硫化氢对PC12细胞存活素表达的影响及其神经保护作用

Objective To explore the effect of hydrogen sulfide (H2S) on the expression of survivin in PC12 cells and the neuroprotective function of H2S on PC12 cells.Methods Different concentrations of sodium hydrosulfide (NaHS) were used to treat the PC12 cells at different times.Dose-effect (50-800 μmol/L) and time-effect (0-180 min) on the expression of survivin were evaluated by Western blotting.Cell viability was tested by using cell counter kit-8.Results NariS treatment at the concentrations from 50 to 200 μmol/L for 30 min could up-regulate the expression of survivin in a dose dependent manner,however,when the concentration of NariS was above that,the expression of survivin decreased gradually;when the concentration of NariS reached 800 μmoi/L,the expression level of survivin was lower than the normal level.Treatment with 400 μmol/L NariS within the range of 0-60 min could promote the expression of survivin in a time dependent manner,but with the extension of time,the expression of survivin was declined.On the other hand,400 μmol/L NaHS preconditioning could enhance the expression of survivin promoted by CoCl2 and reduce the injuries of PC12 cells induced by CoCl2 to increase the cell viability.Conclusion H2S increases the expression ofsurvivin in a dose and time dependent manners at certain degree,which may be related to the protection of PC12 cells against chemical hypoxic damage.     

硫化氢对PC12细胞存活素表达的影响及其神经保护作用

Objective To explore the effect of hydrogen sulfide (H2S) on the expression of survivin in PC12 cells and the neuroprotective function of H2S on PC12 cells.Methods Different concentrations of sodium hydrosulfide (NaHS) were used to treat the PC12 cells at different times.Dose-effect (50-800 μmol/L) and time-effect (0-180 min) on the expression of survivin were evaluated by Western blotting.Cell viability was tested by using cell counter kit-8.Results NariS treatment at the concentrations from 50 to 200 μmol/L for 30 min could up-regulate the expression of survivin in a dose dependent manner,however,when the concentration of NariS was above that,the expression of survivin decreased gradually;when the concentration of NariS reached 800 μmoi/L,the expression level of survivin was lower than the normal level.Treatment with 400 μmol/L NariS within the range of 0-60 min could promote the expression of survivin in a time dependent manner,but with the extension of time,the expression of survivin was declined.On the other hand,400 μmol/L NaHS preconditioning could enhance the expression of survivin promoted by CoCl2 and reduce the injuries of PC12 cells induced by CoCl2 to increase the cell viability.Conclusion H2S increases the expression ofsurvivin in a dose and time dependent manners at certain degree,which may be related to the protection of PC12 cells against chemical hypoxic damage.     

齐拉西酮对PC12细胞的保护作用

目的探讨齐拉西酮对神经生长因子(nerve growth factor,NGF)诱导的PC12细胞活性的影响及其可能的机制。方法以NGF诱导后的PC12细胞作为细胞模型,分为不同剂量齐拉西酮组(5μM、10μM、20μM、50μM)和对照组(含5%胎牛血清的DMEM培养基)分别培养24h。采用细胞计数试剂盒-8(CCK-8)检测PC12细胞活性,免疫细胞化学观察细胞形态学改变以及蛋白质印迹法(Western blotting)检测磷酸化细胞外信号调节激酶(phosphorylated extracellular single-regulated kinase,pERK)1/2的表达水平。结果仅20μM、10μM齐拉西酮组的PC12细胞活性分别与对照组的差异有统计学意义(P0.05);免疫细胞化学观察同样显示,仅10μM、20μM齐拉西酮组的pERK1/2的表达水平分别与对照组的差异有统计学意义(P0.05),20μM齐拉西酮组与其它齐拉西酮组的差异也均有统计学意义(P0.01)。Western blotting进一步证实了该结果。结论齐拉西酮能提高PC12细胞的活性,并上调pERK1/2的表达。提示齐拉西酮可能通过保护神经元活性发挥作用,而这种作用可能是通过细胞外信号调节激酶途径实现的。     

