peroxide was researched, The changes its level of NO and LDH activity were determined.
Major results:
1. Through the ITS barcode identification, CM-m5, CM-m7CM-ml, CM-m3, CM-m8were confirmed for the anamorph of Cordyceps militaris and CS-2and CS-kl were Hepialidae Paecilomyces.
2. The complex nutrient liquid was successfully prepared. Polysaccharide content for22.52mg/ml and the stability, reproducibility and sample recovery of the DNS method were studied.
3. The complex nutrient liquid can effectively inhibit the proliferation of MG63and Hela celsl, showing a dose-dependent. High levels of complex nutrient liquid could inhibit the proliferation of HUVEC, but the low-dose one can promote the growth of HUVEC. The complex nutrient liquid does not reflect the inhibition of HepG-2hepatoma cells within48hours. Fluorescent double staining and DNA ladder showed that complex nutrient liquid induced apoptosis in MG63cells to achieve anti-tumor effect.
4. Hydrogen peroxide was used in PC12cells modeling, the optimal concentration of H2O2was400μM, for24h. The safe concentration of the composite nutrient solution in PC12cells was dosing concentration below5%, confirmed by MTT assay. On this basis, the protective effect of The complex nutrient liquid on the nerve cells to oxidative stress was examined. The results showed that the complex nutrient liquid can effectively reduce the cell supernatant LDH activity and NO content, and reduce the oxidative damage.
Main conclusions:
1. ITS sequence could be used as a barcode to identification and phylogenetic analysis in Cordyceps and its related species. Verify the correspondence between Cordyceps and its anamorphs in the molecular level and confirm that Cordyceps is divided into Hypocreales.
2. The complex nutrient liquid has rich mushroom polysaccharide, which inhibit the proliferation of tumor cells and endothelial cells in tumor growth and induce apoptosis with a certain dose. That lay the foundation for the subsequent study in vivo.
3. The complex nutrient liquid can produce a protective effect on PC12cells to oxidative stress injury by reducing the free radical NO level and the LDH leakage. Also provide an effective experimental basis for the next study, which can be used in anti-neurodegenerative diseases.
引文
[1]Mori K, Inatomi S, Ouchi K, et al. Improving effects of the mushroom Yamabushitake (Hericium erinaceus) on mild cognitive impairment:a double-blind placebo-controlled clinical trial[J]. Phytotherapy Research.2009,23(3):367-372.
[2]潘静,陈生弟.氧化应激与神经退行性疾病[J].国际神经病学神经外科学杂志.2008,35(2)：40-45.
[3]Hiroki Sato & Mitsuaki Shimazu. Stromata production for Cordyceps militaris (Clavicipitales:Clavicipitaceae) by injection of hyphal bodies to alternative host insects. Appl.Entomol.Zool.2002,37(1):85-92.
[4]刘淑艳,张傲,李玉,等.菌物DNA条形码技术的研究进展[J].华中农业大学学报,2012,1(31)：121-126.
[5]Hodge, K.T., S.B. Krasnoff and R.A. Humber, Tolypocladium inflatum is the anamorph of Cordyceps subsessilis. Mycologia,1996:715-719.
[6]蒋毅,姚一建,冬虫夏草无性型研究概况,菌物系统,22(2003)161-76.
[7]李春如,左登平,南圣姬,等.高雄山虫草及其细脚拟青霉无性型[J].菌物学报.2007,26(2)：217-220.
[8]左登平,李春如,黄勃,等.台湾虫草及其无性型关系的分子确证[J].菌物学报.2008,,46(2)：123-129.
[9]Sung, JM, HK Lee, KY Yang, et al. Classification of Cordyceps spp. by morphological characteristics and protein banding pattern[J]. Korean Journal of Mycology, 1995,23(1): 92-104.
[10]张莉,李海生.3.5二硝基水杨酸比色法测定甘草多糖肠溶片中多糖的含量[J].2004天津市第十六届色谱学术交流会论文集.2004.,
[11]庄筱葳,刘秀芳,毛贵元,等.西藏红雪茶多糖含量测定[J].中国实验方剂学杂志.2012,18(004)：103-106.
[12]王晓玉,蒋秋燕,凌沛学,等.猴头菌活性成分及药理作用研究进展[J].中国生化药物杂志.2010(001)：70-72.
