Sargaquinoic acid promotes neurite outgrowth via protein kinase A and MAP kinases-mediated signaling pathways in PC12D cells

We previously isolated a nerve growth factor (NGF)-dependent neurite outgrowth promoting substance MC14 (sargaquinoic acid) from a marine brown alga, Sargassum macrocarpum. In the present study, the NGF-potentiating activity of MC14 to neural differentiation of PC12D cells was investigated in detail. The treatment of cells with 3 microg/ml MC14 in the presence of 1.25-100 ng/ml NGF markedly enhanced the proportion of neurite-bearing cells compared with the NGF-only controls. In addition, MC14 significantly elevated the NGF-induced specific acetylcholinesterase (AchE) activity in PC12D cells, suggesting that MC14 could morphologically and biochemically promote the differentiation of PC12D cells. The mechanism of action of MC14 was further investigated by pharmacological inhibition of several intracellular signaling molecules. Results indicated that the neurite outgrowth promoting activity of MC14 was almost completely blocked by 10 microM PD98059, suggesting that a TrkA-dependent MAP kinases-mediated signaling pathway may play a crucial role in modulating the effect of MC14. Besides, the MC14-enhanced neurite outgrowth was substantially suppressed by the pretreatment with 10 ng/ml protein kinase A (PKA) inhibitor, demonstrating that the adenylate cyclase-PKA signaling cascade was also involved in the action of MC14. In contrast, a PKC inhibitor chelerythrine chloride did not inhibit the neurite outgrowth promoting activity of MC14. Altogether, these results demonstrate that MC14 enhances the neurite outgrowth by cooperating at least two separated signaling pathways, a TrkA-MAP kinases pathway and an adenylate cyclase-PKA pathway, in PC12D cells.     

骨髓基质细胞分泌物促进PC12细胞的分化和存活

目的体外检测骨髓基质细胞分泌物对PC12细胞的活性作用，并探讨其产生的可能机制。方法收集培养至第4代第7天的SD大鼠MSCs培养上清，按不同的体积百分比浓度加入到PC12细胞培养体系中，在倒置相差显微镜下观察1d和4d的细胞形态学改变；用丙二酸钠对PC12细胞造成氧化应激损伤，同时加入不同体积百分比浓度的MSCs培养上清，采用MTT法测定24h后的细胞活性。结果有突细胞／总细胞数、最长突起长度随培养时间及MSC培养上清体积百分比的增加而增加：PC12细胞氧化损伤后．加入一定浓度MSC培养上清组的PC12细胞活性较未加入组增高，差异有显著性。结论MSCs能够合成和分泌具有神经营养活性的物质，该物质能诱导PC12细胞分化并减轻氧化应激对PC12细胞的损伤。     

Changes in levels of microtubule-associated proteins in relation to the outgrowth of neurites from PC12D cells, a forskolin- and nerve growth factor-responsive subline of PC12 pheochromocytoma cells

Immunoblotting analysis and immunofluorescence studies of proteins that react with MAP₁- and MAP₂-specific antibodies in PC12 rat pheochromocytoma cells were carried out. When cells of the PC12D subline of PC12 cells, which rapidly extend neurites in response to NGF or drugs that elevate intracellular levels of cyclic AMP, were examined, they were found to contain a relatively higher level of MAP₁ or of a MAP₁-like protein than conventional PC12 cells. Immunoblotting study showed that levels of MAP₁ and MAP₂ or of MAP₁ or MAP₂-like proteins increased in PC12D cells, but not in conventional PC12 cells, in response to forskolin. Immunofluorescence studies also revealed increases in levels of MAP₁ and MAP₂ or of MAP₁ or MAP₂-like proteins in conjunction with the outgrowth of neurites from the cells. These results support the hypothesis that the induction of MAPs may be one of the first steps required for outgrowth of neurites from PC12 cells. Furthermore, PC12D cells may contain a sufficiently high level of MAP₁ or MAP₁-like protein to permit the extension of neurites in the absence of the lag period normally required by PC12 cells. The MAP₁ or a MAP₁-like protein was localized in the cell soma and neurites. An increase of MAP₂-specific immunoreactivity in perikarya was observed in the differentiated cells. After immunostaining with a monoclonal antibody that reacted with phosphorylated MAP₁, intense fluorescence was seen in the growth cones of neurites. This observation supports the hypothesis that the phosphorylation of MAP₁ or of a MAP₁-like protein may play a regulatory role in the formation of neurites in growth cones.     

