N-乙酰半胱氨酸对脑缺血诱导信号转导与转录激活子-3的磷酸化及DNA结合活性的影响

目的 研究抗氧化剂N-乙酰半胱氨酸对全脑缺血诱导海马组织信号转导与转录激活子-3(STAT3)的激活及DNA结合活性的影响。方法 采用SD雄性大鼠四动脉结扎(4-VO)全脑缺血模型，以及腹腔注射给药的方法。运用抗磷酸化STAT3抗体做免疫印迹(IB)检测海马核抽提物磷酸化STAT3的变化；以及电泳迁移率改变实验(EMSA)分析核内STAT3 DNA结合活性的变化。结果 全脑缺血不同时间导致核内STAT3磷酸化水平及DNA结合活性的持续增高；缺血前20min腹腔注射抗氧化剂N-乙酰半胱氨酸能明显抑制其增高。结论 全脑缺血所致STAT3的磷酸化水平及DNA结合活性的增高与氧化应激有一定的关系。     

Par-4反义寡核苷酸对谷氨酸诱导PC12细胞STAT3活性下调的抑制作用

目的研究Par-4反义寡核苷酸拮抗谷氨酸对PC12细胞中信号转导子和激活子3(STAT3)活性的下调作用及其抗凋亡意义.方法脂质体将Par-4反义寡核苷酸转染PC12细胞.谷氨酸诱导PC12细胞凋亡.吖啶橙/溴化乙锭荧光染色观察PC12细胞形态.流式细胞分析评价凋亡百分率.Western blot测定Par-4的蛋白表达量,凝胶迁移改变实验测定STAT3的DNA结合力.结果谷氨酸诱导PC12细胞中蛋白表达上调,Par-4反义寡核苷酸呈剂量依赖性地拮抗其上调(P＜0.01).谷氨酸诱导PCl2细胞中STAT3的DNA结合力下调,Par-4反义寡核苷酸拈抗其下调(P＜0.01).Par-4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡.如予Jak/STAT3信号通路阻断剂AG-490预处理,则其拮抗作用被下调.结论Par-4反义寡核苷酸拈抗谷氨酸诱导的PC12细胞凋亡,其机制可能与STAT3活化有关.     

受损坐骨神经中STAT3表达和酪氨酸磷酸化的变化及重组CNTF的作用

研究周围神经修复后,受损周围神经中信号转导子和转录激活子3(STAT3)表达和酪氨酸磷酸化的变化及睫状神经营养因子(CNTF)的作用.用硅管套接切断的成年大鼠坐骨神经,术时在受损神经处给予重组CNTF,用免疫组织化学ABC法结合计算机图像分析观测修复侧神经中STAT3、磷酸化酪氨酸(PTyr)的分布和相对含量.在生理盐水组修复侧神经中,轴突、雪旺细胞STAT3的含量升高,轴突、雪旺细胞、单核细胞的PTyr含量升高;CNTF组修复侧神经上述细胞成分的STAT3和PTyr含量更高.提示受损坐骨神经的STAT3表达上调,酪氨酸磷酸化增强,外加重组CNTF有进一步增强的作用.     

受损坐骨神经中STAT3表达和酪氨酸磷酸化的变化及重组CNTF的作用

研究周围神经修复后 ,受损周围神经中信号转导子和转录激活子 3(STAT3)表达和酪氨酸磷酸化的变化及睫状神经营养因子 (CNTF)的作用。用硅管套接切断的成年大鼠坐骨神经 ,术时在受损神经处给予重组CNTF ,用免疫组织化学ABC法结合计算机图像分析观测修复侧神经中STAT3、磷酸化酪氨酸 (PTyr)的分布和相对含量。在生理盐水组修复侧神经中 ,轴突、雪旺细胞STAT3的含量升高 ,轴突、雪旺细胞、单核细胞的PTyr含量升高 ;CNTF组修复侧神经上述细胞成分的STAT3和PTyr含量更高。提示受损坐骨神经的STAT3表达上调 ,酪氨酸磷酸化增强 ,外加重组CNTF有进一步增强的作用。     

L-dopa诱导PC12细胞凋亡及相关基因表达的实验研究

目的 探讨Ｌ－ｄｏｐａ在帕金森氏病（ＰＤ）治疗中的神经毒性作用及其毒性机制。方法以ＰＣ１２细胞为多巴胺神经元的细胞模型,利用ＤＮＡ凝胶电泳及图像分析仪、ＴＵＮＥＬ法、流式细胞术及免疫荧光技术检测不同浓度的Ｌ－ｄｏｐａ对ＰＣ１２细胞的凋亡诱导作用及凋亡相关基因Ｂｃｌ－２、Ｂａｘ表达的改变。结果 ２０μｍｏｌ／Ｌ、５０μｍｏｌ／Ｌ、１００μｍｏｌ／Ｌ、１５０μｍｏｌ／Ｌ处理组凋亡率分别为２．５％、１２     

