吡咯喹啉醌对体外培养的神经干细胞增殖和凋亡的影响
|
摘要
神经退行性疾病(Neurodegenerative disease)是指中枢神经系统组织非正常退变引起的功能缺陷与衰退的一类疾病,病理上表现为脑和脊髓发生神经元变性、丢失。研究发现,机体内过量的自由基产生导致的氧化应激损伤在神经退行性疾病的发病过程中起着重要的作用。近年来,神经干细胞(Neural Stem Cells, NSCs)因其具有自我更新的能力和多向分化潜能,在中枢神经系统再生和重建方面引起广泛关注,但NSCs移植后存活率低和易于受到氧化应激损伤的缺陷,则成为其应用在神经退行性疾病临床治疗上的限制。吡咯喹啉醌(Pyrroloquinline quinone, PQQ)在自然界中广泛存在,作为氧化还原酶的辅基参与机体内的多种氧化还原反应,具有多种生物学功能,尤其是其强大的清除自由基能力和神经营养保护功能已经引起人们的注意。
本文首先通过体外悬浮方式培养NSCs,同时采用H2O2和Glu造成细胞氧化应激,以构建NSCs药物损伤模型。结果表明,二者均能对NSCs产生毒性作用,其毒性作用呈时间依赖和浓度依赖；据此,本文分别采用0.35mM H2O2作用细胞24h建立体外NSCs直接氧化应激模型,15mMGlu作用细胞48h建立间接氧化应激损伤模型。
其次利用CCK-8试剂盒、免疫荧光染色等方法考察PQQ对NSCs自发的增殖、凋亡和分化的影响。结果显示,一定浓度(0.003-3μM) PQQ对NSCs的生长具有促进作用,并存在着浓度依赖,但高浓度的PQQ(30gM或以上)对NSCs的增殖反而有抑制作用,PQQ对NSCs的自发性凋亡和分化均无显著性影响。
通过建立的损伤模型,采用Hoechst33258-PI荧光染色、AV-PI双染检测凋亡以及检测细胞内酶活性等方法,考察PQQ的预处理和后处理对NSCs的神经保护作用。结果表明,在药物诱导之前预加PQQ能够明显提高细胞存活率,并存在浓度-效应关系,而PQQ后处理则无明显效果。PQQ预处理组细胞的形态上的染色质浓集或碎块现象减少,并且活细胞增多,折光度增强,状态明显出现好转；其早期/晚期凋亡细胞的比率均有所减少。对胞内抗氧化酶活力水平检测发现,PQQ的预处理能够显著提高SOD和CAT的活性,但GSH-Px的变化则不明显。说明PQQ对NSCs的氧化应激损伤具有保护作用,并且可能是通过提高胞内抗氧化酶的活力来实现的。
总之,本研究结果表明,PQQ对体外培养的NSCs具有促增殖的作用,PQQ预处理对氧化应激导致的NSCs损伤具有明显的保护作用,为神经退行性疾病的治疗提供了新的手段。
Neurodegenerative disease is a kind of disease which is caused by the abnormally degeneration of the central nervous system organization. The symptoms of the neurodegenerative disease are the degeneration and loss of neurons. Recent research found that idative stress which caused by he production of excessive free radicals in the organism has plays an important role in the process of neurodegenerative disease attacking. In recent years, Neural stem cells (NSCs) have raised a lot of concern in regeneration and reconstruction of the CNS due to its ability of renew and multi-lineage differentiation potential. But the low cell viability and weakness of vulnerable to oxidative stress injury, have limited the application of NSCs in the clinical treatment of neurodegenerative disease. Pyrroloquinline quinine (PQQ) exists widely in the nature. As the coenzyme of oxidoreductase, it takes part in many kinds of redox reactions, and has a variety of ability of biological functions. Especially its powerful free-radicals scavengine capacity and neuro-protection has been attracting attention.
Firstly, NSCs in vitro suspension culture, use H2O2and Glu which induce oxidative stress to establish the injury model. The results show that both can make toxic action on NSCs, and its toxic effects are time-dependency and concentration-dependent. Accordingly, we use H2O20.35mM24h and Glu15mM48h, respectively dispose NSCs, built the direct and the indirect oxidative stress injury model.
Secondly, we use CCK-8kit, immunofluorescence staining method to examine the effect of PQQ on the proliferation, apoptosis and differentiation of NSCs. The results show that certain concentrations (0.003-3μM) of PQQ can promot the growth of NSCs, and there is a concentration-dependent. But high concentrations (30μM or above) may have the inhibition on the proliferation of NSCs. PQQ has no significant impact on the spontaneous apoptosis and differation of NSCs.
Finally, we investigate the neuroprotective effect of pretreatment and aftertreatment of PQQ on NSCs through the use of Hoechst33258-PI staining, AV-PI and the detection of intracellular enzyme activity. The results show that the treatment of PQQ before the drug-induced can significantly improve the cell viability and has the concentration-effect relationship. While the PQQ of aftertreatment had no effect. The morphology of cells in pretreatment of PQQ group has chromatin condensation of fragments, and the number of the living cells increased, the refractive index enhanced. The early/late apoptotic cells ratio also reduced. The activities of antioxidant enzymes in the intracellular level of detection of PQQ pretreatment can significantly increase the activity of SOD and CAT, but not significant changes in GSH-Px. PQQ has a protective effect of NSCs oxidative stress injury, and may be achieved by increasing the intracellular antioxidant enzyme activity.
In summary, the results of this study shows that PQQ can promote the proliferation of NSCs, and the pretreatment of PQQ has a significant protective effect on NSCs damage caused by oxidative stress, provides a new means for the treatment of neurodegenerative diseases.
