Low-dose methylmercury-induced oxidative stress, cytotoxicity, and tau-hyperphosphorylation in human neuroblastoma (SH-SY5Y) cells

Acute neurotoxic effects of high-dose methylmercury (MeHg) in humans have been well documented in the scientific literature. However, low-dose effects are less well described. This study was designed to evaluate the effects of low-dose MeHg (<100 nM) on human brain cells in a tissue culture model. Neuroblastoma (NB) cells (SH-SY5Y) were used in the cell culture model to study low-dose effects of MeHg on cell growth, cell survival, reactive oxygen species (ROS), and the phosphorylation of tau protein, as a measure of potential markers of cellular events associated with tauopathies. When cells were incubated in culture with MeHg (50 and 100 nM), there were significant decreases in cell viability as well as significant increase in ROS generation as determined by fluorescent dye analysis (H(2)DCFDA). Furthermore, a concomitant decrease in glutathione levels to 25% of control was observed at both 50 and 100 nM MeHg. In addition, the level of phosphorylated tau was significantly increased after treatment at both 50 and 100 nM MeHg, compared with controls. Pretreatment of NB cells with the antioxidant, N-acetylcysteine (1.25 mM) and the calpain inhibitor, MDL-28170 (10 μM), significantly attenuated the effects of MeHg (50 and 100 nM) on cell viability as well as on tau phosphorylation. These results indicate that low-dose MeHg toxicity may be related to an induction of tau phosphorylation through an oxidative stress-dependent mechanism and that blockade of this pathway may attenuate the toxic effects of MeHg.     

Isomers and their metabolites of endosulfan induced cytotoxicity and oxidative damage in SH‐SY5Y cells

As an organochlorine insecticide, endosulfan has been widely banned or restricted, but it is still largely used in many developing countries. Previous studies have shown multiple adverse health effects of endosulfan. However, the neurotoxicity of endosulfan has not been fully elucidated. In this study, endosulfan isomers (α‐/β‐endosulfan) and their major metabolites (endosulfan sulfate, endosulfan diol, and endosulfan lactone) were, respectively, exposed to human neuroblastoma SH‐SY5Y cells. Results showed that both α‐endosulfan and β‐endosulfan caused decrease of cell viability and morphological damages in a dose‐dependent manner. Their median effective concentrations (EC_50s) were respectively 79.6 μM (α‐endosulfan) and 50.37 μM (β‐endosulfan) for 72 h exposure. EC_50s of α/β‐endosulfan mixture were lower than that of the single isomer. However, EC_50s of its metabolites were higher than that of technical endosulfan. Endosulfan and its metabolites caused increases of reactive oxygen species and the lipid peroxidation, but decrease of superoxide dismutase in a dose‐dependent manner. These results indicate that α‐endosulfan exhibits higher neurotoxicity than β‐endosulfan. Mixture of endosulfan isomers shows stronger cytotoxicity than the single isomer. After endosulfan is degraded, cytotoxicity of its metabolites decreases gradually. The neurotoxicity of endosulfan and its metabolites is closely related to oxidative damage and antioxidative deficit. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 496–504, 2016.     

Protective effect of C(60) -methionine derivate on lead-exposed human SH-SY5Y neuroblastoma cells

Oxidative stress has been considered as one of the possible mechanisms leading to the neurotoxicity of lead. One of the effective ways to prevent cellular damage after lead exposure is using antioxidants. In this paper, a novel C₆₀‐methionine derivate (FMD), a fullerene molecule modified with methionine, was synthesized. The protective effect of FMD on lead‐exposed human SH‐SY5Y neuroblastoma cells was investigated. In this research, after incubating with 500 µm Pb acetate alone for 72 h, the cells had undergone a series of biological changes including viability loss, apoptotic death, the depletion of glutathione (GSH), the peroxidation of membrane lipid and DNA damage. Pretreatment with FMD before lead exposure could improve cell survival, increase the GSH level, reduce malondialdehyde content and attenuate DNA damage without obvious toxicity. In addition, the protective effects of FMD were proven to be greater than those of other two C₆₀‐amino acid derivates, β‐alanine C₆₀ derivate and cystine C₆₀ derivate, which have been confirmed in our previous work to be able to protect rat pheochromocytoma PC12 cells from hydrogen dioxide‐induced oxidative injuries. These observations suggest that FMD may serve as a potential antioxidative and neuroprotective agent in the prevention of lead intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

