川牛膝多糖对H2O2诱导PC12细胞氧化损伤保护作用的研究

目的:探讨川牛膝多糖组分对H_2O_2诱导PC12细胞产生的氧化和凋亡作用,并对其机制进行深入研究。方法:通过H_2O_2诱导PC12细胞,建立神经氧化损伤模型,通过MTT法测定PC12细胞存活率,丙二醛(MDA)、活性氧(ROS)和超氧化物歧化酶(SOD)检测氧化作用,Hoechst 33342法染色观察川牛膝多糖组分对PC12细胞遭受H_2O_2损伤凋亡状态特征。结果:浓度为100μmol·L~(-1) H_2O_2时,PC12细胞产生明显损伤,为最佳造模浓度;川牛膝多糖组分能够减少H_2O_2诱导PC12的损伤程度,减少MDA含量、降低ROS的活性、提高SOD的活性,并减轻细胞凋亡作用。结论:川牛膝多糖能够减少H_2O_2对PC12细胞的氧化作用,增强细胞保护作用。     

氧糖剥夺条件下PC12细胞RAGE的表达及对其损伤的影响

目的探讨大鼠肾上腺嗜铬细胞瘤(PC12)细胞在氧糖剥夺(OGD)的条件下,晚期糖基化终产物受体(RAGE)的表达及其对PC12细胞损伤的影响。方法PC12细胞随机分为3组:OGD培养组(PC12细胞在无血清无糖的DMEM培养液中厌氧培养);封闭RAGE的OGD培养组(PC12细胞在加5μg/mlRAGE抗体的无血清无糖DMEM培养液中厌氧培养);对照组(PC12细胞在无血清无糖DMEM培养液中培养)。免疫组化法检测RAGE表达。收集细胞上清液,检测乳酸脱氢酶(LDH)活性和一氧化氮(NO)含量,并对细胞死亡率进行检测。结果在OGD条件下PC12细胞培养8、11、20h均有RAGE表达,与对照组相比表达均明显增加,OGD培养组与封闭RAGEOGD培养组相比,LDH活性差异显著。随着厌氧时间的延长,PC12细胞死亡率明显增高,封闭RAGEOGD培养组与OGD培养组相比,PC12细胞死亡率明显降低。NO含量差异无显著意义,但与对照组相比差异显著。结论在OGD条件下,PC12细胞RAGE表达增高。RAGE的表达对细胞的损伤起促进作用。     

PC12细胞体外培养法测定蛇毒神经生长因子活性

目的建立PC12细胞检测蛇毒NGF活性的方法。方法在有血清条件下,观察NGF诱导PC12细胞分化情况。在无血清条件下,以SRB染色定量NGF维持PC12细胞存活数量,计算NGF效价,并与鸡胚背根神经节法比较。结果在有血清条件下,NGF能诱导PC12细胞分化,出现神经突触样生长。在无血清条件下,NGF浓度对数值与PC12细胞数呈直线相关,标准品与样品的生物效应平行性良好,以量反应平行线法可计算样品的NGF平均效价为1108·3U/ml,与鸡胚背根神经节法检测结果相符。结论该法简便易行,定量准确,重复性好,实验周期短。     

嗜酸乳杆菌上清液制备及其对PC12细胞增殖的影响

本实验首先对嗜酸乳杆菌进行相应的处理,使其复苏和活化,在进行生长曲线测定的过程中,发现其生长28h左右达到稳定期.之后将以28h为培养时间的L.a.s按不同体积分数作用于PC12细胞,荧光染色和CCK-8细胞增殖检测结果表明,PC12细胞能够正常生长,研究发现加入L.a.s体积分数为6％时,PC12细胞的存活率提高至对...     