[13]王新民,李宇伟,连瑞丽,等.灵芝液体发酵及产多糖的初步研究[J].农业科学与技术：英文版.2008,8(3)：15-19.
[14]赵丰丽.预处理对水提取醇沉法提取虫草多糖效果的影响研究[J].食品工业科技.2008(1)：140-142.
[15]胡玲爱.梓醇对H202诱导HUVECs和H9c2细胞凋亡的影响及其机制的研究[D].中国医科大学,2010.
[16]Kolotushkina E V, Moldavan M G, Voronin K Y, et al. The influence of Hericium erinaceus extract on myelination process in vitro.[J]. Fiziolohichnyi zhurnal, 2003,49(1): 38.
[17]匡荣.苁蓉总苷和松果菊苷对体内外氧化应激阿尔茨海默病模型的作用及机理研究[D].浙江大学,2009.
[18]李晓峰.红景天苷通过抑制一氧化氮通路对抗MPP-+诱导的PC12细胞凋亡[D].第四军医大学,2010.
[19]唐小卿,冯鉴强,胡弼.H202预处理对多巴胺损伤PC12细胞的适应性保护作用[J],中国药理学通报.2005,12(6)：721-725.
[20]郁利平,闫秀欣.蛹虫草对癌诱变剂—MMS所致BALB/C小鼠脾细胞DNA损伤的拮抗作用[J].实用肿瘤学杂志.1994.8(002)：7-8.
[21]王薇,王利丽,王青龙,李迪强,茆灿泉.虫草ITS基因指纹及生信分析[A].首届中国蕈菌与健康高峰论坛论文集.2010.
[22]I.S. Druzhinina, A.G. Kopchinskiy, M. Komon, et al. An oligonucleotide barcode for species identification in Trichoderma and Hypocrea[J]. Fungal Genet Biol,2005,42(10): 813-828.
[23]Conrad L. Schoch, Joseph W. Spatafora, H, et al. A phylogenetic re-evaluation of Dothideomycetes[M],2009.
[24]Eric W.A. Boehma, Conrad L. Schochb, Joseph W. Spataforab.On the evolution of the Hysteriaceae and Mytilinidiaceae (Pleosporomycetidae, Dothideomycetes, Ascomycota) using four nuclear genes[J], Mycological Research,2009,113(4):461-479.
[25]S. Artjariyasripong,J. I. Mitchell,N. L. Hywel-Joneset al. Relationship of the Genus Cordyceps and related genera, based on parsimony and spectral analysis of partial 18S and 28S ribosomal gene sequences[J]. Mycoscience.2001.42(6):503-517.
[26]A. E. Glenn,C. W. Bacon,R. Priceet al. Molecular phylogeny of Acremonium and its taxonomic implications[J]. Mycologia.1996:369-383.
[27]G. H. Sung,N. L. Hywel-Jones,J. M. Sunget al. Phylogenetic classification of Cordyceps and the clavicipitaceous fungi[J]. Studies in Mycology.2007.57(1):5-59.
[28]Y. Kobayasi. Keys to the taxa of the genera Cordyceps and Torrubiella[J]. Transaction of the Mycological Society of Japan.1982.23:329-364.
[29]G. H. Sung,J. W. Spatafora,R. Zareet al. A revision of Verticillium sect Prostrata. II. Phylogenetic analyses of SSU and LSU nuclear rDNA sequences from anamorphs and teleomorphs of the Clavicipitaceae[J]. Nova Hedwigia.2001.72(3-4):311-328.
[30]S. Artjariyasripong,J. I. Mitchell,N. L. Hywel-Joneset al. Relationship of the genusCordyceps and related genera, based on parsimony and spectral analysis of partial 18S and 28S ribosomal gene sequences[J]. Mycoscience.2001.42(6):503-517.
[31]王宁,章卫民.虫草属多元起源的分子生物学证据[J].中山大学学报：自然科学版.2000.39(4)：70-73.
[32]J. M. Sung, H. K. Lee, K. J. Yang. Classification of Cordyceps spp. by morphological characteristics and protein banding pattern[J]. Korean Journal of Mycology. 1995.23(1): 92-104.
[33]Z. Liu,Z. Liang,A. Liu, et al. Molecular evidence for teleomorph-anamorph connections in Cordyceps based on ITS-5.8 S rDNA sequences[J]. Mycological Research, 2002,106: 1100-1108.