氧化应激在鱼藤素致SH-SY5Y细胞神经毒性中的作用

目的研究氧化应激与鱼藤素致神经毒性的相关性,为后续鱼藤素的结构改造和联合用药提供机制基础。方法将鱼藤素(1.56~100μmol·L-1)与SH-SY5Y细胞共孵育培养24~72 h,采用CCK-8法测定细胞存活率;比色法测定乳酸脱氢酶(lactate dehydrogenase,LDH)漏出量、丙二醛(malondialdehyde,MDA)含量、谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)和超氧化物歧化酶(superoxide dismutase,SOD)的活力;流式细胞术检测活性氧(reactive oxygen species,ROS)含量;免疫印迹法检测细胞中MEK、EGF、RAS、CREB蛋白的表达情况。结果鱼藤素对SH-SY5Y细胞增殖有抑制作用,且呈浓度和时间依赖性(P<0.05)。鱼藤素损伤细胞后,LDH漏出量、MDA和ROS含量明显增加,GPx和SOD的活力下降(P<0.05)。同时MAPK/ERK信号通路中MEK、EGF、RAS、CREB蛋白水平出现不同程度的下调。结论鱼藤素致SH-SY5Y神经细胞的损伤与氧化应激密切相关,MAPK/ERK信号通路可能在其中起介导作用。     

Attenuation of low dose methylmercury and glutamate induced‐cytotoxicity and tau phosphorylation by an N‐methyl‐D‐aspartate antagonist in human neuroblastoma (SHSY5Y) cells

Methylmercury (MeHg), a known neurotoxin, has been reported to alter glutamate homeostasis in the neuronal environment resulting in excitotoxicity. This study was conducted to investigate whether, and if so, under what conditions, that low dose MeHg would enhance the toxicity of glutamate and to what extent that blockade of NMDA receptors would alter MeHg and glutamate's toxicity in cultured neuroblastoma cells. Neuroblastoma cells (SH‐SY5Y) were used in a cell culture model to study effects of MeHg, glutamate (glu), a calcium chelator (BAPTA‐AM), and a noncompetitive NMDA antagonist, MK‐801 on cell growth, cell survival, and phosphorylation of tau protein, as a measure of cellular events associated with tauopathies. Exposure of cells to a combination of MeHg (50 nM) and glutamate (1 mM) resulted in both a greater decrease in cell viability as well as a greater induction in tau phosphorylation, as compared to exposures with MeHg and glutamate alone. MK‐801, an NMDA receptor antagonist, and the intracellular calcium chelator, BAPTA‐AM, both significantly inhibited tau hyperphosphorylation and protected cells from the effects of combination exposures to glutamate and MeHg. These results may indicate that exposure to even nontoxic levels of MeHg may prime neuronal cells to be more susceptible to neuronal injury from excitotoxicants such as glutamate and thus may increase the likelihood of neurological disease states. In conclusion, low‐dose MeHg‐induced toxicity may be related to an increase in the cellular response to glutamate and that NMDA receptor antagonists may provide a potential treatment for MeHg‐associated neurological diseases. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 700–706, 2013.     