本文首先通过体外悬浮方式培养NSCs,同时采用H2O2和Glu造成细胞氧化应激,以构建NSCs药物损伤模型。结果表明,二者均能对NSCs产生毒性作用,其毒性作用呈时间依赖和浓度依赖；据此,本文分别采用0.35mM H2O2作用细胞24h建立体外NSCs直接氧化应激模型,15mMGlu作用细胞48h建立间接氧化应激损伤模型。
其次利用CCK-8试剂盒、免疫荧光染色等方法考察PQQ对NSCs自发的增殖、凋亡和分化的影响。结果显示,一定浓度(0.003-3μM) PQQ对NSCs的生长具有促进作用,并存在着浓度依赖,但高浓度的PQQ(30gM或以上)对NSCs的增殖反而有抑制作用,PQQ对NSCs的自发性凋亡和分化均无显著性影响。
通过建立的损伤模型,采用Hoechst33258-PI荧光染色、AV-PI双染检测凋亡以及检测细胞内酶活性等方法,考察PQQ的预处理和后处理对NSCs的神经保护作用。结果表明,在药物诱导之前预加PQQ能够明显提高细胞存活率,并存在浓度-效应关系,而PQQ后处理则无明显效果。PQQ预处理组细胞的形态上的染色质浓集或碎块现象减少,并且活细胞增多,折光度增强,状态明显出现好转；其早期/晚期凋亡细胞的比率均有所减少。对胞内抗氧化酶活力水平检测发现,PQQ的预处理能够显著提高SOD和CAT的活性,但GSH-Px的变化则不明显。说明PQQ对NSCs的氧化应激损伤具有保护作用,并且可能是通过提高胞内抗氧化酶的活力来实现的。
总之,本研究结果表明,PQQ对体外培养的NSCs具有促增殖的作用,PQQ预处理对氧化应激导致的NSCs损伤具有明显的保护作用,为神经退行性疾病的治疗提供了新的手段。
Neurodegenerative disease is a kind of disease which is caused by the abnormally degeneration of the central nervous system organization. The symptoms of the neurodegenerative disease are the degeneration and loss of neurons. Recent research found that idative stress which caused by he production of excessive free radicals in the organism has plays an important role in the process of neurodegenerative disease attacking. In recent years, Neural stem cells (NSCs) have raised a lot of concern in regeneration and reconstruction of the CNS due to its ability of renew and multi-lineage differentiation potential. But the low cell viability and weakness of vulnerable to oxidative stress injury, have limited the application of NSCs in the clinical treatment of neurodegenerative disease. Pyrroloquinline quinine (PQQ) exists widely in the nature. As the coenzyme of oxidoreductase, it takes part in many kinds of redox reactions, and has a variety of ability of biological functions. Especially its powerful free-radicals scavengine capacity and neuro-protection has been attracting attention.
Firstly, NSCs in vitro suspension culture, use H2O2and Glu which induce oxidative stress to establish the injury model. The results show that both can make toxic action on NSCs, and its toxic effects are time-dependency and concentration-dependent. Accordingly, we use H2O20.35mM24h and Glu15mM48h, respectively dispose NSCs, built the direct and the indirect oxidative stress injury model.
Secondly, we use CCK-8kit, immunofluorescence staining method to examine the effect of PQQ on the proliferation, apoptosis and differentiation of NSCs. The results show that certain concentrations (0.003-3μM) of PQQ can promot the growth of NSCs, and there is a concentration-dependent. But high concentrations (30μM or above) may have the inhibition on the proliferation of NSCs. PQQ has no significant impact on the spontaneous apoptosis and differation of NSCs.
Finally, we investigate the neuroprotective effect of pretreatment and aftertreatment of PQQ on NSCs through the use of Hoechst33258-PI staining, AV-PI and the detection of intracellular enzyme activity. The results show that the treatment of PQQ before the drug-induced can significantly improve the cell viability and has the concentration-effect relationship. While the PQQ of aftertreatment had no effect. The morphology of cells in pretreatment of PQQ group has chromatin condensation of fragments, and the number of the living cells increased, the refractive index enhanced. The early/late apoptotic cells ratio also reduced. The activities of antioxidant enzymes in the intracellular level of detection of PQQ pretreatment can significantly increase the activity of SOD and CAT, but not significant changes in GSH-Px. PQQ has a protective effect of NSCs oxidative stress injury, and may be achieved by increasing the intracellular antioxidant enzyme activity.
In summary, the results of this study shows that PQQ can promote the proliferation of NSCs, and the pretreatment of PQQ has a significant protective effect on NSCs damage caused by oxidative stress, provides a new means for the treatment of neurodegenerative diseases.
引文
[1]马艳,盛卸晃,宋淑亮.神经退行性疾病和脑损伤的细胞疗法[J].生命的化学,2011,5(10)：727-731.
[2]Reiter RJ. Oxidative processes and antioxidative defense mechanisms in the aging brain [J].The FASEB Journal,1995,9(7):526-533.
[3]Yan-qiu Yu, Li-li Du. Stem cells based transplantation for cardiovascular diseases in China [J]. Pathophysiology,2009,16(4):285-290.
[4]Scanlon J M, Aizenman E, Reynolds I J. Effects of pyrroloquinoline quinone on glutamate-induced production of reactive oxygen species in neurons [J]. European Journal of Pharmacology,1997,326(1):67-74.
[5]杨璐,熊向华,汪建华等.吡咯喹啉醌研究进展[J].生物技术通讯,2009,6(20)：874-879.
[6]Andrea C R, Antonio R, Augusto R, et al. Modeling novel quinocofactors:An overview [J]Bioorganic Chemistry,1999,27(4):253-288.
[7]C Heurteaux, C Gandin, M Borsotto. et al. Neuroprotective and neuroproliferative activities of NeuroAid (MLC601, MLC901), a Chinese medicine, in vitro and in vivo [J]. Neuropharmacology,2010,58:987-1001.
[8]He K, Nukada H, Urakami T, et al. Antioxidant and pro-oxidant properties of pyrroloquinoline quinone(PQQ):implications for its function in biological systems [J]. Biochemical Pharmacology,2003,65(1):67-74.