多巴胺抑制PC12细胞增殖和诱导凋亡作用的研究

目的　研究多巴胺(DA)对PC12细胞的增殖抑制和诱导凋亡的作用,探讨帕金森病(PD)神经元的死亡机制。方法　应用免疫组织化学、流式细胞仪、电镜及电泳技术,研究DA对大鼠嗜铬细胞瘤PC12细胞的增殖抑制及诱导凋亡的作用。结果　适当浓度(0.5mmol/L)DA能显著抑制PC12细胞的生长并诱导其凋亡,在作用时间较短时(<12h)表现为对PC12细胞的生长抑制,此时流式细胞仪检测未见凋亡峰,但细胞周期显示S期细胞明显抑制。此时Bcl-2染色呈强阳性,电镜下细胞形态基本正常,可见线粒体、内质网肿胀及核分裂相减少。当作用时间延长时(>24h),流式细胞仪可见典型亚二倍体凋亡峰,此时电泳可见典型DNA“阶梯状”电泳带,电镜可见核浓缩、染色体边聚等凋亡特征性核结构改变,Bcl-2染色阳性率降低。结论　DA具有抑制PC12细胞增殖和诱导凋亡作用,细胞凋亡参与了PD的病变过程。     

胰岛素受体磷酸化增加对MPP＋诱导的PC12细胞凋亡的影响

目的:研究胰岛素抗MPP 诱导PC12细胞凋亡中胰岛素受体(IR)的变化。方法:应用半定量逆转录聚合酶链式反应(RT-PCR)测定胰岛素抗MPP 诱导PC12细胞凋亡过程中IRmRNA的基因表达,免疫沉淀Western印迹分析技术测定相同条件下IR的蛋白表达,应用IR自身磷酸化的特异阻断剂HNMPA-AM3,通过MTT法观察PC12细胞生存率的改变、瞬转IR质粒后再次观察细胞生存率的改变及进一步通过免疫沉淀Western印迹分析技术检测IR蛋白质磷酸化水平。结果:IR磷酸化程度的变化与细胞生存率的变化有关,HNMPA-AM3可阻断胰岛素对MPP 诱导的PC12细胞的抗凋亡作用,IR可增强胰岛素的抗凋亡作用。结论:IR磷酸化增加可抵抗MPP 诱导的PC12细胞的凋亡。     

L—dopa诱导PC12细胞凋亡及Bcl—2、Bax表达的改变

目的：探讨L－dopa治疗帕金森病（PD）疗效减退的机制及其毒性作用机制。方法：以PC12细胞为多巴胺神经元的细胞模型,利用PI／HO33342双染结合荧光显微镜技术、电镜技术、流式细胞术及免疫荧光技术检测不同浓度的L-dopa对CPC12铁凋亡诱导作用及凋亡相关基因Bcl-2、Bax表达的改变。结果50、100、150μmol/L不同浓度L－dopa处理组凋亡率分别为12．4％、24．4％、37．2％、PI／HO33342双染可区别凋亡,坏死和正常细胞,且可以见到染色质碎裂;电镜下可见早期凋亡细胞和晚期凋亡细胞,给予L-dopa处理后,Bcl-2的表达量减少,与凋亡率呈显著负相关;Bax的表达量增加,与凋亡率呈显著正相关。结论：L－dopa诱导PC12细胞凋亡且呈量效关系,提示L－dopa可能是通过凋亡途径损害多巴胺神经元导致疗效减退,其机制可能是通过改变Bcl-2/Bax的比值来介导细胞凋亡。     

增塑剂邻苯二甲酸二丁酯诱导类神经细胞PC12细胞凋亡的相关基因表达调控

目的探讨增塑剂邻苯二甲酸二丁酯对类神经细胞PC12细胞增殖能力的影响及其对PC12细胞凋亡相关基因表达的调控．为接触人群可能产生远期效应的生物学检测提供参考评价指标。方法体外培养类神经细胞株PC12细胞，培养基分别含有0，12．5，25，50mg／L邻苯二甲酸二丁酯，作用24，48，72和96h后，用细胞计数CCK-8法检测细胞的存活和增殖，台盼蓝染色绘制生长曲线，DNA凝胶电泳观察DNA—Ladder，RT—PCR检测p53，bcl-2，Bax等凋亡相关基因表达变化情况。结果邻苯二甲酸二丁酯在12．5mg／L时即表现出显著的抑制PC12细胞增殖的作用（P〈0．01），且随剂量增加抑制作用增强，同时出现DNA断裂阳性；凋亡相关基因均有表达，其中p53基因、Bax基因表达增加,bcl—2基因表达降低，且呈剂量相关关系（P〈0．01）。结论邻苯二甲酸二丁酯暴露可抑制PC12细胞增殖并诱导PC12细胞凋亡，同时通过调节凋亡相关基因p53、Bax、bcl-2基因的表达水平而在其凋亡发生中起重要作用。推测邻苯二甲酸二丁酯长期低剂量暴露的累积效应与阿尔茨海默病以及其他神经细胞凋亡相关性疾病的发生发展可能存在相关关系。     