Neuronatin mRNA in PC12 cells: downregulation by nerve growth factor

Neuronatin was recently cloned from neonatal rat brain (Biochem. Biophys. Res. Commun., 201 (1994) 1227–1234). In subsequent studies, we noted that neuronatin mRNA was brain-specific and that there were two alternatively spliced forms, α and β (Brain Res., 690 (1995) 92–98). Furthermore, on sequencing the human neuronatin gene, it was determined that the α-form was encoded by three exons, and the β-form was encoded by the first and third exons only (Genomics, 33 (1996) 292–297). The middle exon was spliced out in the β-form. The human neuronatin gene is located in single copy on chromosome 20q11.2-12 (Brain Res., 723 (1996) 8–22). These studies called for an understanding of the function of this gene. Therefore, we studied the expression of neuronatin in PC12 cells, an established model of neuronal growth and differentiation. Neuronatin mRNA expression was found to be abundant in undifferentiated PC12 cells. Treatment with nerve growth factor (NGF), resulting in neuronal differentiation, was associated with a downregulation of neuronatin mRNA expression. Removal of NGF was associated with a return of neuronatin mRNA levels towards baseline. These effects appear to be specific for NGF as they were not seen with transforming growth factor, epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate or dexamethasone. Although, basic fibroblast growth factor also reduced neuronatin mRNA levels, the effect was less pronounced than with NGF. The NGF-induced decrease in neuronatin mRNA occurred even in the presence of protein and RNA syntheses inhibitors. Of the two spliced forms, only the α-form was expressed in PC12 cells. In conclusion, we report the presence of neuronatin mRNA in PC12 cells, and that NGF downregulates its expression. These findings provide a basis for investigating the role of neuronatin in neuronal growth and differentiation.     

Metabolites of sesamin,a major lignan in sesame seeds,induce neuronal differentiation in PC12 cells through activation of ERK1/2 signaling pathway

Sesamin, a major lignan in sesame seeds, exhibits various health benefits. Here, we investigated effects of sesamin, its stereoisomer episesamin, and their metabolites on neuronal differentiation in rat pheochromocytoma PC12 cells. Among all compounds tested, primary metabolites of sesamin and episesamin, SC-1 and EC-1 {S- and R-epimer of 2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo [3.3.0]octane}, were the most potent to induce neuronal differentiation. SC-1 alone induced neuronal differentiation through extracellular signal-regulated kinase (ERK) 1/2 activation that is essential for nerve growth factor (NGF)-induced neuronal differentiation, as shown by the suppression with MEK1/2 inhibitors, PD98059 and U0126. However, SC-1 did not increase phosphorylation of TrkA, a high-affinity NGF receptor, and a TrkA inhibitor, K252a, did not affect SC-1-induced neuronal differentiation. Furthermore, SC-1 potentiated neuronal differentiation in cells co-treated with NGF, which was associated with enhanced ERK1/2 activation and increased expression of neuronal differentiation markers. Interestingly, when treated with SC-1 and a high dose of NGF, formation of synaptic connections and synaptophysin accumulation at the neurite terminals were markedly enhanced. These results indicate that (1) SC-1 alone induces neuronal differentiation, (2) SC-1 potentiates neuronal differentiation in NGF-treated cells, (3) SC-1 enhances formation of synaptic connections in cells treated with a high dose of NGF, all of which are associated with ERK1/2 activation. It is therefore concluded that SC-1 may promote neuronal differentiation by tapping into the ERK1/2-MAPK (mitogen-activated protein kinase) signaling pathway downstream from the TrkA receptor in PC12 cells.     