干扰硫氧还蛋白-1对H_2O_2诱导PC12细胞超氧化损伤的保护作用

目的探讨硫氧还蛋白-1(sulfiredoxin-1,Srxn-1)对H2O2诱导的PC12细胞超氧化损伤的保护作用及其可能机制。方法在Lipofectamine 2000的介导下,分别将pLVT574、pLVT575和pLVT576干扰质粒转染PC12细胞,同时设空白对照组(正常培养细胞)及阴性对照组(转染pLVT4质粒),普通显微镜和倒置荧光显微镜下观察转染效率,并采用RT-PCR和Western blot法分别检测转染细胞中Srxn-1基因mRNA转录和蛋白表达的水平。采用不同终浓度(50、100、150、200、250、300μmol/L)的H2O2诱导PC12细胞,建立超氧化细胞损伤模型,MTT法检测Srxn-1对细胞存活率的影响,并采用超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)试剂盒检测细胞SOD活性及MDA含量。结果镜下观察可见,转染后细胞死亡数较多,胞体缩小;有绿色荧光,转染效率达50%。si-Srxn-1-575组细胞中Srxn-1基因mRNA转录及蛋白表达水平较空白对照组明显下降(P0.05)。终浓度为200μmol/L的H2O2处理12 h后,PC12细胞的存活率为66%;H2O2+si-Srxn-1-575组的细胞存活率(49.3%)明显低于空白对照组(65.2%)(P0.05)。H2O2+si-Srxn-1-575组细胞中SOD活性[(10.319±1.362)U/mg pro]明显低于H2O2+空白对照组[(15.7±1.138)U/mg pro],MDA含量[(5.507±0.297)nmol/mg pro]明显高于H2O2+空白对照组[(3.41±0.281)nmol/mg pro](P均0.05)。结论 Srxn-1对H2O2损伤的PC12细胞具有保护作用,其机制可能与其发挥内源性抗氧化作用有关。     

神经干细胞对去血清诱导PC12细胞凋亡的作用

肾上腺嗜铬细胞瘤细胞(PC12 cells)去血清后,其凋亡与多巴胺能神经元的凋亡有许多共同之处,今通过研究神经干细胞(NSCs)对去血清诱导的PC12细胞凋亡的作用,进一步为NSCs移植治疗神经系统疾病提供相应的实验和理论依据.将正常培养的PC12细胞与NSCs以不同的方式进行去血清共培养,观察PC12细胞的形态,检测PC12细胞的活性,计算PC12细胞的存活率,同时检测培养基中胶质细胞源神经营养因子(GDNF)的浓度,分析不同培养方式下NSCs对去血清诱导凋亡的PC12细胞的作用以及NSCs与去血清诱导凋亡的PC12细胞共培养后,其分泌GDNF的能力.结果表明:①去血清诱导PC12细胞凋亡呈时间依赖性,去血清72 h后,PC12细胞存活的比率为44.25%;②NSCs培养基对去血清诱导凋亡的PC12细胞没有明显的保护作用;③NSCs培养上清及NSCs对去血清诱导凋亡的PC12细胞具有保护作用;④NSCs与去血清诱导凋亡的PC12细胞共培养后,分泌GDNF的能力增强.     

硫酸锌对紫外线造成的角朊细胞氧化损伤及胞内抗氧化酶活性的影响

目的探讨硫酸锌对B段紫外线(Ultraviolet radiation B,UVB)造成的角朊细胞(Keratinocytes,KC)氧化损伤及胞内抗氧化酶活性的影响。方法取新生Wistar大鼠背部皮肤,采用中性蛋白酶和胰蛋白酶联合消化,经贴壁培养获得高纯度角朊细胞,在含10%FBS的L-DMEM+F12培养液中扩增培养。将细胞随机分为UVB组、加锌组和正常对照组,UVB组用15W的UVB进行照射,加锌组与50μmol/L硫酸锌共孵育24h后进行照射,正常对照组不照射。照射后24h收集各组细胞,进行氧化损伤指标(MDA、OH-、O2-)及胞内抗氧化酶(SOD和GSH)活性测定。结果 UVB组细胞MDA、OH-和O2-水平显著高于正常对照组(P<0.05),加锌组显著低于UVB组(P<0.05);UVB组细胞SOD和GSH水平显著低于正常对照组(P<0.05),加锌组显著高于UVB组(P<0.05)。结论硫酸锌可拮抗UVB对角朊细胞造成的氧化损伤,并提高胞内抗氧化物酶的水平。     

槲皮素对皮质酮损伤的pc12细胞的保护作用

利用pc12细胞的抗抑郁损伤模型,研究了槲皮素的抗抑郁作用.槲皮素、氯丙咪嗪与0.1 mmol·L-1皮质酮共孵pc12细胞48 h,用MTT比色法判断细胞损伤程度.结果表明,高浓度皮质酮处理pc12细胞后,细胞存活率比正常细胞降低;当给予槲皮素或氯丙咪嗪后,细胞存活率升高,表明细胞损伤减少或接近正常.与经典抗抑郁剂氯丙咪嗪的作用效果一样,槲皮素对皮质酮损伤的pc12细胞有明显的保护作用,有抗抑郁作用.     