[34]Stensrud,N. L. Hywel-Jones,T. Schumacher. Towards a phylogenetic classification of Cordyceps:ITS nrDNA sequence data confirm divergent lineages and paraphyly[J], Mycol. Res,2005,109:41-56.
[35]K. A. Seifert,H. Boulay. Hirsutella uncinata, a new hyphomycete from Australia[J]. Mycologia.2004.96(4):929-934.
[36]Z. Liu,Z. Liang,A. Liuet al. Molecular evidence for teleomorph-anamorph connections in Cordyceps based on ITS-5.8 S rDNA sequences[j], Mycological Research,106(9): 1100-1108.
[37]赵锦,李泰辉.冬虫夏草无性型的分子鉴别[J].中山大学学报：自然科学版.1999.38(1)：121-123.
[38]杨金玲,肖薇,何惠霞等.蝙蝠蛾拟青霉与冬虫夏草关系的分子系统学研究[J].药学学报.2008.43(4)：421-426.
[39]李增智,黄勃,李春如等.确证冬虫夏草无性型的分子生物学证据[J].菌物系统.2000.19(1)：60-64.
[40]张泽文,傅岚,陈作红.古尼虫草无性型的分子鉴别[J].菌物学报.2005.24(3)：344-348.
[41]梁宗琦.蛹虫草无性型—蛹草拟青霉的确证[J].食用菌学报.2001.8(4)：28-32.
[37]F. Driver,R. J. Milner,J. W. H. Trueman. A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data [J]. Mycological Research, 2000,102,134-150.
[43]左登平,李春如,陈名君等.根足虫草琅琊山变种及其无性型关系的分子确证[J].菌物学报.2008.27(3)：469-473
[44]赵雪梅,张辉,姜红霞等.泰山虫草无性型的分子研究[J].中药材.2010.9：35-45.
[45]左登平,李春如,黄勃等.台湾虫草及其无性型关系的分子确证[J]：2008,27(2)：123-128.
[46]N. Nikoh,T. Fukatsu. Interkingdom host jumping underground:phylogenetic analysis of entomoparasitic fungi of the genus Cordyceps[J]. Molecular Biology and Evolution. 2000.17(4):629-638.
[47]N. Nikoh,T. Fukatsu. Evolutionary dynamics of multiple group I introns in nuclear ribosomal RNA genes of endoparasitic fungi of the genus Cordyceps[J]. Molecular Biology and Evolution.2001.18(9):1631-1642.
[48]杨成雄,夏继伟,杨希雄等.虫草洋参胶囊抗疲劳与免疫作用及急性毒性实验[J].中国医院药学杂志.2006.26(1)：10-11.
[49]刘杰麟,费樱,戴氏虫草和粉被虫草多糖对巨噬细胞等活性的影响[m].免疫学杂志,2001,17(3)：189-191.
[50]]肖建辉,梁宗琦,胡锡阶等.古尼虫草多糖及其解聚物的免疫活性[J].免疫学杂志.2005.21(1)：51-53.
[51]唐荣江,闵照华.冬虫夏草及青海虫草菌对小鼠血清溶菌酶水平的影响[J].西北药学杂志.1988(4)：74-81.
[52]刘春泉,宋江峰,李大婧等.虫草素的提取纯化及测定方法研究进展[J].食品科学.2007.28(11)：596-599.
[53]沈新娥,龚珊,蒋星红等.复方虫草精华对小鼠免疫功能的影响[J].中国血液流变学杂志.2004.13(4)：327-330.
[54]王峰,张才军,王玲等.云南虫草菌粉对小鼠迟发型超敏反应影响的实验研究[J].中国误诊学杂志.2010(3)：547-548.
[55]孙万峰,孙荣玲,姜维苓等.复方冬虫夏草多糖脂质体口服液治疗慢性乙肝的临床观察[J].中国药师.2003.6(7)：438-439.
[56]刘蛟,宓伟,李琳等.冬虫夏草对小鼠淋巴细胞增殖和IL-2产生的作用[J].滨州医学院学报.2008.31(3)：196-197.
[57]索志荣,刘效珍.宁心宝治疗室性早搏的临床观察[J].中西医结合心脑血管病杂志.2005,3(007)：643-644.