Modulation of neurotoxicant-induced increases in intracellular calcium by phytoestrogens differ for amyloid beta peptide (Abeta) and 1-methyl-4-phenyl-pyridine (MPP(+))

Neurotoxicant-induced elevation of intracellular calcium (Ca(2+)) and modulation by phystoestrogens were examined in vitro using human neuroblastoma SH-SY5Y cells cultured with amyloid beta-peptide (Abeta) and 1-methyl-4-phenyl-pyridine (MPP+). Although Abeta itself did not increase Ca(2+), it exacerbated the effects of carbachol. The elevation of Ca(2+) caused by the agents in combination could be reduced by pretreatment with the phytoestrogens equol and genistein, as well as by the L-type Ca(2+) channel blocker nifedipine. MPP+ exposure also elevated Ca(2+), an effect blocked by nifedipine but not by the phytoestrogens. As opposed to phytoestrogens, nifedipine was also able to significantly reduce cell death caused by higher concentrations of MPP(+) in the LDH viability assay. The results suggest that phytoestrogens are unlikely to serve as general cellular protectants for neurotoxicants with different mechanisms of action. The concentrations of Abeta and MPP(+) affecting Ca(2+) release did not inhibit cell viability as measured with the LDH release assay. This indicates that mechanisms involved with toxicity can be studied at doses that are not lethal.     

Autophagy potentially protects against 2,3,7,8‐tetrachlorodibenzo‐p‐Dioxin induced apoptosis in SH‐SY5Y cells

The environmental toxicant TCDD may elicit cytotoxic effects by inducing reactive oxygen species (ROS) generation. Autophagy is one of the first lines of defense against oxidative stress damage. Herein, we investigated whether autophagy played a regulatory role in TCDD‐induced neurotoxicity. Here, we showed that TCDD exposure caused marked autophagy in SH‐SY5Y cells, whose dose range was close to that inducing apoptosis. Electron microscopic and Western blot analyses revealed that TCDD induced autophagy at a starting dose of approximate 100 nM. Interestingly, 100–200 nM TCDD exposure resulted in obviously decreased cell viability and evident apoptotic phenotype. Furthermore, the levels of pro‐apoptotic molecules, Bax and cleaved‐PARP, increased significantly, whereas Bcl2 declined after exposed to 100 nM TCDD. In addition, the apoptosis was verified using flow cytometrical analysis. These data strongly suggested that TCDD induced both autophagy and apoptosis at a similar dose range in SH‐SY5Y cells. Interestingly, pretreatment with ROS scavenger, N‐acetyl‐cysteine (NAC), could effectively block both TCDD‐induced apoptosis and autophagy. More surprisingly, inhibition of autophagy with 3‐methyladenine (3MA), remarkably augmented TCDD‐induced apoptosis. The findings implicated that the onset of autophagy might serve as a protective mechanism to ameliorate ROS‐triggered cytotoxic effects in human SH‐SY5Y neuronal cells under TCDD exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1068–1079, 2016.     

Apoptosis induced by acrylamide in SH-SY5Y cells

Acrylamide (1–5 mM) dose-dependently decreased cell viability in human neuroblastoma cells (SH-SY5Y). The caspase-3 activity and cell population in sub-G₁ phase were elevated and peaked on exposure to 3 mM acrylamide, while both were less so at higher dose (4 and 5 mM). Z-VAD-fmk, a pan-caspase inhibitor, lowered the apparent cytotoxicity of acrylamide. U0126, a specific inhibitor of extracellular signal-regulated protein kinase (ERK) kinase, suppressed the elevation of caspase-3 activities as well as that of sub-G₁ population. Thus, although mechanisms other than caspase-dependent apoptosis may be involved, apoptotic process seems to take place in the genesis of toxicity of acrylamide in SH-SY5Y cells through ERK pathway and activation of caspase-3.     

20-羟基蜕皮甾酮对H_2O_2诱导SH-SY5Y细胞损伤的保护作用

本文旨在研究一种类固醇激素即20-羟基蜕皮甾酮对H2O2诱导的SH-SY5Y细胞损伤的保护作用及其作用机制。预孵育20-羟基蜕皮甾酮可以明显提高H2O2诱导的SH-SY5Y细胞的生存率,减少乳酸脱氢酶（LDH）漏出率,抑制脂质过氧化水平（MDA）升高,提高超氧化物歧化酶（SOD）活性。此外,20-羟基蜕皮甾酮可以通过降低Bax/Bcl-2的比值,延缓caspase-3的活化来抑制H2O2诱导的细胞凋亡。本研究表明20-羟基蜕皮甾酮对H2O2诱导的SH-SY5Y细胞具有保护作用,有可能用于由氧化应激和凋亡导致的神经退行性疾病的治疗。     