PC12h-R cell,a subclone of PC12 cells,shows EGF-induced neuronal differentiation and sustained signaling

Unlike nerve growth factor (NGF), epidermal growth factor (EGF) does not induce neuronal differentiation but promotes proliferation of the rat pheochromocytoma PC12 cells. We found that PC12h-R, a subclone of PC12 cells, differentiated into neuron-like cells in response to EGF as well as to NGF. PC12h-R cells treated with EGF extended neurites, attenuated cell proliferation, and increased the levels of tyrosine hydroxylase protein synthesis and of acetylcholinesterase activity as those treated with NGF. The EGF-induced differentiation of PC12h-R cells was not mediated by the indirect activation of p140^trkA by EGF. In addition, EGF induced the sustained tyrosine phosphorylation of the EGF receptor, mitogen-activated protein (MAP) kinases, and 46 and 52 kDa proteins, and the prolonged activation of MAP kinases in PC12h-R cells compared with the parent PC12h, which does not show EGF-induced differentiation. The response of PC12h-R cells to EGF was not simply due to an increase in the level of EGF receptor protein. These results indicated that the duration of EGF-induced signaling might determine the cellular response of PC12 cells between cell proliferation and neuronal differentiation. © 1996 Wiley-Liss, Inc.     

1-Methyl-4-phenylpyridinum kills differentiated PC12 cells with a concomitant change in protein phosphorylation

1-Methyl-4-phenylpyridinum (MPP⁺), a selective neurotoxin, destroys the dopaminergic nigrostriatal pathway and results in a parkinsonian syndrome. Exposure of differentiated PC12 cells with nerve growth factor for 5 days to MPP⁺ (100 μM) for 4 h induced DNA fragmentation which is typical for the programmed cell death. MPP⁺ treatment (100 μM) concomitantly stimulates S6 kinase activity and resultant phosphorylation of S6 protein of 40S ribosomal subunits in the cells. Cycloheximide treatment prevents the MPP⁺-induced DNA fragmentation and enhancement of the phosphorylation of S6 protein. The present data demonstrate that neurotoxin, MPP⁺, kills differentiated PC12 cells by the apparent involvement of apoptotic process. Furthermore, the data strongly suggest that a change in protein phosphorylation might be involved in the signal transduction of MPP⁺ neurotoxicity and/or the protection from its toxicity.     

Activation of mitogen-activated protein kinases is not required for the extension of neurites from PC12D Cells triggered by nerve growth factor

Numerous studies with PC12 cells have suggested that the mitogen-activated protein (MAP) kinase pathway might play a major role in the neuronal differentiation that is induced by nerve growth factor (NGF). Cells of the PC12D subline extend neurites within several hours in response to NGF in the presence of inhibitors of the synthesis of RNA and protein. We examined the effects of a specific inhibitor 2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD98059) of the MAP kinase kinase (MEK)/MAP kinase pathway on the NGF-induced outgrowth of neurites in PC12D cells. The increase in MAP kinase activity in response to NGF was reduced by 80% upon treatment of PC12D cells with 50 μM PD98059, whereas the NGF-dependent formation of ruffles and the subsequent outgrowth of neurites were not blocked by PD98059 at this concentration. The outgrowth of neurites from conventional PC12 cells by NGF was suppressed by the addition of 50 μM PD98059 as reported by Pang et al. [L. Pang, T. Sawada, J. Stuart,S.J. Decker, A.R. Saltiel, Inhibition of MAP kinase kinase blocks the differentiation of PC12 cells induced by nerve growth factor, J. Biol. Chem. 270 (1995) 13585–13588]. In contrast, the rapid regeneration of neurites from PC12 cells primed with NGF, was not altered in the presence of the same dose of the inhibitor of MEK. It appeared, therefore, that the activation of the MAP kinase pathway was not necessarily required for the NGF-dependent extension of neurites. When PC12D cells were transfected with the dominant inhibitory Ha-ras Asn-17 gene, the induction of the mutant Ras protein led the suppression of the rapid outgrowth of neurites in response to NGF but not to dibutyryl cyclic AMP (dbcAMP). The result implies a direct involvement of Ras protein in the NGF-induced signal transduction that lead to the formation of neurites in PC12D cells. We can conclude that the activation of MAP kinase and selective gene expression are required for the differentiation of conventional PC12 cells to sympathetic neuron-like cells and that activation of Ras protein and, subsequently, of a MAP kinase-independent pathway might be involved in the extension of neurites from PC12D cells or in the regeneration of neurites from primed PC12 cells in response to NGF.     