[58]李雪芹,刘建云.冬虫夏草对垂体后叶素所致大鼠缺血心肌的保护作用[J].河北医药.2004,26(012)：934-935.
[59]王赫,周宇宏,单宏丽,等.冬虫夏草水提液对单个心室肌细胞钾通道的影响[J].中国药理学通报.2004,20(5)：536-539.
[60]龚晓健,李绍平.人工虫草提取物抗心律失常作用的研究[J].中国药科大学学报.2001,32(003)：221-223.
[61]韩冰,王泽君,王天,等.人工虫草提取物抗急性心肌缺血作用的实验研究[J].时珍国医国药.2005,16(8)：745-746.
[62]冯鸣国,周前贵,冯高闳,等.人工培养虫草菌菌丝体对麻醉犬血管的扩张作用[J].中国中药杂志.1987,12(12)：41-45.
[63]吴秀香,李淑云,马克玲,等.冬虫夏草对肾性高血压大鼠血管重构的影响[J].数理医药学杂志.2005,18(001)：46-47.
[64]申大伟,朱海波,全吉淑,等.虫草素对HepG2培养细胞降脂作用机制初探[J].东北三省生物化学与分子生物学学会2008年学术交流会论文摘要.2008.
[65]Guo P, Kai Q, Gao J, et al. Cordycepin prevents hyperlipidemia in hamsters fed a high-fat diet via activation of AMP-activated protein kinase[J]. Journal of pharmacological sciences.2010,113(4):395-403.
[66]Kim H G, Shrestha B, Lim S Y, et al. Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kB through Akt and p38 inhibition in RAW 264.7 macrophage cells[JJ. European journal of pharmacology.2006, 545(2-3):192-199.
[67]李宪花,管益君.虫草肾康胶囊对慢性肾小球肾炎疗效的研究[J].深圳中西医结合杂志.1999,6.
[68]魏冬梅,高山林,张红梅,等.百令胶囊治疗慢性肾炎的临床对照研究[J].中国综合临床.2006,22(6)：514-515.
[69]杨楠.蛹虫草提取物对大鼠系膜增生型肾小球肾炎模型的防治作用[D].华中科技大学,2009.
[70]王静凤,李晓林,张殉,等.海参虫草复剂对糖尿病大鼠肾脏保护作用机制的研究[J].中国药理学通报.2010,26(009)：1238-1242.
[71]韩亚莉,傅余芹,方华伟,等.虫草菌丝对糖尿病大鼠肾组织中基质金属蛋白酶2及其组织抑制因子2表达的影响[J].中国老年学杂志.2008,28(17)：1668-1671.
[72]孙纳新.蛹虫草多糖降血糖的机理研究[D].山东师范大学,2005.
[73]Hekerman P, Zeidler J, Korfmacher S, et al. Leptin induces inflammation-related genes in RINm5F insulinoma cells[J]. BMC molecular biology. 2007,8(1):41-46.
[74]郑倩,刘红,曹弟勇,等.虫草菌丝提取物对白介素1β损伤的胰岛细胞损伤保护作用的实验研究[J].中国中医基础医学杂志.2009,14(10)：765-767.
[75]张小强,浦跃朴,尹立红,等.冬虫夏草及人工虫草菌丝体对超氧阴离子自由基和羟自由基清除作用的实验研究[J].中国老年学杂志.2004,23(11)：773-775.
[76]董秀英,吕青涛,张国英,等.DPPH法测定九州虫草不同极性部位抗氧化活性[J].中国实验方剂学杂志.2011,17(10)：70-73.
[77]沈齐英.北虫草抗活性氧自由基作用的研究[J].锦州医学院学报.1997,18(005)：11-13.
[78]朱振元,原静,李盛峰,等.古尼虫草多糖硫酸酯化修饰及其抗氧化活性[J].现代食品科技.2011,27(001)：1-5.
[79]武忠伟,许桂芳,曹蓬勃,等.虫草与富硒虫草多糖的体外抗氧化活性[J].食品科学.2011,32(09)：76-78.
[80]李春如,吴茜茜,严景华,等.几种虫草无性型及其相关真菌SOD比较研究[J].安徽农业大学学报.2006,33(001)：90-93.
[81]Li S P, Zhao K J, Ji Z N, et al. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC 12 cells against hydrogen peroxide-induced injury[J]. Life sciences.2003,73(19):2503-2513.