蛇床子素对MPP~＋损伤PC12细胞的保护作用研究

目的观察蛇床子素（osthole）对1-甲基-4-苯基吡啶离子（MPP＋）诱导PC12细胞损伤的神经保护作用。方法将MPP＋加入培养的PC12细胞中,建立多巴胺能神经元损伤模型,加入不同浓度的蛇床子素预处理细胞（0.01、0.05、0.1mmol/L）。处理24h后用噻唑蓝（MTT）比色法检测细胞活性;以乳酸脱氢酶（LDH）活性测定反映细胞的损伤程度;采用Westernblot法检测Bax、Bcl-2蛋白的表达,分析Bax/Bcl-2比值变化,以及检测细胞色素C的改变。结果蛇床子素可以明显减少MPP＋诱导的PC12细胞活性的降低,LDH的释放,Bax/Bcl-2比值的增高以及细胞色素C的释放（P〈0.05）。结论蛇床子素对MPP＋诱导的PC12细胞损伤具有保护作用。     

黄芩苷通过上调lncRNAH19表达来减轻MPP+诱导的PC12细胞神经毒性

目的 探讨黄芩苷是否通过上调长链非编码RNA(Long noncoding RNA,lncRNA)H19表达来减轻1-甲基4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP⁺)诱导的PC12细胞神经毒性。方法 分别采用0.5、0.75、1、2 mmol/L MPP⁺和10、20、50和70 μmol/L黄芩苷作用PC12细胞,后续试验使用水平分别选择0.75 mmol/L MPP⁺和50 μmol/L黄芩苷; 以0.75 mmol/L MPP⁺损伤PC12细胞,体外模拟帕金森病,并加入黄芩苷; 采用四甲基偶氮唑盐(Methyl thiazolyl tetrazolium,MTT)进行细胞活力测定,Annexin V-异硫氰酸荧光素(Fluorescein isothiocyanate isomer,FITC)/碘化丙啶(Propidium iodide,PI)双染法进行凋亡定量分析,蛋白质印迹分析(Western blot)检测Pro-天冬氨酸特异性半胱氨酸蛋白酶(Cysteinyl aspartate specific proteinase,Caspase)-3,Cleaved-caspase-3,B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)蛋白和Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)的相对表达水平,实时定量聚合酶链式反应(Quantitative real-time polymerase chain reaction,qPCR)进行lncRNA H19的相对表达水平检测; 在PC12细胞中转染lncRNA H19小干扰RNA(lncRNA H19 siRNA,si-H19),并加入MPP⁺和黄芩苷,考察PC12细胞增殖、凋亡等变化。结果 与对照组比较,MPP⁺降低PC12细胞的存活率、Bcl-2蛋白、lncRNA H19相对表达水平,提高PC12细胞的凋亡率、Cleaved-caspase-3,Bax蛋白相对表达水平(P<0.05),而Pro-caspase-3蛋白相对表达水平无明显变化(P>0.05)。与MPP⁺组比较,黄芩苷显著提高MPP⁺诱导的PC12细胞的存活率、Bcl-2蛋白、lncRNA H19相对表达水平,降低PC12细胞的凋亡率、Cleaved-caspase-3,Bax蛋白相对表达水平(P<0.05),而Pro-caspase-3蛋白相对表达水平无明显变化(P>0.05)。转染si-H19后黄芩苷和MPP⁺诱导的PC12细胞的存活率、Bcl-2蛋白相对表达水平降低,PC12细胞的凋亡率、Cleaved-caspase-3,Bax蛋白相对表达水平升高(P<0.05),而Pro-caspase-3蛋白相对表达水平无明显变化(P>0.05)。结论 黄芩苷上调lncRNA H19表达,提高MPP⁺诱导的PC12细胞活力和降低其凋亡,减轻MPP⁺诱导的PC12细胞神经毒性。     