姜黄素对SH-SY5Y细胞血红素加氧酶同工酶表达的影响

目的研究姜黄素在神经母细胞瘤细胞(SH-SY5Y)中对血红素加氧酶(HO)同工酶表达的影响,探讨姜黄素通过维持HO-1和HO-2的平衡对神经细胞的保护作用。方法体外培养人神经母细胞瘤细胞(SH-SY5Y细胞),以不同浓度的姜黄素(0、1.25、5.0、20μmol·L-1)处理SH-SY5Y细胞24h,及用浓度为5.0μmol·L-1的姜黄素分别处理SH-SY5Y细胞0、12、24、48h。利用荧光探针DCFH-DA和荧光分光光度计检测细胞内活性氧的水平。通过RT-PCR检测HO-1、HO-2mRNA的水平,Western blot检测HO-1、HO-2蛋白的表达。结果姜黄素作用于SH-SY5Y细胞后活性氧水平降低(P<0.05)。HO-1mRNA和蛋白的表达增加(P<0.05),而HO-2mRNA和蛋白的表达水平明显减弱(P<0.05),并呈浓度-时间依赖性。结论姜黄素通过抗氧化应激保护SH-SY5Y细胞,并促进SH-SY5Y细胞中HO-1的表达,下调HO-2的表达(P<0.05),姜黄素对HO-1和HO-2的这种反向调节,可能是姜黄素抗氧化应激、发挥神经细胞保护作用的一个机制。     

MPP+和6-羟基多巴胺对C6胶质瘤细胞摄取谷氨酸的影响

目的：研究1-甲基4-苯基-吡啶离子(MPP^ )和6-羟基多巴胺(6-OHDA)对C6胶质瘤细胞摄取谷氨酸(Glu)的影响。方法：应用放射免疫法测定C6胶质瘤细胞对培养液中[^3H]-D，L-谷氨酸的摄取；应用四氮唑(MTY)比色法检测胶质瘤细胞的活性。结果：6-OHDA和MPP^ 均不同程度地抑制C6胶质瘤细胞摄取Glu；6-OHDA显抑制C6胶质瘤细胞活性，而MPP^ 却不影响细胞活性；蛋白激酶C(PKC)激动剂TPA显增强C6细胞对Glu的摄取，并拮抗：MPP^ 对C6细胞摄取Glu的抑制。结论：6-OHDA抑制C6胶质瘤细胞对Glu的摄取，可能与其抑制细胞活性有关，而MPP^ 的抑制作用可能与直接影响转运体的摄取功能有关，并可能由PKC途径介导。     

利福平抑制MPP^＋所致PC12细胞凋亡的研究

目的探讨利福平对MPP＋（1-甲基4-苯基吡啶离子）诱导的分化大鼠嗜铬细胞瘤细胞株（ratphaeochmmocytoma,PC12）细胞活性、细胞形态、调亡率的影响及其影响的机制。方法（1）利用MPP^＋诱导分化PCI2细胞建立帕金森病细胞模型;（2）MTT法检测细胞活性,（3）Tunel原位末端标记法检测细胞形态及半定量细胞凋亡率,（4）流式细胞术检测caspase－3激活率。结果（1）MPP＋作用后,细胞生长活性明显受到抑制,凋亡细胞数量增多,调亡率增加,caspase－3激活率明显增高;（2）而经100、200和300μmol／L各浓度利福平预处理后,利福平各浓度组内细胞活性增高,细胞凋亡程度、凋亡率及caspase－3激活率降低,且与利福平作用浓度存在剂量一效应关系。结论利福平可抑制MPP＋所致PCI2细胞的凋亡,这一作用是通过caspase-3途径起作用的,且存在剂量一效应关系。     