[82]李伯勤.虫草多糖对皮肤成纤维细胞抗氧化能力的影响[J].2010(4),34-38.
[83]Nakamura K, Yamaguchi Y, Kagota S, et al. Activation of in vivo Kupffer cell function by oral administration of Cordyceps sinensis in rats[J]. The Japanese Journal of Pharmacology. 1999,79(4):505-508.
[84]李元青,马成杰,李晓明,等.三七虫草混合粉抑制小鼠黑色素瘤效应研究[J].亚太传统医药.2007(004)：64-67.
[85]李婧,何肇晴,龚皓,等.国产虫草素对Hela细胞MMP-9, TIMP-1及TIMP-1 mRNA表达的调节[J].现代肿瘤医学.2009,17(009)：1615-1618.
[86]Yoo H S, Shin J W, Cho J H, et al.Effects of Cordyceps militaris extract on angiogenesis and tumor growth[J]. Acta Pharmacologica Sinica.2004,25(5):657-665.
[87]Price P J,Suk W A, Peters R L, et al. Cordycepin inhibition of 3-methylcholanthrene-induced transformation in vitro[C]. Royal Society of Medicine, 1975.150(3):650-653.
[2]潘静,陈生弟.氧化应激与神经退行性疾病[J].国际神经病学神经外科学杂志.2008,35(2)：40-45.
[3]Hiroki Sato & Mitsuaki Shimazu. Stromata production for Cordyceps militaris (Clavicipitales:Clavicipitaceae) by injection of hyphal bodies to alternative host insects. Appl.Entomol.Zool.2002,37(1):85-92.
[4]刘淑艳,张傲,李玉,等.菌物DNA条形码技术的研究进展[J].华中农业大学学报,2012,1(31)：121-126.
[5]Hodge, K.T., S.B. Krasnoff and R.A. Humber, Tolypocladium inflatum is the anamorph of Cordyceps subsessilis. Mycologia,1996:715-719.
[6]蒋毅,姚一建,冬虫夏草无性型研究概况,菌物系统,22(2003)161-76.
[7]李春如,左登平,南圣姬,等.高雄山虫草及其细脚拟青霉无性型[J].菌物学报.2007,26(2)：217-220.
[8]左登平,李春如,黄勃,等.台湾虫草及其无性型关系的分子确证[J].菌物学报.2008,,46(2)：123-129.
[9]Sung, JM, HK Lee, KY Yang, et al. Classification of Cordyceps spp. by morphological characteristics and protein banding pattern[J]. Korean Journal of Mycology, 1995,23(1): 92-104.
[10]张莉,李海生.3.5二硝基水杨酸比色法测定甘草多糖肠溶片中多糖的含量[J].2004天津市第十六届色谱学术交流会论文集.2004.,
[11]庄筱葳,刘秀芳,毛贵元,等.西藏红雪茶多糖含量测定[J].中国实验方剂学杂志.2012,18(004)：103-106.
[12]王晓玉,蒋秋燕,凌沛学,等.猴头菌活性成分及药理作用研究进展[J].中国生化药物杂志.2010(001)：70-72.
[13]王新民,李宇伟,连瑞丽,等.灵芝液体发酵及产多糖的初步研究[J].农业科学与技术：英文版.2008,8(3)：15-19.
[14]赵丰丽.预处理对水提取醇沉法提取虫草多糖效果的影响研究[J].食品工业科技.2008(1)：140-142.
[15]胡玲爱.梓醇对H202诱导HUVECs和H9c2细胞凋亡的影响及其机制的研究[D].中国医科大学,2010.
[16]Kolotushkina E V, Moldavan M G, Voronin K Y, et al. The influence of Hericium erinaceus extract on myelination process in vitro.[J]. Fiziolohichnyi zhurnal, 2003,49(1): 38.
[17]匡荣.苁蓉总苷和松果菊苷对体内外氧化应激阿尔茨海默病模型的作用及机理研究[D].浙江大学,2009.
[18]李晓峰.红景天苷通过抑制一氧化氮通路对抗MPP-+诱导的PC12细胞凋亡[D].第四军医大学,2010.
[19]唐小卿,冯鉴强,胡弼.H202预处理对多巴胺损伤PC12细胞的适应性保护作用[J],中国药理学通报.2005,12(6)：721-725.