[9]熊顺华,王君,龚坚,等.吡咯喹啉醌对培养的鼠海马神经元的促生长作用[J].陕西医学杂志,2007,35(5)：527-572.
[10]Qi Zhang, Mi Shen, Mei Ding, et al. Protective action of pyrroloquinoline quinone against glutamate-induced apoptosis in hippocampal neurons is mediated through the activation of PI3K Akt pathway [J]. Toxicology and Applied Pharmacology,2001,252(1):62-72.
[11]Anderson DJ, Michelsohn A. Role of glucocorticoids in the chromaffin-neuron developmental decision [J]. International Journal of Developmental Neuroscience,1989,7(5):475-487.
[12]Cattaneo E, McKay R. Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor [J].Nature,1990,347(6295):762-765.
[13]Reynolds B A, Tetzlaff W, Weiss S. A Multipotent Egf-Responsive Striatal Embryonic Progenitor-Cell Produces Neurons and Astrocytes [J]. Journal of Neuroscience,1992,12(11):4565-4574.
[14]McKay R. Stem cells in the central nervous system [J]. Science,1997,276(5309):66-71.
[15]Temple S. The development of neural stem cells [J].Nature,2001,414(6859):112117.
[16]Alliot F, Rutin J, Leenen PJ, et al. Pericytes and periendothelial cells of brain parenchyma vessels co-express aminopeptidase N, aminopeptidase A, and nestin [J]. Journal of neuroscience research,1999,58(3):367-378.
[17]Vescovi AL, Snyder EY. Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo[J]. Brain pathology,1999,9(3):569-598.
[18]Gage F H. Stem cells of the central nervous system [J]. Current opinion in neurobiology,1998,8(5):671-676.
[19]田增民,孙君昭.干细胞在神经系统疾病治疗中的应用[J].中国实用内科杂志,2007,27(10)：399-401.
[20]Rennstam Karin, McMichael Nicole, Berglund Pontus et al. Numb protein expression correlates with a basal-like phenotype and cancer stem cell markers in primary breast cancer[J]. Breast cancer research and treatment,2010,122(2)315-324.
[21]Flax J D, Aurora S, Yang C, et al. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes [J]. Nature biotechnology,1998,16(11):1033-1039.
[22]Hori J, Ng TF, Shatos M, et al. Neural progenitor cells lack immunogenicity and resist destruction as allografts [J]. Stem cells,2003,21(4)405-416.
[23]Snyder EY. Review:Neural Stem-Like Cells Developmental Lessons with Therapeutic Potential [J].The Neuroscientist,1998,4(6):408-425.
[24]Moeller M L, Dinitrijecich S D. A new strategy for analysis of phenotype marker antigens in hollow neurospheres [J]. Journal of Neuroscience Methods,2004,139(1):43-50.
[25]Jin M, Kayoko N I, Akiko S et al. Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors [J]. Journal of anatomy,2012,220(6):612-621.
[26]Sakakibara S, Imai T, Hamaguchi K et al. Mouse-Musashi-1:A Neural RNA-Binding Protein Highly Enriched in the Mammalian CNS Stem Cell [J]. Developmental Biology,1996,176(2):230-242.
[27]Sakakibara S, Okano H. Expression of neural RNA-binding proteins in the postnatal CNS:implications of their roles in neuronal and glial cell development [J]. Neuroscience,1997,17(21):8300-8312.
[28]Capela A, Temple S. LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal [J]. Neuron,2002,35(5):865-875.
[29]Lang R J, Haynes J M, Kelly J et al. Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-l lineage selection and directed differentiation [J].European Journal of Neurosience,2004,20(12):3209-3221.
[30]张舸.神经干细胞分化及诱导的研究进展[J].医学研究生学报,2004,17(9)：847-851
[31]Eng L F. Glial fibrillary acidic protein (GFAP):the major protein of glial intermediate filaments in differentiated astrocytes [J]. Journal of Neurommunol,1985,8(4-6):203-214.
[32]刘天庆,戴明舒,葛丹等.神经球内神经干/祖细胞活性与代谢研究[J].大连理工大学学报,2008,48(6)：811-818.
[33]Ray J, Peterson D A, Schinstine M, Gage F H. Proliferation, Differentiation, and Long-term culture of primary hippocampal-neurons [J].Proceedings of the national academy of science of the United States of America,1993,90(8):3602-3606.
[34]Kallos M S, Behie L A. Inoculation and growth conditions for high-cell-density expansion of mammalian neural stem cells in suspension bioreactors [J]. Biotechnology and Bioengineering,1999,63(4):473-483.
[35]Lu L, Vacanti JP, Langer R et al. In vitro and in vivo degradation of porous poly(D, L-lactic-co-glycolic acid) foams [J]. Biomaterials,2000,21(18):1837-1845.
[36]林建平,吴礼光.医用可生物降解高分子材料.功能高分子学报.1996,9(4)：631-638.
[37]McDonald J W, Liu X Z, Qu Y, et al. Transplanted embryonie stem cells survive, differentiate and promote recoverty in injured rat spinal cord [J]. Nature Medicine, 1999,5 (12):1410-1412.
[38]Ostenfeld T, Caldwell M A, Prowse K R, et al. Human neural precursor cells express low levels of of Telomerase in Vitro and Show Diminishing Cell Proliferation with Extensive Axonal Outgrowth following Transplantation [J]. Experimental Neurology,2004,164(1):215-226.
[39]Li Y, Zhang W M, Wang T H. Optimal Location and Time for Neural Stem Cell Transplantation into Transected Rat Spinal Cord [J]. Cellular and molecular neurobiology,2011,31(3):407-414.
[40]Abematsu M, Tsujimura K, Yamano M, et al. Neurons derived from transplanted neural stem cell restore_disrupted_neuronal_circuitry_in_a_mouse_model_of_spinal_cord_injury [J]. Journal of Clinical Investigation,2010,120(9):3255-3266.
[41]P. Lu, Jones EY, Snyder, etal. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury [J]. Experimental Neurology,2003,181(1):15-129.