明胶水解物对冰淇淋中冰晶生长的抑制作用

本文研究了明胶水解物对冰淇淋中冰晶生长的抑制作用。明胶水解物是用明胶以木瓜酶水解制得。冰晶生长控制在-14℃～-12℃热循环中,变温速率为每3min一个循环。实验指出,含肽的水解物级分分子量范围约在2000～5000Da,它与分子量大于7000Da的含肽水解物相比,对冰淇淋中冰晶的生长具有最高的抑制活力。水解物中的明胶肽的大小分布受水解时的pH的影响。要制备具有最高冰晶生长抑制活力的肽的最佳水解条件是37℃、pH7、10min,木瓜酶:明胶=1:100。此条件取决于明胶的类型与来源。本文讨论了明胶肽对冰晶生长抑制作用的可能的机理。     

蛋白酶体抑制剂诱导的PC12细胞帕金森病模型中血红素加氧酶-1的差异表达

目的探讨蛋白酶体抑制剂(Proteasome inhibitor,PSI)诱导的PC12细胞帕金森病(Parkinson disease,PD)模型中血红素加氧酶-1(Hemeoxygenase-1,HO-1)的差异表达,为深入研究PD的发病机制提供理论依据。方法取培养的PC12细胞,加入终浓度为10μmol/L的PSI,建立PSI诱导的PC12细胞模型,以加入终浓度为10μmol/L的二甲基亚砜(DMSO)为对照组。经HE、AO&EB及α-SYN染色进行鉴定;PSI作用48h后提取蛋白,应用荧光差异凝胶电泳(DIGE)系统获得差异蛋白点,运用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOFProMS)鉴定差异蛋白。结果模型组与对照组比较,PSI作用48h可见细胞内嗜酸性类Lewy小体形成及细胞凋亡,凋亡率达(24.74±4.55)%。模型组与对照组比较,HO-1表达量显著增加。结论在泛素-蛋白酶体系统(Ubiquitin-proteasome system,UPS)功能障碍诱发PD过程中,氧化应激反应发挥着一定的作用。     

Synthetic Chalcones with Potent Antioxidant Ability on H2O2-Induced Apoptosis in PC12 Cells

Chalcone derivatives (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl) prop-2-en-1-one and (E)-3-(4-hydroxyphenyl)-1-(4-methoxyphenyl) prop-2-en-1-one (Compounds 1 and 2) have been demonstrated to be potent anti-inflammatory agents in our previous study. In light of the relationship of intracellular mechanisms between anti-inflammatories and antioxidants, we further designed and synthesized a series of chalcone derivatives based on 1 and 2, to explore their antioxidant efficacy. The majority of the derivatives exhibited strong protective effects on PC12 (PC12 rat pheochromocytoma) cells exposed to H₂O₂, and all compounds were nontoxic. A preliminary structure-activity relationship was proposed. Compounds 1 and 1d ((E)-2-methoxy-4-(3-(4-methoxyphenyl)-3-oxoprop-1-en-1-yl) phenyl acrylate) exerted the action in a good dose-dependent manner. Quantitative RT-PCR (qRT-PCR) and western blot analysis showed that 1 and 1d significantly improve the expression of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-dependent antioxidant genes g-Glutamylcysteine Ligase Catalytic Subunit (GCLC) and heme oxygenase-1 (HO-1) and their corresponding proteins (γ-glutamyl cysteine synthase (γ-GCS) and HO-1) in PC12 cells. Inhibition of GCLC and HO-1 by specific inhibitors, l-buthionine-S-sulfoximine (BSO) and zinc protoporphyrin (ZnPP), respectively, partially reduce the protective effect of 1 and 1d. These data present a series of novel chalcone analogs, especially compounds 1 and 1d, as candidates for treating oxidative stress-related disease by activating the Nrf2-antioxidant responsive element (ARE) pathway.     

Ginsenoside-Rd Promotes Neurite Outgrowth of PC12 Cells through MAPK/ERK- and PI3K/AKT-Dependent Pathways

Panax ginseng is a famous herbal medicine widely used in Asia. Ginsenosides have been identified as the principle active ingredients for Panax ginseng’s biological activity, among which ginsenoside Rd (Rd) attracts extensive attention for its obvious neuroprotective activities. Here we investigated the effect of Rd on neurite outgrowth, a crucial process associated with neuronal repair. PC12 cells, which respond to nerve growth factor (NGF) and serve as a model for neuronal cells, were treated with different concentrations of Rd, and then their neurite outgrowth was evaluated. Our results showed that 10 μM Rd significantly increased the percentages of long neurite- and branching neurite-bearing cells, compared with respective controls. The length of the longest neurites and the total length of neurites in Rd-treated PC12 cells were much longer than that of respective controls. We also showed that Rd activated ERK1/2 and AKT but not PKC signalings, and inhibition of ERK1/2 by PD98059 or/and AKT by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 effectively attenuated Rd-induced neurite outgrowth. Moreover, Rd upregulated the expression of GAP-43, a neuron-specific protein involved in neurite outgrowth, while PD98059 or/and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 decreased Rd-induced increased GAP-43 expression. Taken together, our results provided the first evidence that Rd may promote the neurite outgrowth of PC12 cells by upregulating GAP-43 expression via ERK- and ARK-dependent signaling pathways.     