[20]郁利平,闫秀欣.蛹虫草对癌诱变剂—MMS所致BALB/C小鼠脾细胞DNA损伤的拮抗作用[J].实用肿瘤学杂志.1994.8(002)：7-8.
[21]王薇,王利丽,王青龙,李迪强,茆灿泉.虫草ITS基因指纹及生信分析[A].首届中国蕈菌与健康高峰论坛论文集.2010.
[22]I.S. Druzhinina, A.G. Kopchinskiy, M. Komon, et al. An oligonucleotide barcode for species identification in Trichoderma and Hypocrea[J]. Fungal Genet Biol,2005,42(10): 813-828.
[23]Conrad L. Schoch, Joseph W. Spatafora, H, et al. A phylogenetic re-evaluation of Dothideomycetes[M],2009.
[24]Eric W.A. Boehma, Conrad L. Schochb, Joseph W. Spataforab.On the evolution of the Hysteriaceae and Mytilinidiaceae (Pleosporomycetidae, Dothideomycetes, Ascomycota) using four nuclear genes[J], Mycological Research,2009,113(4):461-479.
[25]S. Artjariyasripong,J. I. Mitchell,N. L. Hywel-Joneset al. Relationship of the Genus Cordyceps and related genera, based on parsimony and spectral analysis of partial 18S and 28S ribosomal gene sequences[J]. Mycoscience.2001.42(6):503-517.
[26]A. E. Glenn,C. W. Bacon,R. Priceet al. Molecular phylogeny of Acremonium and its taxonomic implications[J]. Mycologia.1996:369-383.
[27]G. H. Sung,N. L. Hywel-Jones,J. M. Sunget al. Phylogenetic classification of Cordyceps and the clavicipitaceous fungi[J]. Studies in Mycology.2007.57(1):5-59.
[28]Y. Kobayasi. Keys to the taxa of the genera Cordyceps and Torrubiella[J]. Transaction of the Mycological Society of Japan.1982.23:329-364.
[29]G. H. Sung,J. W. Spatafora,R. Zareet al. A revision of Verticillium sect Prostrata. II. Phylogenetic analyses of SSU and LSU nuclear rDNA sequences from anamorphs and teleomorphs of the Clavicipitaceae[J]. Nova Hedwigia.2001.72(3-4):311-328.
[30]S. Artjariyasripong,J. I. Mitchell,N. L. Hywel-Joneset al. Relationship of the genusCordyceps and related genera, based on parsimony and spectral analysis of partial 18S and 28S ribosomal gene sequences[J]. Mycoscience.2001.42(6):503-517.
[31]王宁,章卫民.虫草属多元起源的分子生物学证据[J].中山大学学报：自然科学版.2000.39(4)：70-73.
[32]J. M. Sung, H. K. Lee, K. J. Yang. Classification of Cordyceps spp. by morphological characteristics and protein banding pattern[J]. Korean Journal of Mycology. 1995.23(1): 92-104.
[33]Z. Liu,Z. Liang,A. Liu, et al. Molecular evidence for teleomorph-anamorph connections in Cordyceps based on ITS-5.8 S rDNA sequences[J]. Mycological Research, 2002,106: 1100-1108.
[34]Stensrud,N. L. Hywel-Jones,T. Schumacher. Towards a phylogenetic classification of Cordyceps:ITS nrDNA sequence data confirm divergent lineages and paraphyly[J], Mycol. Res,2005,109:41-56.
[35]K. A. Seifert,H. Boulay. Hirsutella uncinata, a new hyphomycete from Australia[J]. Mycologia.2004.96(4):929-934.
[36]Z. Liu,Z. Liang,A. Liuet al. Molecular evidence for teleomorph-anamorph connections in Cordyceps based on ITS-5.8 S rDNA sequences[j], Mycological Research,106(9): 1100-1108.
[37]赵锦,李泰辉.冬虫夏草无性型的分子鉴别[J].中山大学学报：自然科学版.1999.38(1)：121-123.
[38]杨金玲,肖薇,何惠霞等.蝙蝠蛾拟青霉与冬虫夏草关系的分子系统学研究[J].药学学报.2008.43(4)：421-426.
[39]李增智,黄勃,李春如等.确证冬虫夏草无性型的分子生物学证据[J].菌物系统.2000.19(1)：60-64.