[42]Akerud P, Alberch JS, Wagner J, et al. Differential effects of GDNF and Neurturin on developing and adult substantia nigra dopaminergic neurons [J]. Neurochem,1999,73(1):70-78
[44]Lee S J, Kim Y, Jo M Y, et al. Combined treatment of tumor-tropic human neural stem cells containing the CD suicide gene effectively targets brain tumors provoking a mild immune response [J]. Oncology Reports,2011,25 (1):63-68.
[45]Hauge J G. Purification and properties of glucose dehydrogenase and cytochrome from Bacterium anitratum [J]. Biochemica et Biophysica Acta,1960,45 (2):250-262.
[46]Anthony C, Zatman L J. The microbial oxidation of methanol, the prosthetic group ofthe alcohol dehydrogenase of Pseudomonas sp. M27:a new oxidoreductase prosthetic group [J].Biochemical Journal,1967,104(3):960-969.
[47]Westerling J, Frank J, Duine J A. The prosthetic group ofmethanol dehydrogenase from Hyphomicrobium X:electron spin resonance evidence for a quinine structure [J]. Biochemical Biophysical Research Communications,1979,87(3):719-724.
[48]Salisbury S A, Forrest H S, Cruse W B et al. A novel coenzyme from bacterial primary alcohol dehydrogenases [J]. Nature,1979,280(5725):843-844.
[49]Duine J A. Cofactor diversity in biological oxidations:implications and applications [J].Chem Reviews,2001,1(1):74-83.
[50]Xiong L B, Sekiya J, Shimose N. Occurrence of PQQ in pistils and pollen grains of higher plant [J]. Agricultural Biology Chemistry,1990,54(l):249-250.
[51]Kumazawa T, Seno H, Urakami T et al. Trace levels of pyrroloquinoline quinone in human and rat samples deteceted by gas chromatography/mass spectrometry [J].Biochimica et Biophysica Acta,1992,1156(1):62-66.
[52]王歆,张惟材.吡咯喹啉醌生物合成研究进展[J].生物技术通讯,2007,18(3)：534-539.
[53]Smidt C R, Unkefer C J, Houck D R et al. Intestinal absorption and tissue distribution of [14C] pyrroloquinoline quinone in mice [J]. Proceedings of the Society for Experimental Biology and Medicine,1991,197(1):27-31.
[54]Kumazawa T, Sato K, Seno H et al. Levels of pyrroloquinoline quinone in various foods [J]. Biochemistry Journal,1995,307(2):331-333.
[55]Mitchell A E, JonesAD, Mercer R S et al. Characterization of pyrroloquinoline quinone amino acid derivatives by electrospray ionization mass spectrometry and detection in human milk [J]. Analytical Biochemistry,1999,269(2):317-325.
[56]Hamagishi Y, Murata S Kamei H et al. New Biological properties of Pyrroloquinoline Quinone and its related compounds:Inhibition of Chemiluminescence, Lipid peroxidation and rat paw edema [J].The journal of Pharmacology and experimental,1990,255(3):980-985.
[57]ShimaoM, Yamamoto H, Ninomiya K et al. Pyrroloquinoline quinone as an essential growth factor for a poly(vinyl alcohol)-degrading symbiont, Pseudomonas sp. VM 15C [J].Agricultural Biology Chemistry,1984,48(11):2873-2876.
[58]Ameyama M, Matsushita K, Shinagawa E et al. Pyrroloquinoline quinine:excretion by methylotrophs and growth stimulation for microorganisms [J]. Biofactors,1988,1(1):51-53.
[59]Xiong L B, Sekiya J, Shimose N et al. Stimulation ofLilium pollen germination by pyrroloquinoline quinine [J]. Agricultural Biology Chemistry,1990,52(4):1065-1066.
[60]刘卫群,韩锦峰,彭文博等.吡咯喳啉醌对烟草种子萌发的生理效应[J].河南农业大学学报,1993,27(3)：223-226.
[61]卫秀英,李秀菊,朱坤华等.吡咯喹啉醌对苹果花粉萌发和花粉管生长的影响简报[J].植物生理学通讯,2000,36(4)：320-321.
[62]Choi O, Kim J, Kim J G et al. Pyrroioquinoline quinone is aplant growth promotion factor produced by Pseudomonasfluorescens B16 [J]. Plant Physiology,2008,146(2):657-668.
[63]Han S H, Kim C H, Lee J H, et al. Inactivation of pqq genes of Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistance [J].FEMS Microbiology Lettters,2008,282(1):140-146.
[64]Naito Y, Kumazawa T, Kino I et al. Effects of pyrroloquinoline quinone(PQQ)and PQQ-oxazole on DNA synthesis ofcultured human fibroblasts [J].Life Science,1993,52(24):1909-1915.
[65]熊顺华,王军,龚坚等.吡咯喹啉醌对培养的鼠海马神经元的促生长作用[J].陕西医学杂志,2007,36(5)：527-572.
[66]贺斌,刘世清,李浩恒.P13K/Akt信号通路在吡咯喹啉醌促雪旺细胞增殖中的作用[J].中华整形外科杂志,2010,26(1)：53-56
[67]李浩恒,刘世清,彭浩等.吡咯喹啉醌对许旺细胞合成与分泌神经生长因子的影响[J].中华显微外科杂志,2005,28(3)：242-243.
[68]Killgore J, Smidt C, Duich L et al. Nutritional importance of pyrroloquinoline quinine [J]. Science,1989,245(4920):850-852.
[69]Steinberg F M, Gershwin ME, Rucker R B. Dietary pyrroioquinoline quinone:growth and immune response in BALB/c mice [J]. Journal of Nutrition,1994,124(5):744-753.
[70]Akaike T, Sato K. PQQ as a generator and scavenger of oxygen radicals determination with ESR spectroscopy using a spin trap agent. In:Fukui T, Kagamiyama H, Soda K et al eds. Enzymes dependent on pyfidoxal phosphate and other carbonyl compounds as cofactors. Oxford, New York, Seoul, Tokyo:Pergamon Press,1991,511-513.