Nerve growth factor-induced differentiation of PC12 cells employs the PMA-insensitive protein kinase C-ζ isoform

To elucidate the role of protein kinase C (PKC) in nerve growth factor (NGF)-induced differentiation, PMA downregulation of pheochromocytoma (PC12) cells was undertaken. Prolonged treatment (2 d) of PC12 cells with PMA (1 μM) resulted in depleting the cells of α, β, δ, and ɛ-PKC isoforms, but had no effect on the expression of the atypical PKC isoform ζ. PC12 cells, which expressed only PKC ζ, were evaluated for their responses to NGF. Removal of the PMA-sensitive PKC isoforms enhanced the ability of NGF to promote neurite extension. Both the percentage cells with neurites and length of neurites were increased in the PMA-treated cells, whereas no effect was observed on the number of neurites per cell or branching of individual neurites. In addition, PMA downregulation resulted in an increase in the incorporation of³H-thymidine without any significant effect on the expression of c-fos. Addition of NGF to PC12 cells depleted of the PMA-sensitive PKC isoforms resulted in the activation of PKC ζ (Wooten et al., 1994). To test whether the transient activation of PKC ζ is a necessary component of the neuritogenetic pathway, antisense oligonucleotide strategy was utilized to remove this particular PKC isoform. The addition of a 20-bp antisense oligonucleotide directed against the 5′ coding sequence of PKC ζ attenuated NGF-induced neurite outgrowth in PC12 cells lacking PMA-sensitive PKC isoforms. Sense oligonucleotide directed at the same site was without effect on NGF responses. These data indicate that PKC ζ comprises a portion of the NGF pathway and underscores the importance of this isoform in neuronal differentiation. Moreover, these findings demonstrate that the PMA-insensitive pathway, which was previously characterized as PKC-independent, and the neurite induction pathway are synonymous and mediated by PKC ζ.     

Staurosporine induces the outgrowth of neurites from the dorsal root ganglion of the chick embryo and PC12D cells

Staurosporine, a potent inhibitor of protein kinases, caused the rapid outgrowth of neurites from cultured dorsal root ganglia of chick embryos and from PC12D cells, a subline of PC12 cells. Treatment of dorsal root ganglia with 1 to 20 nM staurosporine resulted in the extensive outgrowth of neurites that were indistinguishable from those induced by NGF, as assessed by phase-contrast microscopy, electron microscopy and cytochemical staining of actin and tubulin. However, neurites generated from the ganglia in response to the higher concentrations of staurosporine (40–100 nM) seemed to have different characteristics, possible as a result of the inhibition of cell migration from ganglia. The sequential changes in morphology of PC12D cells in response to staurosporine and to NGF were revealed by staining of actin. Ruffling membranes emerged at the margins of PC12D cells within 4 min after the addition of staurosporine or of NGF. From 10 min to 24 h after the addition of either compound, the ruffles were transformed into several projections that became growing neurites. The formation of ruffles and the outgrowth of neurites were both apparent at a concentration of staurosporine of 10 nM. The neurites that emerged from PC12D cells in response to staurosporine and in response to NGF were indistinguishable under the phase-contract microscope and after staining of actin and tubulin. However, staurosporine never promoted survival of PC12D cells in serum-free conditions as that promoted by NGF. The observations indicate that staurosporine at nanomolar concentrations may reproduce the neurogenic changes that induced by NGF in primed neuronal cells, although it can not mimic the action of NGF that supports survival of neurons.     

HMG-CoA reductase inhibitor induces a transient activation of high affinity nerve growth factor receptor, trk, and morphological differentiation with fatal outcome in PC12 cells

The present study was aimed at investigating the possible toxicity of simvastatin on a neuronal cell line, PC12 cells. Simvastatin clearly induced a transient morphological differentiation as evidenced by the occurrence of neurite outgrowth with a transient activation of the high affinity nerve growth factor receptor, Trk, but died at 36 h after its addition. Tyrosine autophosphorylation of the Trk protein also disappeared at 36 h after addition. During the morphological differentiation, NGF mRNA expression was upregulated transiently and returned to the basal level at 36 h after addition of simvastatin. These results suggest that simvastatin is neurotoxic and PC12 cells elicited a protective response, involving a transient activation of a Trk-mediated intracellular signal transduction pathway by an autocrine secretion of NGF, although these responses did not persist against pro-apoptotic signals and resulted in an apoptosis of the PC12 cells.     