[40]张泽文,傅岚,陈作红.古尼虫草无性型的分子鉴别[J].菌物学报.2005.24(3)：344-348.
[41]梁宗琦.蛹虫草无性型—蛹草拟青霉的确证[J].食用菌学报.2001.8(4)：28-32.
[37]F. Driver,R. J. Milner,J. W. H. Trueman. A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data [J]. Mycological Research, 2000,102,134-150.
[43]左登平,李春如,陈名君等.根足虫草琅琊山变种及其无性型关系的分子确证[J].菌物学报.2008.27(3)：469-473
[44]赵雪梅,张辉,姜红霞等.泰山虫草无性型的分子研究[J].中药材.2010.9：35-45.
[45]左登平,李春如,黄勃等.台湾虫草及其无性型关系的分子确证[J]：2008,27(2)：123-128.
[46]N. Nikoh,T. Fukatsu. Interkingdom host jumping underground:phylogenetic analysis of entomoparasitic fungi of the genus Cordyceps[J]. Molecular Biology and Evolution. 2000.17(4):629-638.
[47]N. Nikoh,T. Fukatsu. Evolutionary dynamics of multiple group I introns in nuclear ribosomal RNA genes of endoparasitic fungi of the genus Cordyceps[J]. Molecular Biology and Evolution.2001.18(9):1631-1642.
[48]杨成雄,夏继伟,杨希雄等.虫草洋参胶囊抗疲劳与免疫作用及急性毒性实验[J].中国医院药学杂志.2006.26(1)：10-11.
[49]刘杰麟,费樱,戴氏虫草和粉被虫草多糖对巨噬细胞等活性的影响[m].免疫学杂志,2001,17(3)：189-191.
[50]]肖建辉,梁宗琦,胡锡阶等.古尼虫草多糖及其解聚物的免疫活性[J].免疫学杂志.2005.21(1)：51-53.
[51]唐荣江,闵照华.冬虫夏草及青海虫草菌对小鼠血清溶菌酶水平的影响[J].西北药学杂志.1988(4)：74-81.
[52]刘春泉,宋江峰,李大婧等.虫草素的提取纯化及测定方法研究进展[J].食品科学.2007.28(11)：596-599.
[53]沈新娥,龚珊,蒋星红等.复方虫草精华对小鼠免疫功能的影响[J].中国血液流变学杂志.2004.13(4)：327-330.
[54]王峰,张才军,王玲等.云南虫草菌粉对小鼠迟发型超敏反应影响的实验研究[J].中国误诊学杂志.2010(3)：547-548.
[55]孙万峰,孙荣玲,姜维苓等.复方冬虫夏草多糖脂质体口服液治疗慢性乙肝的临床观察[J].中国药师.2003.6(7)：438-439.
[56]刘蛟,宓伟,李琳等.冬虫夏草对小鼠淋巴细胞增殖和IL-2产生的作用[J].滨州医学院学报.2008.31(3)：196-197.
[57]索志荣,刘效珍.宁心宝治疗室性早搏的临床观察[J].中西医结合心脑血管病杂志.2005,3(007)：643-644.
[58]李雪芹,刘建云.冬虫夏草对垂体后叶素所致大鼠缺血心肌的保护作用[J].河北医药.2004,26(012)：934-935.
[59]王赫,周宇宏,单宏丽,等.冬虫夏草水提液对单个心室肌细胞钾通道的影响[J].中国药理学通报.2004,20(5)：536-539.
[60]龚晓健,李绍平.人工虫草提取物抗心律失常作用的研究[J].中国药科大学学报.2001,32(003)：221-223.
[61]韩冰,王泽君,王天,等.人工虫草提取物抗急性心肌缺血作用的实验研究[J].时珍国医国药.2005,16(8)：745-746.
[62]冯鸣国,周前贵,冯高闳,等.人工培养虫草菌菌丝体对麻醉犬血管的扩张作用[J].中国中药杂志.1987,12(12)：41-45.
[63]吴秀香,李淑云,马克玲,等.冬虫夏草对肾性高血压大鼠血管重构的影响[J].数理医药学杂志.2005,18(001)：46-47.
[64]申大伟,朱海波,全吉淑,等.虫草素对HepG2培养细胞降脂作用机制初探[J].东北三省生物化学与分子生物学学会2008年学术交流会论文摘要.2008.