[71]Urakami T, Yoshida C, Akaike T et al. Synthesis of monoesters of pyrroloquinoline ouinone and imidazopyrroloquinoline, and radical scavenging activities using electron spin resonance in vitro and pharmacological activity in vivo [J]. Journal of Nutritional Science Vitaminology,1997,43(1):19-33.
[72]Christopher W. M. Kay, Bina Mennenga, Helmut Go risch. Substrate binding in quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa studied by electron-nuclear double resonance [J]. Biophysics Chemistry,2006,103(14)5267-5272.
[73]Fu Y, Buryanovskyy L, and Zhang Z. Quinone Reductase 21s a Catechol Quinone Reductase [J]. Journal of Biological Chemistry,2008,283(35):23829-23935.
[74]Kulys J, Tetianec L, Bratkovskaj I. Pyrroloquinoline quinone-dependent carbohydrate dehydrogenase:Activity enhancement and the role of artificial electron acceptors [J].Biotechology Journal,2010,5(66):822-828
[75]Zhu B Q, Zhou H Z, Teerlink J R et al. Pyrroloquinoline quinone(PQQ)decreases myocardial infarct size and improves cardiac function in rat models of ischemia and ischemia/eperfusion [J]. Journal of Cardiovasc Pharmacology and Therapeutics,2004,18(6):421-431.
[76]Zhu B Q, Simonis U, Cecchini G et al. Comparison of pyrroloquinoline quinone and/or metoprolol on myocardial infarct size and mitochondfial damage in arat model of ischemia/eperfusion injury [J]. Journal of Cardiovasc Pharmacology and Therapeutics,2006,11(2):119-128.
[77]Oubrie, A, Rozeboom, H. J, Dijkstra, B. W., Active-site structure of the soluble quinoprotein glucose dehydrogenase complexed with methylhydrazine:A covalent cofactor-inhibitor complex [J]. PNAS,1999,96(2:11787-11791.
[78]Tao R, Karliner J S, Simonis U, et al. Pyrroloquinoline quinine preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes [J]. Biochemical and Biophysical Research Communications,2007,363(2):257-262.
[79]Yamaguchi K, Sasano A, Urakami T et al. Stimulation of nerve growth factor production by pyrroioquinoline quinone and its derivatives in vitro and in vivo [J]. Bioscience Biotechnology Biochemistry,1993,57(7):1231-1233.
[80]Jensen F E, Gardner G J, Williams A P et al. The putative essential nutrient pyrroloquinoline quinine is neuroprotective in a rodent model of hypoxic/ischemic brain injury [J]. Neuroscience,1994,62 (2):399-406.
[81]李皓桓,刘世清,彭吴等.毗咯喹啉醌对许旺细胞合成与分泌神经生长因子的影响[J].中华显微外科杂志,2005,28(3)：242-243.
[82]Hara H, Hiramatsu H, Adachi T et al. Pyrroloquinoline quinone is a potent neuroprotective nutrientagainst 6-hydroxydopamine induced neurotoxicity [J]. Neurochemistry Research,2007,32(3):489-495.
[83]Kobayashi M, Kim J, Kobayashi N, et al. Pyrroloquinoline quinine (PQQ) prevents fibril formation of alpha-synuclein [J].Biochemical and Biophysical Research Communications,2006,349 (3):1139-1144.
[84]Harman D. Ageing:A theory based on free radical and radition chemistry [J].Gerontol,1956,11:298-300.
[85]Sohal RS, Allen RG. Oxidative stress as a causal factor in differentiation and aging: a unifying hypothesis [J]. Experimental Gerontology,1990,25(6):499-522.
[86]Smith M A, Hirai K, Hsiao K et al. Amyloid-beta deposition in Alzheimer transgenic mice is associated with oxidative stress [J].Journal of Neurochemistry,1998,70(5):2212-2215.
[87]Dexter D T, Holley A E, Flitter W D et al. Increased levels of lipid hydroperoxides in the Parkinsonian substantia nigra:an HPLC and ESR study [J]. Movement Disorders,1994,99(1):92-97.
[88]Yoritaka A, Hattori N, Mori H et al. An immunohistochemical study on manganese superoxide dismutase in Parkinson's disease [J].Journal Neurology Science,1997,148(2):181-186.
[89]Petros All, Medek A, Nettesheim DG et al. Solution structure of the anti-apoptotic protein Bcl-2 [J].PNAS,2001,98(6):3012-3017.
[90]方晓阳,盛伟.细胞凋亡的概念来源[J].生命中的化学,2003,23(2)：121-123.
[91]Grimm S, Baeuerle PA. The inducible transcription factor NF-kB:Structure-function relationship of its protein subunits [J]. Biochemistry Journal,1993,290:297-308.
[92]Floury C, Mignotte B, Vayssiere JL. Mitochondrial reactive oxygen species in cell death signaling [J]. Biochimie,2002,84(2-3):131-141.
[93]Tan S, Sagara T, Liu Y et al. The regulation of reactive oxygen species production uring programmed cell death [J].The Journal of Cell Biology,1998,141(6):1423-1432.
[2]Reiter RJ. Oxidative processes and antioxidative defense mechanisms in the aging brain [J].The FASEB Journal,1995,9(7):526-533.
[3]Yan-qiu Yu, Li-li Du. Stem cells based transplantation for cardiovascular diseases in China [J]. Pathophysiology,2009,16(4):285-290.
[4]Scanlon J M, Aizenman E, Reynolds I J. Effects of pyrroloquinoline quinone on glutamate-induced production of reactive oxygen species in neurons [J]. European Journal of Pharmacology,1997,326(1):67-74.
[5]杨璐,熊向华,汪建华等.吡咯喹啉醌研究进展[J].生物技术通讯,2009,6(20)：874-879.
