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Axon Guidance and Growth Cone Collapse in Vitro 轴突诱导与体外生长锥塌陷
Neuroprotocols Pub Date : 1994-04-01 DOI: 10.1006/NCMN.1994.1012
H. Baier, S. Klostermann
{"title":"Axon Guidance and Growth Cone Collapse in Vitro","authors":"H. Baier, S. Klostermann","doi":"10.1006/NCMN.1994.1012","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1012","url":null,"abstract":"Abstract Important information about the mechanisms of axon guidance in the developing and regenerating brain has been obtained from in vitro experiments. One particular system, the retinotectal projection of vertebrates, has been analyzed by several in vitro assays, three of which are described here in detail: (i) the stripe choice assay, (ii) the collapse assay, and (iii) the gradient assay. Each of these has revealed position-specific behavior of retinal axons in response to cell membranes derived from different regions of the optic tectum. The stripe choice assay tests the ability of growing axons to discriminate between two membrane substrates offered as alternating stripes. The gradient assay assesses whether growth cones can detect (and be guided by) smooth transitions from one substrate type to another. The collapse assay reveals instantaneous reactions of growth cones to inhibitory or repellent factors present in their environment. The protocols describe the preparation of retinal explants and tectal membranes, as well as the assays proper. Particular emphasis is placed on the gradient assay, which has not yet been described in detail. All of the approaches discussed here have in common that they are applicable to axon guiding components bound to cell membranes. By a few modifications, however, it should be possible to extend this type of investigation to a wider range of related questions, including cell migration and guidance. We do not consider the important aspect of chemotropic guidance of axons in response to diffusible factors.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"173 1","pages":"96-105"},"PeriodicalIF":0.0,"publicationDate":"1994-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77611726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 17
Axon Regeneration in Vitro on Physiologically Relevant Substrata 轴突在生理相关基质上的体外再生
Neuroprotocols Pub Date : 1994-04-01 DOI: 10.1006/ncmn.1994.1018
Shewan Derryck A., Bedi Kuldip S., Berry Martin, Winter Janet, Cohen James
{"title":"Axon Regeneration in Vitro on Physiologically Relevant Substrata","authors":"Shewan Derryck A.,&nbsp;Bedi Kuldip S.,&nbsp;Berry Martin,&nbsp;Winter Janet,&nbsp;Cohen James","doi":"10.1006/ncmn.1994.1018","DOIUrl":"https://doi.org/10.1006/ncmn.1994.1018","url":null,"abstract":"<div><p>The study of axon growth in culture is limited by a poor understanding of the relative contribution of each of a complex array of factors, which include diffusible, axon growth-modulating molecules and substrate-bound guidance cues available to developing and regenerating neurons <em>in vivo</em>. With the objective of more closely mimicking <em>in vivo</em> conditions, one approach we have exploited employs thin cryosections of appropriate regions of unfixed nervous tissue as culture substrata for the growth of regenerating neurons. By using this technique it is possible to culture different populations of neurons on substrata in which environmental growth-modulating factors are preserved. This form of bioassay has facilitated the study of the different neurite outgrowth responses of neurons both from different sources and at different developmental ages on varying native substrata. Using this method we have demonstrated that mature dorsal root ganglion neurons (DRG) will regrow axons only on predegenerated sciatic nerve <em>in vitro</em>, while immature DRG extend neurites on both intact and degenerated sciatic nerve. In contrast, both mature and neonatal DRG fail to regenerate on either fully myelinated mature optic nerve or unmyelinated embryonic optic nerve. Moreover, neonatal retinal ganglion cells do not regenerate on any of these substrata.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"4 2","pages":"Pages 142-145"},"PeriodicalIF":0.0,"publicationDate":"1994-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1994.1018","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72113434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 8
Growth Cone Behavior at Borders between Different Extracellular Matrix Molecules 不同细胞外基质分子边界上的生长锥行为
Neuroprotocols Pub Date : 1994-04-01 DOI: 10.1006/NCMN.1994.1020
Joanne Taylor