Effect of Cuscuta chinensis glycoside on the neuronal differentiation of rat pheochromocytoma PC12 cells

Exposure of rat pheochromocytoma PC12 cells to Cuscuta chinensis glycoside induced neuronal differentiation with resulting outgrowth of neurites and increase of acetylcholinesterase activity. A specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, PD98059, prevented this effect of C. chinensis on PC12 cells. These results suggested that C. chinensis glycoside induced neuronal differentiation in PC12 cells linked to the mitogen-activated protein kinase signaling cascade.     

PC12细胞分化的神经元与离体培养的大鼠原代皮质神经元之间功能性突触的形成

目的　观察由PC12细胞分化成的神经元样细胞与原代皮质神经元之间突触的形成情况,探讨移植细胞和宿主细胞形成神经连接的可能性及其机制。方法　将增强型绿色荧光蛋白标记的PC12细胞种植到原代神经元和胶质细胞中,建立模拟细胞移植环境的共培养系统,以神经生长因子(NGF)诱导其中的PC12细胞分化为神经元样细胞。然后通过FM1 -43荧光造影研究共培养系统中神经元突触小泡活动,电镜及胶体金标记免疫电镜两种方法观察两种神经元间的超微结构。结果与原代皮质细胞共培养的PC12细胞受NGF诱导后,分化的比例更高(由57 .1%提高到67. 8%, P<0 .01);神经突起的长度明显高于对照组(由146 .3μm提高到272 2μm, P<0 .01)。FM1 43造影显示,PC12细胞分化的神经元与原代神经元之间存在突触小泡活动;电镜下通过精确定位和胶体金标记,显示两种细胞间形成了比较典型的突触样连接。结论　与原代皮质细胞共培养的PC12细胞在NGF诱导下可以分化为成熟的神经元,并与宿主来源的神经元形成功能性突触样连接,这为通过细胞移植治疗中枢神经系统疾病提供了一定线索。     

Treatment time influences the effects of a low-frequency pulsed electric field on synthesis of tyrosine hydroxylase and dopamine in PC12 cells

BACKGROUND: Electromagnetic radiation can influence dopamine (DA) synthesis in brain tissues or cells, but electromagnetic frequencies, intensities, and radiation time can produce different effects. In addition, the signal pathway by which electromagnetic radiation influences DA synthesis remains controversial. OBJECTIVE: To determine tyrosine hydroxylase (TH) expression in PC12 cells and DA levels in cell culture media after different periods of low-frequency pulsed electric field (LF-PEF) stimulation, and to determine how LF-PEF signaling stimulates TH synthesis using inhibitors. DESIGN, TIME AND SETTING: A parallel, controlled, cell experiment was performed at the Laboratory of Cell Biology, School of Life Science, East China Normal University, between January and October 2006. MATERIALS: PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. Nerve growth factor was purchased from PeproTech, USA. The protein kinase A inhibitor, H-89, and mitogen-activated protein kinase kinase inhibitor, U0126, were purchased from Sigma, USA. METHODS: (1) Following routine culture in Dulbecco's modified eagle medium, primary PC12 cells were stimulated under LF-PEF (pulse frequency 50 Hz, pulse width 20 us, peak field strength 1 V/m) for 5,10,15,20, and 30 minutes. (2) Inhibitors (H-89 or U0126,1 umol/L) were added 30 minutes before LF-PEF stimulation for 10 minutes. MAIN OUTCOME MEASURES: (1) TH expression was determined by Western blot in PC12 cells at 0.5,1, 2, 3, and 4 days after LF-PEF stimulation. Similarly, DA was measured by high-performance liquid chromatography in media at 2,3,4, or 5 days after LF-PEF. (2) TH expression was detected 1 day after H-89 or U0126 treatment and LF-PEF. RESULTS: (1) Short-term LF-PEF stimulation (5 and 10 minutes) increased TH expression and media DA levels after short-term culture (2 days) (P < 0.01), but both parameters decreased with longer culture (3-4 days) (P< 0.01). Long-term LF-PEF stimulation (15, 20, or 30 minutes) decreased TH and DA synthesis, followed by a rapid increase (P< 0.01). (2) H89 could completely inhibit TH expression in PC12 cells stimulated by LF-PEF for 10 minutes, while the inhibition rate of U0126 was 53.2%. CONCLUSION: Short-term LF-PEF first promotes then inhibits, while long-term LF-PEF first inhibits then promotes, TH and DA synthesis. LF-PEF stimulation regulates TH expression primarily by activating protein kinase A to regulate DA synthesis.     