Bcl-2家族参与β-淀粉样蛋白对PC12细胞的致凋亡作用

目的：研究bcl一2家族(主要是bcl-xl和bax)基因及相关蛋白是否参与水溶性淀粉样蛋白对培养的大鼠嗜铬细胞瘤细胞的毒性作用。方法：应用电镜和DNA电泳判断细胞损伤途径，应用RT-PCR，免疫细胞化学和免疫印迹判断bcl-2家族及其相关蛋白是否参与淀粉样蛋白的损伤作用。结果：10μmol／LAβ25-35作用24h后，电镜和DNA电泳均显示Aβ25-35可以导致PCI2细胞凋亡；RT-PCR表明药物作用后6h，bcl-xl mRNA表达量减少而bax mRNA表达量增加；免疫印迹和免疫细胞化学结果显示药物作用后24h，Bax蛋白增加而Bcl-xl无明显改变。结论：水溶性淀粉样蛋白可以导致神经细胞凋亡，bcl-2家族参与了作用环节，对阿尔茨海默病的发病起着重要的作用。     

人参皂甙RD预处理对谷氨酸所致PC12细胞损伤的保护作用

目的探讨人参皂甙RD预处理对谷氨酸所致PC12细胞损伤的影响。方法将PC12细胞分为对照组、谷氨酸损伤组和人参皂甙RD预处理组。对照组细胞正常培养;谷氨酸损伤组细胞置于含10 mmon/L谷氨酸的DMEM培养基中培养24 h;人参皂甙RD预处理组细胞经50μmol/L人参皂甙RD预处理30 min后,再加谷氨酸继续培养24 h。采用噻唑蓝比色法检测细胞活力;按试剂盒检测培养液乳酸脱氢酶（LDH）释放量;流式细胞仪检测胞内活性氧（ROS）水平;按试剂盒检测细胞内超氧化物歧化酶（SOD）含量。结果人参皂甙RD预处理最佳浓度为50μmol/L。与谷氨酸损伤组相比,人参皂甙RD预处理PC12细胞活力明显增高（P〈0.05）,培养液LDH释放量明显降低（P〈0.05）,胞内ROS含量明显降低（P〈0.05）,胞内SOD含量明显增高（P〈0.05）。结论人参皂甙RD预处理可减轻谷氨酸引起的PC12细胞损伤。     

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。     

Need for caspase-2 in apoptosis of growth-factor-deprived PC12 cells

Previous studies have shown that caspases (proteases related to interleukin-1β converting enzyme) are needed for the death of trophic factor-deprived PC12 cells. However, the protease involved in this process has not been identified. The results presented here strongly suggest that caspase-2 (Nedd2/Ich-1) plays a major role in the death of serum-deprived PC12 cells. We show that in PC12 cells overexpression of caspase-2 induces cell death, serum deprivation induces processing (i.e., activation) of the 48-kDa pro-caspase-2, and stable expression of caspase-2 antisense RNA inhibits apoptosis induced by serum deprivation. In addition, overexpression of bcl-2, which prevents this death process, also inhibits the processing of pro-caspase-2, suggesting that bcl-2 acts upstream of pro-caspase-2 activation. J. Neurosci. Res. 52:491–497, 1998. © 1998 Wiley-Liss, Inc.     

木犀草素对鱼藤酮诱导损伤PC12细胞保护作用机制

目的：探讨木犀草素对鱼藤酮诱导的帕金森病细胞模型的保护作用及机制。方法：运用鱼藤酮诱导损伤PC12细胞,经100mol/L木犀草素预处理后,观察细胞形态学改变,流式细胞仪检测细胞凋亡率,彗星电泳分析DNA损伤情况,Western blot检测Cleaved Caspase-3的表达,比较各组的差异。结果：1.6mol/L鱼藤酮作用24h后,PC12细胞形态明显改变且凋亡率为36.1%,彗星电泳出现典型拖尾;木犀草素预处理后,细胞形态明显改善,凋亡率降至11.8%,显著低于鱼藤酮组（p<0.01）,彗星头平均光密度值增加（p<0.01）,彗星尾距缩短（p<0.01）,Cleaved Caspase-3的表达降低（p<0.05）。结论：木犀草素可保护PC12细胞免于鱼藤酮的损伤以及抗凋亡,下调Cleaved Caspase-3的表达可能是其作用的机制之一。     