Growth‐promoting effects of low‐level butyl benzyl phthalate exposure on human neuroblastoma SH‐SY5Y cells

The purpose of present study was to investigate the impact of butyl benzyl phthalate (BBP) on SH‐SY5Y neuroblastoma cells in vitro. The cell counting kit‐8 was used to measure cell proliferation and flow cytometry was utilized to study cell cycle phases and apoptosis. Western blotting and quantitative real‐time polymerase chain reaction were used to detect levels of aromatase, estrogen receptors (ERs) and some apoptosis and cell cycle‐related genes. Results showed BBP‐stimulated SH‐SY5Y cells in a dose‐dependent manner and produced a reverted U‐shaped dose‐response curve. BBP at lower concentrations (0.01 and 0.1 μm ) significantly induced cell proliferation while inhibited cell growth at 300 μm . The promoting effect of estradiol could be entirely blocked by administration of ICI182 780, a pure antagonist of ERs, while the effect of BBP could be partly blocked. Additionally, we confirmed 0.1 μm BBP‐induced cell proliferation caused the arrest of cells in S phase and inhibited apoptosis, which might be partially explained by the decreased expression of p53, the increased expression of proliferating cell nuclear antigen, Bcl‐2 and cell cycle regulator cyclin‐D1, and the activation of aromatase. The addition of ICI182 780 had no effect on BBP‐induced ERβ mRNA expression, whereas ICI182 780 could effectively counteract the effect of estradiol. Moreover, pretreatment with ICI182 780 could block the induction of aromatase protein expression and activity by BBP, showing an involvement of ERs. Except for the ER pathway, these results showed there might be other pathways involved in promoting the effects of low‐level BBP on SH‐SY5Y cells, which require further investigation.     

类叶升麻苷对鱼藤酮致SH-SY5Y细胞凋亡的保护作用

目的探讨类叶升麻苷对鱼藤酮致多巴胺能神经元SH-SY5Y细胞凋亡的保护作用及其机制。方法采用MTT法检测细胞存活率,以荧光染料Hoechst33342染色分析细胞核的形态学变化,用流式细胞仪定量分析细胞凋亡峰,以2,′7′-二氢二氯荧光黄双乙酸钠(DCFH-DA)为标记探针检测细胞内活性氧的产生。结果①0.5μmol.L-1的鱼藤酮处理SH-SY5Y细胞48 h能引起细胞存活率的显著下降;诱导细胞发生凋亡,凋亡率达47.39%;大部分细胞胞体皱缩,突起缩短消失或断裂;染色质皱缩、浓缩、断裂及形成凋亡小体;细胞内活性氧水平上升。②预先用盐生肉苁蓉提取物类叶升麻苷(10,20或40 mg.L-1)处理细胞6 h,可提高细胞存活率;明显改善鱼藤酮引起的细胞形态学变化;流式细胞仪检测凋亡率分别降低到25.87%,23.97%,10.45%;以DCFH-DA为标记探针检测到20 mg.L-1类叶升麻苷可明显抑制鱼藤酮引起的细胞内活性氧产生。结论类叶升麻苷能抑制鱼藤酮诱导的多巴胺能神经元SH-SY5Y细胞凋亡,其神经细胞保护作用可能与降低细胞内活性氧水平有关。     

Paraquat induces cyclooxygenase-2 (COX-2) implicated toxicity in human neuroblastoma SH-SY5Y cells