雷帕霉素对PC12细胞氧糖剥夺损伤的保护作用

目的探讨雷帕霉素对PC12细胞氧糖剥夺损伤(oxygen and glucose deprivation,OGD)的作用。方法选取处于对数生长期的PC12细胞,分为正常组、OGD组、溶剂组和1 nmol、10 nmol、100 nmol、500 nmol雷帕霉素组,OGD同时给予雷帕霉素处理,8 h后通过倒置显微镜观察细胞形态学的变化、MTT法检测细胞存活率的变化以及LDH活性检测细胞受损程度。结果与正常组相比,单纯OGD组细胞活性明显下降,且具有统计学意义(P0.05);与溶剂组相比,1、10、100、500 nmol雷帕霉素组细胞存活率均存在不同程度的增高,其中10、100、500 nmol组具有统计学意义(P0.05)。结论雷帕霉素对PC12细胞OGD损伤具有保护作用。     

PC12细胞缺氧后细胞凋亡与DNA损伤相关基因表达关系的研究

目的 探讨PC12细胞缺氧后细胞凋亡与DNA损伤的关系。方法 采用TUNEL法,结合应用流式细胞术观察PC12细胞缺氧培养不同时间点细胞凋亡现象,以及DNA损伤相关基因表达的改变。结果 在缺氧0.5h开始出现凋亡细胞,引时P53、P21^waf1/cip1蛋白表达开始增高。至缺氧1h凋亡细胞达高峰,此时P53、P21蛋白表达最高（P＜0.05）。至缺氧6－12h,则以坏死为主,此时以上基因表达均减弱。结论 PC12细胞在缺氧早期（0.5-2h）主要出现以凋亡为主的细胞死亡,伴有DNA损伤相关基因表达的动脉改变,提示缺氧后PC12细胞凋亡部分是由于DNA损伤严重、损伤不能及时修复所致。     

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。     

雄激素干预对PC12细胞Seladin-1表达的影响

目的观察雄激素干预对体外培养PC12细胞Seladin-1水平的影响。方法将培养的PC12细胞分为5组：正常对照组、药物处理组（雄激素终浓度分别为1×10-3mol/L、1×10-4mol、1×10-5mol/L、1×10-6mol/L）。采用Western-blot和Real-time PCR方法对PC12细胞Seladin-1的表达进行检测。结果各药物组处理后,PC12细胞Seladin-1的蛋白质与mRNA的表达水平与正常对照组比较均明显提高,差异具有统计学意义（P〈0.05,P〈0.001）。结论雄激素对PC12细胞Seladin-1基因的表达具有正性调控作用。雄激素的神经保护效应可能与上调seladin-1的表达有关。     

银杏叶提取物对百草枯诱导PC12细胞凋亡的保护作用

目的:探讨银杏叶提取物(EGb761)对百草枯(PQ)诱导PC12细胞凋亡的保护作用。方法:4-甲基偶氮唑蓝(MTT)检测PC12细胞活性;流式细胞仪(FCM)测定PC12细胞凋亡百分率;Hoechst33342染色观察细胞凋亡;罗丹明123(Rh123)染色检测细胞线粒体膜电位(MMP)。结果:EGb76120,40μg·mL-1可明显减轻PQ对PC12细胞的细胞毒性,EGb761可使细胞活性增强,降低凋亡百分率,减少细胞凋亡数目,抑制细胞MMP的下降。结论:EGb761对PQ诱导的PC12细胞凋亡具有保护作用,其机制可能与抑制PQ引起的细胞MMP下降有关。     

PC12细胞化学缺氧复氧损伤与缺氧诱导因子1表达的关系

目的 研究缺氧诱导因子1(HIF1)在PC12细胞化学缺氧复氧损伤中的作用。方法 在培养液中加入和去除氯化钴模拟化学缺氧和复氧，以乳酸脱氢酶(LDH)漏出和细胞超微结构改变作为细胞损伤指标，观察化学缺氧和复氧后不同时间细胞损伤和HIF1 α蛋白变化。结果 在氯化钴模拟化学缺氧实验中，LDH漏出明显增加，8h达高峰，随后逐渐下降，电镜结果与LDH改变相一致，HIF1 α蛋白表达在化学缺氧后2，4，8和12h均明显增加，8h达高峰，提示化学缺氧8h后细胞损伤逐渐减轻可能与HIF1 α蛋白水平升高有关。在模拟复氧实验中，LDH和细胞形态学改变都显示化学复氧8h细胞损伤最为严重，而HIF1 α蛋白表达在化学复氧4和8h均明显下降，提示细胞化学复氧损伤可能与HIF1 α蛋白水平下降有关。结论 HIF1对神经细胞化学缺氧复氧损伤具有保护作用。     