{"title":"Growth Cone Behavior at Borders between Different Extracellular Matrix Molecules","authors":"Joanne Taylor","doi":"10.1006/NCMN.1994.1020","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1020","url":null,"abstract":"Abstract The reaction of growth cones in in vitro assays to substrate-bound molecules might yield important clues to the roles that these molecules play in growth cone guidance in vivo. Janusin and tenascin are glia-derived, extracellular matrix molecules that are expressed in the nervous system at times and in locations that suggest that they might act as barriers to neurite outgrowth. To test this hypothesis we have used video time-lapse microscopy to observe the behavior of growth cones, growing on a substrate permissive for neurite outgrowth, when they are confronted with janusin or tenascin as sharp, substrate boundaries. Here we describe the method for offering growth cones a choice between two substrates, in which the border between the two molecules can be clearly visualized in the phase-contrast microscope during the period of observation. We have learned from these observations that growth cones avoid advancing onto janusin or tenascin substrates, but do not undergo gross morphological changes, such as complete collapse, when they contact these molecules.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"38 1","pages":"158-166"},"PeriodicalIF":0.0,"publicationDate":"1994-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81405073","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
In Vitro Assays for Molecules That Inhibit Growth Cone Motility during Neural Development and Regeneration 在神经发育和再生过程中抑制生长锥运动分子的体外测定
Neuroprotocols Pub Date : 1994-04-01 DOI: 10.1006/NCMN.1994.1015
Alan R. Johnson, G. Cook, R. Keynes
{"title":"In Vitro Assays for Molecules That Inhibit Growth Cone Motility during Neural Development and Regeneration","authors":"Alan R. Johnson, G. Cook, R. Keynes","doi":"10.1006/NCMN.1994.1015","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1015","url":null,"abstract":"Abstract There is increasing evidence that molecules that inhibit growth cone motility are involved in the guidance of axons to their appropriate targets during neural development and contribute to the suppression of axon regeneration in the mammalian CNS. Two tissue culture phenomena have been used to detect and monitor these molecules: inhibition of neurite outgrowth and growth cone collapse. In neurite outgrowth assays the inhibitory material is used as a culture substratum. It can be presented to neurons either as a continuous layer or in a form that growing axons will encounter, such as an explant or a stripe. Tissue explants or sections, monolayer cultures of cells, membrane fractions, and purified or partially purified material have all been used. In the growth cone collapse assay, the growth cones of axons extending on a permissive substratum are treated with liposomes incorporating the putative inhibitory material. This method is particularly useful for testing the inhibitory effects of membrane-derived molecules. The relevance of results obtained with these in vitro assays to axon growth phenomena in vivo must always be established. Their principal value lies in the provision of a means of monitoring biochemical purification procedures aimed at identifying and characterizing molecules that inhibit nerve growth.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"11 1","pages":"121-128"},"PeriodicalIF":0.0,"publicationDate":"1994-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88729374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Neuronal Polarity and Morphogenesis 神经元极性和形态发生
Neuroprotocols Pub Date : 1994-04-01 DOI: 10.1006/NCMN.1994.1016
F. Lafont, A. Joliot, F. Perez, A. Prochiantz
{"title":"Neuronal Polarity and Morphogenesis","authors":"F. Lafont, A. Joliot, F. Perez, A. Prochiantz","doi":"10.1006/NCMN.1994.1016","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1016","url":null,"abstract":"Abstract For a given neuron, the development of its axonal and dendritic arborizations depends on many external factors, such as matrix molecules, growth factors, depolarization, electric fields, and adhesion molecules. In this paper, we summarize and comment on several protocols that can be used to modulate axonal or dendritic elongation and/or modify the shape of the neurites. A first series of protocols is based on the modulation of neuron-substratum adhesion by the addition of extra cellular matrix molecules. Indeed, axons initiate and elongate under low adhesion conditions, whereas dendrites grow only on highly adhesive substrata. A second series of protocols involves the use of drugs affecting the organization of the cytoskeleton. They suggest that the different behaviors of the axonal and dendritic compartments, in particular under low adhesion conditions, are due partly to the organization of the microtubule and actin networks. Third, we describe a protocol based on the internalization of Antennapedia homeodomain that translocates through the cell membrane and is conveyed to neuronal nuclei. Using this technique, we demonstrated that homeoproteins are involved in the morphological differentiation of postmitotic neurons and, in the case of the motoneurons, in axonal elongation. Furthermore, fusion polypeptides up to 109 amino acids and encompassing the 60-amino-acid translocating homeodomain are also transported through the membrane, thus offering a way to introduce exogenous biologically active peptides into live neurons.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"22 1","pages":"129-133"},"PeriodicalIF":0.0,"publicationDate":"1994-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82648592","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Micromethods for Analyzing Axon-Target Interactions in Vitro 体外分析轴突-靶标相互作用的显微方法
Neuroprotocols Pub Date : 1994-04-01 DOI: 10.1006/NCMN.1994.1013
D. Baird, M. Hatten, N. Heintz, C. Mason