壳聚糖对氧化损伤HepG2细胞的修复作用

采用体外细胞评价法,以抗坏血酸(Vc)为参照物,分别从细胞形态、细胞活力、细胞内超氧化物歧化酶(SOD)、丙二醛(MDA)含量和谷胱甘肽过氧化酶(GSH-Px)活性等指标来研究不同剂量壳聚糖(Cs)对HepG2细胞氧化损伤的干预效果.结果表明壳聚糖对氧化受损HepG2细胞的形态和活力具有修复作用,且在降低MDA水平的同时提高SOD含量和GSH-Px的活性.与参照物Vc相比,壳聚糖对HepG2细胞氧化损伤的修复效果更好.     

Single Cell Analysis of Reversibility of the Cell Death Program in Ethanol-Treated Neuronal PC12 Cells

Neurodegenerative diseases are generally characterized clinically by the selective loss of a distinct subset of neurons and a slow progressive course. Mounting evidence in vivo indicates that large numbers of neurons pass through a long period of injury and dysfunction before the actual death of the cells. Whether these dying neurons can be rescued and return to a normal, functional state is uncertain. In the present study, we explored the reversibility of the neuronal cell death pathway at various stages by monitoring the dynamics of single cells with high-resolution live-cell spinning disk confocal microscopy in an in vitro neuronal cell death model. We exposed differentiated neuronal PC12 cells to ethanol as our cell death model. Results showed that exposure to 5% ethanol for 24 h induced cell death in >70% of the cells. Ethanol treatment for 3 h already induced cellular changes and damage such as reactive oxygen species generation, elevation of intracellular Ca²⁺ level, phosphatidylserine exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation and membrane potential loss, and retraction of neurites. These phenomena are often associated with programmed cell death. Importantly, after removing ethanol and further culturing these damaged cells in fresh culture medium, cells recovered from all these cell injuries and generated new neurites. Moreover, results indicated that this recovery was not dependent on exogenous NGF and other growth factors in the cell culture medium. Overall, our results suggest that targeting dying neurons can be an effective therapeutic strategy in neurodegenerative diseases.     

Crosstalk between Neuron and Glial Cells in Oxidative Injury and Neuroprotection

To counteract oxidative stress and associated brain diseases, antioxidant systems rescue neuronal cells from oxidative stress by neutralizing reactive oxygen species and preserving gene regulation. It is necessary to understand the communication and interactions between brain cells, including neurons, astrocytes and microglia, to understand oxidative stress and antioxidant mechanisms. Here, the role of glia in the protection of neurons against oxidative injury and glia–neuron crosstalk to maintain antioxidant defense mechanisms and brain protection are reviewed. The first part of this review focuses on the role of glia in the morphological and physiological changes required for brain homeostasis under oxidative stress and antioxidant defense mechanisms. The second part focuses on the essential crosstalk between neurons and glia for redox balance in the brain for protection against oxidative stress.     

Generation of Cochlear Hair Cells from Sox2 Positive Supporting Cells via DNA Demethylation

Regeneration of auditory hair cells in adult mammals is challenging. It is also difficult to track the sources of regenerated hair cells, especially in vivo. Previous paper found newly generated hair cells in deafened mouse by injecting a DNA methyltransferase inhibitor 5-azacytidine into the inner ear. This paper aims to investigate the cell sources of new hair cells. Transgenic mice with enhanced green fluorescent protein (EGFP) expression controlled by the Sox2 gene were used in the study. A combination of kanamycin and furosemide was applied to deafen adult mice, which received 4 mM 5-azacytidine injection into the inner ear three days later. Mice were followed for 3, 5, 7 and 14 days after surgery to track hair cell regeneration. Immunostaining of Myosin VIIa and EGFP signals were used to track the fate of Sox2-expressing supporting cells. The results show that (i) expression of EGFP in the transgenic mice colocalized the supporting cells in the organ of Corti, and (ii) the cell source of regenerated hair cells following 5-azacytidine treatment may be supporting cells during 5–7 days post 5-azacytidine injection. In conclusion, 5-azacytidine may promote the conversion of supporting cells to hair cells in chemically deafened adult mice.