人野生型parkin基因真核表达载体的构建及其在PC12细胞中的表达

目的 克隆人野生型parkin基因并构建真核表达载体pCDNA3．1—parkin，将重组质粒转染PC12细胞获得高表达人野生型parkin基因的PC12细胞克隆。方法 从胎脑组织中提取总RNA，用RT—PCR方法获得人野生型parkin基因的全长cDNA，插入pCR2．1—TA克隆载体中进行序列测定，测序正确后将其亚克隆至表达载体pCD—NA3．1，利用脂质体将重组质粒转染PC12细胞，经G418筛选获得抗性细胞克隆，采用RT—PCR和Western Blot方法鉴定人野生型parkin基因在PC12细胞中的过表达。结果 经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒，RT—PCR和Western Blot证明经G418筛选得到的转基因PC12细胞克隆中存在人野生型parkin基因的表达。结论 成功构建了人野生型parkin基因的真核表达载体，获得了稳定表达人野生型parkin基因的PC12细胞克隆，为进一步研究parkin的生物学功能以及parkin在帕金森病发病机制中的作用奠定了良好的基础。     

稳定表达人CNTF诱导PC12细胞分化作用的研究

目的:探讨CNTF对PC12细胞的诱导分化作用。方法:应用转基因技术将pcDNA-S-hCNTF质粒转入COS7细胞进行表达,并对CNTF表达进行检测,然后将hCNTF修饰过的COS7细胞与PC12细胞进行联合培养,4天后对CNTF作用后的PC12进行形态学观察,β-tubulin免疫细胞化学染色及不同培养时间4天,5天后阳性细胞的指数进行分析,同时通过MTF实验对诱导培养后的PC12细胞内增殖进行检测。结果:PC12细胞与hCHTF修饰过的COS7细胞经过4天的联合培养,细胞大部分形态呈现出类似于神经元的形态,胞体立体感较强,并且细胞长出较长的突起。另外细胞呈现β-tubulin反应阳性,并且随培养时间延长,β-tubulin阳性细胞的数目有增加的趋势(从76.6％增加到88.3％),最后,反映细胞增殖程度的OD值为0.328±0.019,明显低于对照组0.586±0.028,0.597±0.032。结论:hCNTF对PC12细胞向神经元分化有明显的促进作用。     

6—羟基多巴胺诱导PC12细胞凋亡及其可能分子机制

探讨神经毒素 6 羟基多巴胺 (6 OHDA)诱导PC12细胞死亡本质及其可能分子机制。不同剂量 6 OHDA处理PC12细胞 2 4h后 ,分别用流式细胞仪、透射电镜、TUNEL染色和DNA电泳检测细胞凋亡 ;5 0 μmol/L 6 OH DA处理PC12细胞 2 0h后 ,用RT PCR检测caspase 3表达 ;用westernblot检测Caspase 3p32蛋白酶原的切割。结果如下 :①流式细胞仪检测PC12细胞凋亡百分率 ,对照组为 1.10 %± 1.14% ,30 ,4 0 ,5 0 μmol/L 6 OHDA处理组分别为 4 .73 %± 1.0 6% ,10 .15 %± 1.2 1%和 19.94 %± 2 .5 1% ,较对照组均有显著性差异 (P <0 .0 1)。 4 0 ,5 0 μmol/L 6 OHDA处理组透射电镜、TUNEL染色均证实存在大量凋亡细胞 ;5 0 μmol/L 6 OHDA处理组DNA电泳呈现一定间隔的“梯状”条带。② 5 0 μmol/L 6 OHDA处理组caspase 3mRNA水平增高约一倍 ,并且检测到Cas pase 3p2 0活性片段。本研究提示 6 OHDA能诱导PC12细胞凋亡 ,并呈剂量依赖性 ;激活Caspase 3蛋白酶可能参与 6 OHDA诱导PC12细胞凋亡过程     