{"title":"Micromethods for Analyzing Axon-Target Interactions in Vitro","authors":"D. Baird, M. Hatten, N. Heintz, C. Mason","doi":"10.1006/NCMN.1994.1013","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1013","url":null,"abstract":"Abstract In vitro methods for studying interactions between axons and their target cells are presented. The methods maximize the number of cultures that can be produced by limiting the volume and area of the cultures. Small cultures promote cell-cell Interactions and permit rapid conditioning of medium. In addition, valuable reagents added to these microcultures are conserved. The methods include: (a) the manufacture of 40-μl well-volume, coverslip-bottomed culture dishes with plating area of less than 24 mm 2 the dishes allow the small working distances of high-resolution light microscopy; (b) a micromethod to test for the Involvement of secreted factors in cell-cell interactions; cells on different surfaces are cocultured in shared medium; (c) a method to plate explant sources of neurites at a controlled distance from target cells to facilitate neurite identification and to control the timing of growth cone-target cell contacts; and (d) nonisotopic in situ hybridization for chamber-slide cultures combined with immunolabeling of cells in the hybridized culture. These methods can be used in culture assays to identify cell types or molecules involved in a variety of neuronal or, more generally, cell-cell interactions.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"6 1","pages":"106-115"},"PeriodicalIF":0.0,"publicationDate":"1994-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78413065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
Growth Cone Behavior at Borders between Different Extracellular Matrix Molecules 不同细胞外基质分子边界处的生长锥行为
Neuroprotocols Pub Date : 1994-04-01 DOI: 10.1006/ncmn.1994.1020
Taylor Joanne
{"title":"Growth Cone Behavior at Borders between Different Extracellular Matrix Molecules","authors":"Taylor Joanne","doi":"10.1006/ncmn.1994.1020","DOIUrl":"https://doi.org/10.1006/ncmn.1994.1020","url":null,"abstract":"<div><p>The reaction of growth cones in <em>in vitro</em> assays to substrate-bound molecules might yield important clues to the roles that these molecules play in growth cone guidance <em>in vivo</em>. Janusin and tenascin are glia-derived, extracellular matrix molecules that are expressed in the nervous system at times and in locations that suggest that they might act as barriers to neurite outgrowth. To test this hypothesis we have used video time-lapse microscopy to observe the behavior of growth cones, growing on a substrate permissive for neurite outgrowth, when they are confronted with janusin or tenascin as sharp, substrate boundaries. Here we describe the method for offering growth cones a choice between two substrates, in which the border between the two molecules can be clearly visualized in the phase-contrast microscope during the period of observation. We have learned from these observations that growth cones avoid advancing onto janusin or tenascin substrates, but do not undergo gross morphological changes, such as complete collapse, when they contact these molecules.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"4 2","pages":"Pages 158-166"},"PeriodicalIF":0.0,"publicationDate":"1994-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1994.1020","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72113213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Quantitative Analysis of Low-Abundance mRNA: Application to Adrenergic Receptors 低丰度信使核糖核酸的定量分析:在肾上腺素受体中的应用
Neuroprotocols Pub Date : 1994-02-01 DOI: 10.1006/ncmn.1994.1010
Hughes Richard J.
{"title":"Quantitative Analysis of Low-Abundance mRNA: Application to Adrenergic Receptors","authors":"Hughes Richard J.","doi":"10.1006/ncmn.1994.1010","DOIUrl":"https://doi.org/10.1006/ncmn.1994.1010","url":null,"abstract":"<div><p>The adrenergic receptors belong to a family of receptors that is postulated to span the plasma membrane seven times and is linked to regulatory GTP-binding proteins. These receptors mediate a wide variety of physiological responses through activation of several distinct second-messenger systems. Belying the importance of the adrenergic receptors in regulating physiological responses in target cells is their paucity. These receptors are present in very low numbers, in some cases only a thousand copies per cell. In addition, the adrenergic receptors are relatively stable proteins. Thus, cells have no need to synthesize large amounts of these proteins and, in consequence, the level of mRNA coding for these receptors is very low. In this paper, I examine some of the ways in which quantitation of these scarce mRNA species has been approached, with particular emphasis on the use of polymerase chain reaction.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"4 1","pages":"Pages 88-94"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1994.1010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72106493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Development of Novel Photoaffinity Ligands for the β-Adrenergic Receptor β-肾上腺素能受体新型光亲和配体的研制
Neuroprotocols Pub Date : 1994-02-01 DOI: 10.1006/ncmn.1994.1007
Ruoho Arnold E., Rashidbaigi Abbas, Hockerman Gregory H., Larsen Martha J., Resek John F., Malbon Craig C.