[6]Andrea C R, Antonio R, Augusto R, et al. Modeling novel quinocofactors:An overview [J]Bioorganic Chemistry,1999,27(4):253-288.
[7]C Heurteaux, C Gandin, M Borsotto. et al. Neuroprotective and neuroproliferative activities of NeuroAid (MLC601, MLC901), a Chinese medicine, in vitro and in vivo [J]. Neuropharmacology,2010,58:987-1001.
[8]He K, Nukada H, Urakami T, et al. Antioxidant and pro-oxidant properties of pyrroloquinoline quinone(PQQ):implications for its function in biological systems [J]. Biochemical Pharmacology,2003,65(1):67-74.
[9]熊顺华,王君,龚坚,等.吡咯喹啉醌对培养的鼠海马神经元的促生长作用[J].陕西医学杂志,2007,35(5)：527-572.
[10]Qi Zhang, Mi Shen, Mei Ding, et al. Protective action of pyrroloquinoline quinone against glutamate-induced apoptosis in hippocampal neurons is mediated through the activation of PI3K Akt pathway [J]. Toxicology and Applied Pharmacology,2001,252(1):62-72.
[11]Anderson DJ, Michelsohn A. Role of glucocorticoids in the chromaffin-neuron developmental decision [J]. International Journal of Developmental Neuroscience,1989,7(5):475-487.
[12]Cattaneo E, McKay R. Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor [J].Nature,1990,347(6295):762-765.
[13]Reynolds B A, Tetzlaff W, Weiss S. A Multipotent Egf-Responsive Striatal Embryonic Progenitor-Cell Produces Neurons and Astrocytes [J]. Journal of Neuroscience,1992,12(11):4565-4574.
[14]McKay R. Stem cells in the central nervous system [J]. Science,1997,276(5309):66-71.
[15]Temple S. The development of neural stem cells [J].Nature,2001,414(6859):112117.
[16]Alliot F, Rutin J, Leenen PJ, et al. Pericytes and periendothelial cells of brain parenchyma vessels co-express aminopeptidase N, aminopeptidase A, and nestin [J]. Journal of neuroscience research,1999,58(3):367-378.
[17]Vescovi AL, Snyder EY. Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo[J]. Brain pathology,1999,9(3):569-598.
[18]Gage F H. Stem cells of the central nervous system [J]. Current opinion in neurobiology,1998,8(5):671-676.
[19]田增民,孙君昭.干细胞在神经系统疾病治疗中的应用[J].中国实用内科杂志,2007,27(10)：399-401.
[20]Rennstam Karin, McMichael Nicole, Berglund Pontus et al. Numb protein expression correlates with a basal-like phenotype and cancer stem cell markers in primary breast cancer[J]. Breast cancer research and treatment,2010,122(2)315-324.
[21]Flax J D, Aurora S, Yang C, et al. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes [J]. Nature biotechnology,1998,16(11):1033-1039.
[22]Hori J, Ng TF, Shatos M, et al. Neural progenitor cells lack immunogenicity and resist destruction as allografts [J]. Stem cells,2003,21(4)405-416.
[23]Snyder EY. Review:Neural Stem-Like Cells Developmental Lessons with Therapeutic Potential [J].The Neuroscientist,1998,4(6):408-425.
[24]Moeller M L, Dinitrijecich S D. A new strategy for analysis of phenotype marker antigens in hollow neurospheres [J]. Journal of Neuroscience Methods,2004,139(1):43-50.
[25]Jin M, Kayoko N I, Akiko S et al. Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors [J]. Journal of anatomy,2012,220(6):612-621.
[26]Sakakibara S, Imai T, Hamaguchi K et al. Mouse-Musashi-1:A Neural RNA-Binding Protein Highly Enriched in the Mammalian CNS Stem Cell [J]. Developmental Biology,1996,176(2):230-242.
[27]Sakakibara S, Okano H. Expression of neural RNA-binding proteins in the postnatal CNS:implications of their roles in neuronal and glial cell development [J]. Neuroscience,1997,17(21):8300-8312.
[28]Capela A, Temple S. LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal [J]. Neuron,2002,35(5):865-875.
[29]Lang R J, Haynes J M, Kelly J et al. Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-l lineage selection and directed differentiation [J].European Journal of Neurosience,2004,20(12):3209-3221.
[30]张舸.神经干细胞分化及诱导的研究进展[J].医学研究生学报,2004,17(9)：847-851
[31]Eng L F. Glial fibrillary acidic protein (GFAP):the major protein of glial intermediate filaments in differentiated astrocytes [J]. Journal of Neurommunol,1985,8(4-6):203-214.
[32]刘天庆,戴明舒,葛丹等.神经球内神经干/祖细胞活性与代谢研究[J].大连理工大学学报,2008,48(6)：811-818.
[33]Ray J, Peterson D A, Schinstine M, Gage F H. Proliferation, Differentiation, and Long-term culture of primary hippocampal-neurons [J].Proceedings of the national academy of science of the United States of America,1993,90(8):3602-3606.
[34]Kallos M S, Behie L A. Inoculation and growth conditions for high-cell-density expansion of mammalian neural stem cells in suspension bioreactors [J]. Biotechnology and Bioengineering,1999,63(4):473-483.
[35]Lu L, Vacanti JP, Langer R et al. In vitro and in vivo degradation of porous poly(D, L-lactic-co-glycolic acid) foams [J]. Biomaterials,2000,21(18):1837-1845.
[36]林建平,吴礼光.医用可生物降解高分子材料.功能高分子学报.1996,9(4)：631-638.
[37]McDonald J W, Liu X Z, Qu Y, et al. Transplanted embryonie stem cells survive, differentiate and promote recoverty in injured rat spinal cord [J]. Nature Medicine, 1999,5 (12):1410-1412.
[38]Ostenfeld T, Caldwell M A, Prowse K R, et al. Human neural precursor cells express low levels of of Telomerase in Vitro and Show Diminishing Cell Proliferation with Extensive Axonal Outgrowth following Transplantation [J]. Experimental Neurology,2004,164(1):215-226.