Fucosyl-GM1 expression and amyloid-beta protein accumulation in PC12 cells

Gangliosides, sialic acid-containing glycosphingolipids, are ubiquitously expressed in all eukaryotic cells and are localized primarily in the plasma membrane. For a rat pheochromocytoma cell line, PC12, which has been used frequently as a model for investigating events leading to neuronal differentiation, it is generally thought that GM1 is a major ganglioside, based on reactivity with the probe cholera toxin B subunit (Ctxb). From a series of biochemical studies, however, it has been reported that no GM1 is expressed in PC12 cells. In this study, we have reevaluated GM1 expression and Ctxb reactivity in PC12 cells and a subcloned line, PC12D cells. Flow cytometric analysis with Ctxb revealed that about 30-50% of PC12 cells were reactive with Ctxb. However, a detailed biochemical analysis showed that PC12 cells express abundantly a different ganglioside, fucosyl-GM1, instead of GM1, and the reactivity of Ctxb in the PC12 cells actually arose from its interaction with fucosyl-GM1, which also interacts with this ligand. Because it has been claimed that amyloid-beta protein (Abeta) interacts with GM1 in PC12 cells to provide "seeding" for amyloid to accumulate, we further evaluated this possibility and found that Abeta is mostly likely interacting with fucosyl-GM1 in this cell line. Our data thus suggest that a specific interaction may occur between Abeta and fucosyl-GM1 for the accumulation of amyloid in PC12 cells.     

6-OHDA诱导PC12细胞凋亡及作用机制

目的 探讨6-羟基多巴胺(6-OHDA)诱导PC12细胞损伤的可能作用机制.方法 不同剂量6-OHDA加入培养的大鼠肾上腺嗜铬细胞瘤细胞(pheochromocytoma cell,PC12)24h后,用四甲基偶氮唑盐法(MTT法)检测细胞的活力,流式细胞仪检测细胞凋亡率以及Bax、Bcl-2的蛋白表达.结果 在加入不同浓度的6-OHDA时PC12细胞活力显著下降,细胞凋亡百分率对照组为8.73±1.09,不同浓度的6-OHDA处理组显著上升,分别为10.97±1.52、25.77±0.95、57.94±1.23,较对照组有显著性差异(P<0.01),并且Bcl-2蛋白表达下降,Bax蛋白表达上升,Bcl-2/Bax比值和正常组比较有显著性差异(P<0.01).结论 6-OHDA能显著诱导PC12细胞损伤,并呈剂量依赖性,其作用机制涉及到促进细胞内bax,以及抑制Bcl-2的表达.     

Apoptotic characteristics of cell death and the neuroprotective effect of homocarnosine on pheochromocytoma PC12 cells exposed to ischemia

We recently improved an in vitro ischemic model, using PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) for 3 hr in a special device, followed by 18 hr of reoxygenation. The cell death induced in this ischemic model was evaluated by a series of markers: lactate dehydrogenase (LDH) release, caspase-3 activation, presence of cyclin D1, cytochrome c leakage from the mitochondria, BAX cellular redistribution, cleavage of poly (ADP-ribose) polymerase (PARP) to an 85-kDa apoptotic fragment, and DNA fragmentation. The OGD insult, in the absence of reoxygenation, caused a strong activation of the mitogen-activated protein kinase (MAPK) isoforms extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and stress-activated protein kinase (SAPK), also known as p-38. The detection of apoptotic markers and activation of MAPKs during the ischemic insult strongly suggest that apoptosis plays an important role in the PC12 cell death. Homocarnosine, a neuroprotective histidine dipeptide, present in high concentrations in the brain, was found to provide neuroprotection, as expressed by a 40% reduction in LDH release and caspase-3 activity at 1 mM. Homocarnosine reduced OGD activation of ERK 1, ERK 2, JNK 1, and JNK 2 by 40%, 46%, 55%, and 30%, respectively. These results suggest that apoptosis is an important characteristic of OGD-induced neuronal death and that antioxidants, such as homocarnosine, may prevent OGD-induced neuronal death by inhibiting the apoptotic process and/or in relation to the differential attenuation of activity of MAPKs.