{"title":"Development of Novel Photoaffinity Ligands for the β-Adrenergic Receptor","authors":"Ruoho Arnold E.,&nbsp;Rashidbaigi Abbas,&nbsp;Hockerman Gregory H.,&nbsp;Larsen Martha J.,&nbsp;Resek John F.,&nbsp;Malbon Craig C.","doi":"10.1006/ncmn.1994.1007","DOIUrl":"https://doi.org/10.1006/ncmn.1994.1007","url":null,"abstract":"<div><p>The successful synthesis and use of carrier-free radioiodinated β<sub>2</sub>-adrenergic receptor competitive antagonist photoaffinity labels (±)-[<sup>125</sup>I]IABP, (±)-[<sup>125</sup>I]MAPIT, (−)-[<sup>125</sup>I]IAPTA, and (±)-[<sup>125</sup>I]IAPCGP-12177, are described. In addition, the synthesis and use of two carrier-free radioiodinated β-adrenergic receptor agonist photoaffinity labels (±)-[<sup>125</sup>I]iodoazidoprenalterol ((±)-[<sup>125</sup>I]IAPr) and (−)-<em>N</em>-(<em>p</em>-azido-<em>m</em>-[<sup>125</sup>I]iodophenethylamidoisobutyl)norepinephrine ((−)-[<sup>125</sup>I]NAIN), are described. All antagonist photolabels were capable of highly specific derivatization of the purified recombinant hamster lung β<sub>2</sub>-adrenergic receptor. Tryptic cleavage of the photolabeled receptor into a 30-kDa radiolabeled fragment (transmembrane 1-5) and an 8-kDa radiolabeled fragment (transmembrane 6,7) showed variable Insertion ratios between the two juxtaposed domains, depending on the structure of the photolabel. Unique synthetic strategies were used for the agonist photolabels. The phenolic hydroxyl of (±)-IAPr was protected as the glucoside and deprotected enzymatically in the final step. The final coupling step in the synthesis of (−)-[<sup>125</sup>I]NAIN was accomplished by reductive alkylation without protection of the catechol hydroxyls of norepinephrine using sodium cyanoborohydride. (±)-IAPr was found to be a partial agonist for the turkey erythrocyte β-adrenergic receptor and an effective photoaffinity label for the avian β-adrenergic receptor. (−)-NAIN was found to be a full agonist for the β<sub>2</sub>-adrenergic receptor in guinea pig lung membranes and a highly effective agonist photoaffinity label for the β<sub>2</sub>-adrenergic receptor. These photolabels will be useful for probing the β-adrenergic receptor binding site In order to \"map\" this site under nonactivated (antagonist photolabels) or activated states (agonist photolabels).</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"4 1","pages":"Pages 50-65"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1994.1007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72105688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Site-Directed Mutagenesis of α-Adrenergic Receptors α-肾上腺素能受体的定点突变
Neuroprotocols Pub Date : 1994-02-01 DOI: 10.1006/ncmn.1994.1004
Lee Norman H., Pellegrino Susan M., Fraser Claire M.
{"title":"Site-Directed Mutagenesis of α-Adrenergic Receptors","authors":"Lee Norman H.,&nbsp;Pellegrino Susan M.,&nbsp;Fraser Claire M.","doi":"10.1006/ncmn.1994.1004","DOIUrl":"https://doi.org/10.1006/ncmn.1994.1004","url":null,"abstract":"<div><p>The application of <em>in vitro</em> site-directed mutagenesis has led to the identification of conserved amino acids that play important roles in receptor structure and function. Precise amino acid substitutions can be obtained and then correlated with changes in receptor phenotype. Here, we describe several techniques commonly employed to Introduce site-specific mutations. The benefits and potential drawbacks of each method are discussed. Site-directed mutagenesis of the human α<sub>2A</sub>-adrenergic receptor (α<sub>2A</sub>AR) has been successfully employed to identify conserved amino acids involved in agonist binding and receptor activation. Aspartate residues in the second and intracellular side of the third transmembrane domain of the α<sub>2A</sub>AR are implicated in receptor/G-protein interactions. Since these aspartate residues are highly conserved among all G-protein-coupled receptors, and elimination of these residues has been shown to abolish the ability of other receptors in this class to activate their respective intracellular signaling pathways, It seems likely that these residues are critical for agonist-induced conformational changes that underlie receptor/G-protein interactions. In contrast to the role played by the conserved residues mentioned above, a conserved aspartate residue situated near the extracellular side of the third transmembrane domain plays a pivotal role in adrenergic ligand binding. Genetic analysis of the fifth transmembrane domain of the α<sub>2A</sub>AR suggests that a conserved serine residue in this region participates in hydrogen binding to the <em>meta</em>-hydroxyl group of catecholamines. These findings point to the utility of site-directed mutagenesis in identifying structure-function relationships among G-protein-coupled receptors.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"4 1","pages":"Pages 20-31"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1994.1004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72105691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
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