[39]Li Y, Zhang W M, Wang T H. Optimal Location and Time for Neural Stem Cell Transplantation into Transected Rat Spinal Cord [J]. Cellular and molecular neurobiology,2011,31(3):407-414.
[40]Abematsu M, Tsujimura K, Yamano M, et al. Neurons derived from transplanted neural stem cell restore_disrupted_neuronal_circuitry_in_a_mouse_model_of_spinal_cord_injury [J]. Journal of Clinical Investigation,2010,120(9):3255-3266.
[41]P. Lu, Jones EY, Snyder, etal. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury [J]. Experimental Neurology,2003,181(1):15-129.
[42]Akerud P, Alberch JS, Wagner J, et al. Differential effects of GDNF and Neurturin on developing and adult substantia nigra dopaminergic neurons [J]. Neurochem,1999,73(1):70-78
[44]Lee S J, Kim Y, Jo M Y, et al. Combined treatment of tumor-tropic human neural stem cells containing the CD suicide gene effectively targets brain tumors provoking a mild immune response [J]. Oncology Reports,2011,25 (1):63-68.
[45]Hauge J G. Purification and properties of glucose dehydrogenase and cytochrome from Bacterium anitratum [J]. Biochemica et Biophysica Acta,1960,45 (2):250-262.
[46]Anthony C, Zatman L J. The microbial oxidation of methanol, the prosthetic group ofthe alcohol dehydrogenase of Pseudomonas sp. M27:a new oxidoreductase prosthetic group [J].Biochemical Journal,1967,104(3):960-969.
[47]Westerling J, Frank J, Duine J A. The prosthetic group ofmethanol dehydrogenase from Hyphomicrobium X:electron spin resonance evidence for a quinine structure [J]. Biochemical Biophysical Research Communications,1979,87(3):719-724.
[48]Salisbury S A, Forrest H S, Cruse W B et al. A novel coenzyme from bacterial primary alcohol dehydrogenases [J]. Nature,1979,280(5725):843-844.
[49]Duine J A. Cofactor diversity in biological oxidations:implications and applications [J].Chem Reviews,2001,1(1):74-83.
[50]Xiong L B, Sekiya J, Shimose N. Occurrence of PQQ in pistils and pollen grains of higher plant [J]. Agricultural Biology Chemistry,1990,54(l):249-250.
[51]Kumazawa T, Seno H, Urakami T et al. Trace levels of pyrroloquinoline quinone in human and rat samples deteceted by gas chromatography/mass spectrometry [J].Biochimica et Biophysica Acta,1992,1156(1):62-66.
[52]王歆,张惟材.吡咯喹啉醌生物合成研究进展[J].生物技术通讯,2007,18(3)：534-539.
[53]Smidt C R, Unkefer C J, Houck D R et al. Intestinal absorption and tissue distribution of [14C] pyrroloquinoline quinone in mice [J]. Proceedings of the Society for Experimental Biology and Medicine,1991,197(1):27-31.
[54]Kumazawa T, Sato K, Seno H et al. Levels of pyrroloquinoline quinone in various foods [J]. Biochemistry Journal,1995,307(2):331-333.
[55]Mitchell A E, JonesAD, Mercer R S et al. Characterization of pyrroloquinoline quinone amino acid derivatives by electrospray ionization mass spectrometry and detection in human milk [J]. Analytical Biochemistry,1999,269(2):317-325.
[56]Hamagishi Y, Murata S Kamei H et al. New Biological properties of Pyrroloquinoline Quinone and its related compounds:Inhibition of Chemiluminescence, Lipid peroxidation and rat paw edema [J].The journal of Pharmacology and experimental,1990,255(3):980-985.
[57]ShimaoM, Yamamoto H, Ninomiya K et al. Pyrroloquinoline quinone as an essential growth factor for a poly(vinyl alcohol)-degrading symbiont, Pseudomonas sp. VM 15C [J].Agricultural Biology Chemistry,1984,48(11):2873-2876.
[58]Ameyama M, Matsushita K, Shinagawa E et al. Pyrroloquinoline quinine:excretion by methylotrophs and growth stimulation for microorganisms [J]. Biofactors,1988,1(1):51-53.
[59]Xiong L B, Sekiya J, Shimose N et al. Stimulation ofLilium pollen germination by pyrroloquinoline quinine [J]. Agricultural Biology Chemistry,1990,52(4):1065-1066.
[60]刘卫群,韩锦峰,彭文博等.吡咯喳啉醌对烟草种子萌发的生理效应[J].河南农业大学学报,1993,27(3)：223-226.
[61]卫秀英,李秀菊,朱坤华等.吡咯喹啉醌对苹果花粉萌发和花粉管生长的影响简报[J].植物生理学通讯,2000,36(4)：320-321.
[62]Choi O, Kim J, Kim J G et al. Pyrroioquinoline quinone is aplant growth promotion factor produced by Pseudomonasfluorescens B16 [J]. Plant Physiology,2008,146(2):657-668.
[63]Han S H, Kim C H, Lee J H, et al. Inactivation of pqq genes of Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistance [J].FEMS Microbiology Lettters,2008,282(1):140-146.
[64]Naito Y, Kumazawa T, Kino I et al. Effects of pyrroloquinoline quinone(PQQ)and PQQ-oxazole on DNA synthesis ofcultured human fibroblasts [J].Life Science,1993,52(24):1909-1915.
[65]熊顺华,王军,龚坚等.吡咯喹啉醌对培养的鼠海马神经元的促生长作用[J].陕西医学杂志,2007,36(5)：527-572.
[66]贺斌,刘世清,李浩恒.P13K/Akt信号通路在吡咯喹啉醌促雪旺细胞增殖中的作用[J].中华整形外科杂志,2010,26(1)：53-56
[67]李浩恒,刘世清,彭浩等.吡咯喹啉醌对许旺细胞合成与分泌神经生长因子的影响[J].中华显微外科杂志,2005,28(3)：242-243.