Re-examination of the local control by nerve growth factor of the outgrowth of neurites in PC12D cells

We have examined the local control by nerve growth factor (NGF) of the outgrowth of neurites from clonal cells, PC12D, a subline whose phenotype resembles that of the parent PC12 cell line in the NGF-primed state. We show here that (i) the outgrowth of neurites and their survival can be induced by NGF in enucleated PC12D cells, (ii) individual neurites of a single ‘giant cell’, produced by cell fusion of PC12D cells, can respond independently to the NGF in the local environment, (iii) dissected neurites from giant cells survive for longer in medium that contains NGF than in medium that does not, (iv) in PC12D cells, the rapid formation of ruffles in response to NGF, which appears to be based on increased cell-substratum adhesion, leads to the subsequent formation of neurites, and (v) upon addition of NGF, the movement of short processes displaces polylysine-coated beads in the vicinity of neurites. These observations suggest that the NGF-dependent maintenance or extension of neurites might be controlled within the neurites themselves and might not require the direct involvement of the cell body, even in PC12 cells. It seems possible that any NGF-induced changes that promote an increase in cell-substratum adhesion might be responsible for the initiation and elongation of neurites. It also seems possible that the growth of neurites towards a source of NGF might be based on repeated rounds of extension and retraction of filopodia and neurites in a manner that depends on the concentration of NGF.     

Serum vulnerability and time-dependent stabilization of neurites induced by nerve growth factor in PC12 pheochromocytoma cells

Cultures of PC12 pheochromocytoma cells were established on a polyornithine substratum in medium supplemented with the chemically defined N1 mixture in the presence or absence of Nerve Growth Factor (NGF). Normal cell proliferation in the absence of NGF was equally competent when fetal calf serum (FCS) was replaced with N1-supplemented medium. The differentiation of PC12 cells, which occurs upon NGF treatment, ultimately results in cell death without the addition of 0.1% FCS to the N1-supplemented medium. The combination of N1, 0.1% FCS, and NGF permits the PC12 cells to develop a neuritic outgrowth much earlier than when higher (1-10%) FCS levels are used. Neurite retraction is caused in a dose-dependent manner by a delayed presentation of FCS. Within 2 days of serum presentation, however, neurites regrow to achieve that percentage of neurite-bearing cells which is seen without a serum challenge. Moreover, the retraction response becomes less pronounced with time over the 8-day culture period for any given serum concentration. Among the N1 ingredients, only insulin and transferrin are needed by PC12 cells for survival whether in the dividing state or not. Neurite growth was not dependent on any of the N1 components.     

Axl和Tyro-3受体表达受神经生长因子调控并参与PC12细胞的分化

目的 Axl和Tyro-3受体在脑内有区域性的分布,但两者在中枢神经系统中的生物学功能尚不明确.本研究旨在探讨Axl和Tyro-3受体在神经元分化中的作用.方法 PC12细胞分别转染CMV-Axl-eGFP、CMV-Tyro-3-eCFP和CMV-eGFP质粒后,给予神经生长因子(nerve growth factor...     

Axl和Tyro-3受体表达受神经生长因子调控并参与PC12细胞的分化(英文)

目的Axl和Tyro-3受体在脑内有区域性的分布,但两者在中枢神经系统中的生物学功能尚不明确。本研究旨在探讨Axl和Tyro-3受体在神经元分化中的作用。方法PC12细胞分别转染CMV-Axl-eGFP、CMV-Tyro-3-eCFP和CMV-eGFP质粒后,给予神经生长因子(nerve growth factor,NGF)诱导,观察绿色荧光蛋白和青色荧光蛋白的表达和分布。结果Axl-eGFP和Tyro-3-eCFP的表达随着NGF作用时间的延长而逐渐上调,并且荧光蛋白在细胞内的定位也发生变化。作为对照组,CMV-eGFP转染的PC12细胞并没有出现此变化。此外,过表达Axl和Tyro-3能够促进PC12细胞的突起生长。结论Axl和Tyro-3受体的表达受NGF调控,其过表达可能参与了PC12细胞的分化。