Paraquat produces dopaminergic pathologies of Parkinson’s disease, in which cyclooxygenase-2 (COX-2) is implicated. However, it is unclear whether paraquat induces toxicity within dopaminergic neurons through COX-2. To address this, human neuroblastoma SH-SY5Y cells were treated with paraquat and then the involving mechanism of COX-2 was investigated. We initially examined the involvement of COX-2 in paraquat-induced toxicity. Data suggest that COX-2 is implicated in paraquat-induced reduction of viability in SY5Y cells. Then, to confirm the presence of COX-2 in SY5Y cells, we examined COX-2 mRNA and protein levels, which are regulated by NF-κB. Data indicate that paraquat activates NF-κB and up-regulates COX-2. We then checked quinone-bound proteins as quinones produced by COX-2 bind to intracellular proteins. Paraquat obviously forms quinone-bound proteins, in particular, quinone-bound DJ-1 and this formation is attenuated by meloxicam. Finally, we investigated antioxidant system including nuclear factor erythroid-related factor 2 (Nrf2), gamma glutamylcysteine synthetase (γGCS), and glutathione (GSH) as DJ-1 is linked to Nrf2 and Nrf2 regulates γGCS expression and γGCS is a GSH synthesis enzyme. Paraquat decreases protein levels of Nrf2 and γGCS and intracellular GSH level and these decreases are alleviated by meloxicam. Therefore, collectively, our data indicate that paraquat induces COX-2 implicated toxicity in SY5Y cells. In conclusion, current findings support the idea that paraquat might produce toxicity in dopaminergic neurons through COX-2.     

Characterization of three human cell line models for high‐throughput neuronal cytotoxicity screening

More than 75 000 man‐made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high‐throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH‐SY5Y neuroblastoma cells, LUHMES conditionally‐immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7‐day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH‐SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl‐mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti‐apoptotic genes BCL2 and BIRC5 /survivin , whereas SH‐SY5Y cells may be resistant to apoptosis because they express high levels of BCL2 , BIRC5 /survivin , and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro‐cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd.     

突变型人胰高血糖素样肽-1对神经细胞损伤的保护

目的研究突变型人胰高血糖素样肽-1(mutated human glucagon-like peptide-1,mGLP-1)对谷氨酸诱导的人神经母细胞瘤细胞SH-SY5Y细胞损伤的影响。方法环磷酸腺苷(cAMP)试剂盒检测细胞内cAMP含量,比较mGLP-1和天然人胰高血糖素样肽-1(natural human glucagon-like peptide-1,nGLP-1)与GLP-1受体结合的能力;以谷氨酸诱导SH-SY5Y细胞损伤,同时用mGLP-1处理SH-SY5Y细胞,采用MTT法检测细胞存活率;乳酸脱氢酶(LDH)试剂盒检测细胞LDH的释放量;DAPI染色法观察细胞凋亡形态学特征;钙流法检测胞内钙离子浓度变化;通过检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、总谷胱甘肽(total GS)含量的变化判断细胞氧化损伤程度。结果 mGLP-1与nGLP-1类似,都能提高细胞内cAMP水平;mGLP-1能缓解谷氨酸诱导的细胞损伤,增加细胞存活率,降低LDH释放量;mGLP-1对谷氨酸导致的细胞内钙离子变化没有修复作用;mGLP-1能抑制氧化损伤指标MDA含量升高,促进抗氧化系统指标SOD活性增强和total GS含量增加。结论 mGLP-1可导致细胞内cAMP含量增加。mGLP-1对谷氨酸所致神经细胞损伤具有保护作用,这可能是mGLP-1通过对神经细胞的抗氧化损伤作用实现的,而不是改善谷氨酸导致的钙稳态失衡。mGLP-1的保护作用与nGLP-1类似,而mGLP-1在体内稳定性更强,半衰期更长;提示mGLP-1可能对相关神经退行性疾病的治疗具有更佳的潜在价值。     

维甲酸诱导分化对SH-SY5Y细胞阿片受体表达的影响

目的研究维甲酸诱导SH-SY5Y细胞分化后对阿片受体表达的影响。方法 1×10-5mol.L-1维甲酸诱导SH-SY5Y细胞分化6天,[3H-diprenorphine]放射性配体受体结合试验确定阿片受体表达量。结果维甲酸诱导分化6天后,与对照细胞比较,SH-SY5Y细胞突起增多增长类似轴突,且细胞间形成明显神经纤维网络,细胞生长明显减慢,阿片受体表达提高1.6倍以上。结论维甲酸明显诱导SH-SY5Y细胞向神经元分化,且分化后细胞阿片受体表达明显上调。