[68]Killgore J, Smidt C, Duich L et al. Nutritional importance of pyrroloquinoline quinine [J]. Science,1989,245(4920):850-852.
[69]Steinberg F M, Gershwin ME, Rucker R B. Dietary pyrroioquinoline quinone:growth and immune response in BALB/c mice [J]. Journal of Nutrition,1994,124(5):744-753.
[70]Akaike T, Sato K. PQQ as a generator and scavenger of oxygen radicals determination with ESR spectroscopy using a spin trap agent. In:Fukui T, Kagamiyama H, Soda K et al eds. Enzymes dependent on pyfidoxal phosphate and other carbonyl compounds as cofactors. Oxford, New York, Seoul, Tokyo:Pergamon Press,1991,511-513.
[71]Urakami T, Yoshida C, Akaike T et al. Synthesis of monoesters of pyrroloquinoline ouinone and imidazopyrroloquinoline, and radical scavenging activities using electron spin resonance in vitro and pharmacological activity in vivo [J]. Journal of Nutritional Science Vitaminology,1997,43(1):19-33.
[72]Christopher W. M. Kay, Bina Mennenga, Helmut Go risch. Substrate binding in quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa studied by electron-nuclear double resonance [J]. Biophysics Chemistry,2006,103(14)5267-5272.
[73]Fu Y, Buryanovskyy L, and Zhang Z. Quinone Reductase 21s a Catechol Quinone Reductase [J]. Journal of Biological Chemistry,2008,283(35):23829-23935.
[74]Kulys J, Tetianec L, Bratkovskaj I. Pyrroloquinoline quinone-dependent carbohydrate dehydrogenase:Activity enhancement and the role of artificial electron acceptors [J].Biotechology Journal,2010,5(66):822-828
[75]Zhu B Q, Zhou H Z, Teerlink J R et al. Pyrroloquinoline quinone(PQQ)decreases myocardial infarct size and improves cardiac function in rat models of ischemia and ischemia/eperfusion [J]. Journal of Cardiovasc Pharmacology and Therapeutics,2004,18(6):421-431.
[76]Zhu B Q, Simonis U, Cecchini G et al. Comparison of pyrroloquinoline quinone and/or metoprolol on myocardial infarct size and mitochondfial damage in arat model of ischemia/eperfusion injury [J]. Journal of Cardiovasc Pharmacology and Therapeutics,2006,11(2):119-128.
[77]Oubrie, A, Rozeboom, H. J, Dijkstra, B. W., Active-site structure of the soluble quinoprotein glucose dehydrogenase complexed with methylhydrazine:A covalent cofactor-inhibitor complex [J]. PNAS,1999,96(2:11787-11791.
[78]Tao R, Karliner J S, Simonis U, et al. Pyrroloquinoline quinine preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes [J]. Biochemical and Biophysical Research Communications,2007,363(2):257-262.
[79]Yamaguchi K, Sasano A, Urakami T et al. Stimulation of nerve growth factor production by pyrroioquinoline quinone and its derivatives in vitro and in vivo [J]. Bioscience Biotechnology Biochemistry,1993,57(7):1231-1233.
[80]Jensen F E, Gardner G J, Williams A P et al. The putative essential nutrient pyrroloquinoline quinine is neuroprotective in a rodent model of hypoxic/ischemic brain injury [J]. Neuroscience,1994,62 (2):399-406.
[81]李皓桓,刘世清,彭吴等.毗咯喹啉醌对许旺细胞合成与分泌神经生长因子的影响[J].中华显微外科杂志,2005,28(3)：242-243.
[82]Hara H, Hiramatsu H, Adachi T et al. Pyrroloquinoline quinone is a potent neuroprotective nutrientagainst 6-hydroxydopamine induced neurotoxicity [J]. Neurochemistry Research,2007,32(3):489-495.
[83]Kobayashi M, Kim J, Kobayashi N, et al. Pyrroloquinoline quinine (PQQ) prevents fibril formation of alpha-synuclein [J].Biochemical and Biophysical Research Communications,2006,349 (3):1139-1144.
[84]Harman D. Ageing:A theory based on free radical and radition chemistry [J].Gerontol,1956,11:298-300.
[85]Sohal RS, Allen RG. Oxidative stress as a causal factor in differentiation and aging: a unifying hypothesis [J]. Experimental Gerontology,1990,25(6):499-522.
[86]Smith M A, Hirai K, Hsiao K et al. Amyloid-beta deposition in Alzheimer transgenic mice is associated with oxidative stress [J].Journal of Neurochemistry,1998,70(5):2212-2215.
[87]Dexter D T, Holley A E, Flitter W D et al. Increased levels of lipid hydroperoxides in the Parkinsonian substantia nigra:an HPLC and ESR study [J]. Movement Disorders,1994,99(1):92-97.
[88]Yoritaka A, Hattori N, Mori H et al. An immunohistochemical study on manganese superoxide dismutase in Parkinson's disease [J].Journal Neurology Science,1997,148(2):181-186.
[89]Petros All, Medek A, Nettesheim DG et al. Solution structure of the anti-apoptotic protein Bcl-2 [J].PNAS,2001,98(6):3012-3017.
[90]方晓阳,盛伟.细胞凋亡的概念来源[J].生命中的化学,2003,23(2)：121-123.
[91]Grimm S, Baeuerle PA. The inducible transcription factor NF-kB:Structure-function relationship of its protein subunits [J]. Biochemistry Journal,1993,290:297-308.
[92]Floury C, Mignotte B, Vayssiere JL. Mitochondrial reactive oxygen species in cell death signaling [J]. Biochimie,2002,84(2-3):131-141.
[93]Tan S, Sagara T, Liu Y et al. The regulation of reactive oxygen species production uring programmed cell death [J].The Journal of Cell Biology,1998,141(6):1423-1432.