[65]Guo P, Kai Q, Gao J, et al. Cordycepin prevents hyperlipidemia in hamsters fed a high-fat diet via activation of AMP-activated protein kinase[J]. Journal of pharmacological sciences.2010,113(4):395-403.
[66]Kim H G, Shrestha B, Lim S Y, et al. Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kB through Akt and p38 inhibition in RAW 264.7 macrophage cells[JJ. European journal of pharmacology.2006, 545(2-3):192-199.
[67]李宪花,管益君.虫草肾康胶囊对慢性肾小球肾炎疗效的研究[J].深圳中西医结合杂志.1999,6.
[68]魏冬梅,高山林,张红梅,等.百令胶囊治疗慢性肾炎的临床对照研究[J].中国综合临床.2006,22(6)：514-515.
[69]杨楠.蛹虫草提取物对大鼠系膜增生型肾小球肾炎模型的防治作用[D].华中科技大学,2009.
[70]王静凤,李晓林,张殉,等.海参虫草复剂对糖尿病大鼠肾脏保护作用机制的研究[J].中国药理学通报.2010,26(009)：1238-1242.
[71]韩亚莉,傅余芹,方华伟,等.虫草菌丝对糖尿病大鼠肾组织中基质金属蛋白酶2及其组织抑制因子2表达的影响[J].中国老年学杂志.2008,28(17)：1668-1671.
[72]孙纳新.蛹虫草多糖降血糖的机理研究[D].山东师范大学,2005.
[73]Hekerman P, Zeidler J, Korfmacher S, et al. Leptin induces inflammation-related genes in RINm5F insulinoma cells[J]. BMC molecular biology. 2007,8(1):41-46.
[74]郑倩,刘红,曹弟勇,等.虫草菌丝提取物对白介素1β损伤的胰岛细胞损伤保护作用的实验研究[J].中国中医基础医学杂志.2009,14(10)：765-767.
[75]张小强,浦跃朴,尹立红,等.冬虫夏草及人工虫草菌丝体对超氧阴离子自由基和羟自由基清除作用的实验研究[J].中国老年学杂志.2004,23(11)：773-775.
[76]董秀英,吕青涛,张国英,等.DPPH法测定九州虫草不同极性部位抗氧化活性[J].中国实验方剂学杂志.2011,17(10)：70-73.
[77]沈齐英.北虫草抗活性氧自由基作用的研究[J].锦州医学院学报.1997,18(005)：11-13.
[78]朱振元,原静,李盛峰,等.古尼虫草多糖硫酸酯化修饰及其抗氧化活性[J].现代食品科技.2011,27(001)：1-5.
[79]武忠伟,许桂芳,曹蓬勃,等.虫草与富硒虫草多糖的体外抗氧化活性[J].食品科学.2011,32(09)：76-78.
[80]李春如,吴茜茜,严景华,等.几种虫草无性型及其相关真菌SOD比较研究[J].安徽农业大学学报.2006,33(001)：90-93.
[81]Li S P, Zhao K J, Ji Z N, et al. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC 12 cells against hydrogen peroxide-induced injury[J]. Life sciences.2003,73(19):2503-2513.
[82]李伯勤.虫草多糖对皮肤成纤维细胞抗氧化能力的影响[J].2010(4),34-38.
[83]Nakamura K, Yamaguchi Y, Kagota S, et al. Activation of in vivo Kupffer cell function by oral administration of Cordyceps sinensis in rats[J]. The Japanese Journal of Pharmacology. 1999,79(4):505-508.
[84]李元青,马成杰,李晓明,等.三七虫草混合粉抑制小鼠黑色素瘤效应研究[J].亚太传统医药.2007(004)：64-67.
[85]李婧,何肇晴,龚皓,等.国产虫草素对Hela细胞MMP-9, TIMP-1及TIMP-1 mRNA表达的调节[J].现代肿瘤医学.2009,17(009)：1615-1618.
[86]Yoo H S, Shin J W, Cho J H, et al.Effects of Cordyceps militaris extract on angiogenesis and tumor growth[J]. Acta Pharmacologica Sinica.2004,25(5):657-665.
[87]Price P J,Suk W A, Peters R L, et al. Cordycepin inhibition of 3-methylcholanthrene-induced transformation in vitro[C]. Royal Society of Medicine, 1975.150(3):650-653.