Need for caspases in apoptosis of trophic factor-deprived PC12 cells

PC12 cells are a useful model system for studying neuronal apoptosis. Like neurons, they undergo apoptosis when deprived of trophic support. Involvement of caspases [interleukin 1β-converting enzyme (ICE)-related proteases] has been implicated in apoptosis induced by various stimuli in many cell types, including neurons. In the present study we investigated the need for caspases participation in apoptosis induced by growth factor deprivation in naive and neuronal PC12 cells. For this purpose we generated PC12 cell lines that consistently express the viral caspases inhibitor genes p35 or crmA, and analyzed their susceptibility to trophic factor deprivation. We also examined the effects of cell-permeable peptide inhibitors of caspases. Our results showed that broad-spectrum inhibitors of the caspases, namely the baculovirus p35 gene and the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, effectively inhibit the death of both naive and neuronal PC12 cells. However, caspase-1 (ICE)-specific inhibitors, namely the peptides Ac-Try-Val-Ala-Asp-chloromethylketone and Ac-Try-Val-Ala-Asp-aldehyde, as well as crmA, were much less effective. These findings demonstrate that caspases, but not caspase-1, are needed for apoptosis induced by trophic factor deprivation in both naive and neuronal PC12 cells. Northern and Western blot analyses showed that PC12 cells express caspase-3. We therefore examined the involvement of caspase-3 in the death process of trophic factor-deprived PC12 cells. Our results showed that the pro-caspase-3 and its substrate poly(ADP-ribose)polymerase are cleaved at similar rates in serum-deprived PC12 cells. Moreover, cell lysates prepared from these cells possess caspase-3-like activity, as determined by their ability to cleave the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin. These findings strongly suggest that caspase-3 or caspase-3-like proteases are activated in trophic factor-deprived PC12 cells. J. Neurosci. Res. 50:69–80, 1997. © 1997 Wiley-Liss, Inc.     

S-adenosyl-methionine-induced apoptosis in PC12 cells

Our previous studies showed that S-adenosyl-methionine (SAM) induced Parkinson's disease-like changes in rat. It caused death to dopamine neurons in the substantia nigra, which appeared shrunken and fragmented, indicative of apoptosis-like changes (Charlton and Crowell [1995] Mol. Chem. Neuropathol. 26:269-284; Charlton [1997] Life Sci. 61:495-502). In this study, we investigated whether SAM causes apoptosis in both undifferentiated PC12 (PC12) cells and nerve growth factor (NGF)-differentiated PC12 (D-PC12) cells. S-adenosyl-homocysteine (SAH), the nonmethyl analog of SAM, was also tested. SAM and SAH (1.0 nM to 10.0 microM) caused lactate dehydrogenase (LDH) release from the PC12 cells and D-PC12 cells; cells with morphological changes and fluorescent DNA fragmentation staining were detected among both PC12 cell and D-PC12 cell. Compared with the PC12 cell, the D-PC12 cell, a postmitotic cell, was more sensitive to the toxic effects of SAM or SAH and presented much greater LDH release, suggesting a lethal effect; surprisingly, the amounts of apoptotic cells did not differ significantly between the two kinds of cells. In medium deprived of exogenous methionine, a decline in LDH release was observed in PC12 and D-PC12 cells. Also, lower levels of intracellular SAM and SAH were observed in the methionine-deleted media, which were reversed by the addition of either SAM or SAH. An antivitamin B(12) monoclonal antibody was added to methionine-depleted medium, resulting in deficiency of both endogenous and exogenous methionine, which caused further decreases in LDH release and reduction in the levels of intracellular SAM and SAH. The preliminary data showed different sensitivities to SAM or SAH between PC12 cell and D-PC12 cells, which suggests that PC12 cell may be more stable as a metabolic model. Apoptosis of PC12 cells was also assessed by PARP cleavage detection, Western blot analysis of Bax and Bcl-2 proteins, and DNA laddering on agarose gel electrophoresis. The proapoptoic protein Bax was dominantly expressed, whereas Bcl-2 was slightly down-regulated by SAM. SAH weakly induced the expression of Bax and slightly decreased Bcl-2 levels. The effects of SAM and its analog, SAH, were demonstrated conclusively to induce apoptosis in PC12 cells.     

α-tocopherol protects PC12 cells From hyperoxia-induced apoptosis

A rat clonal pheochromocytoma cell line (PC12) was cultured under normoxic (21% O₂) and hyperoxic (50% O₂) conditions. PC12 cells underwent apoptotic cell death when they were cultured in charcoal-stripped medium in a high-oxygen atmosphere. Vitamin E homologs, α-tocopherol (αT), β-tocopherol (βT), γ-tocopherol (γT), and δ-tocopherol (δT), were added to the culture medium to study their biological activities. αT was more effective than γT and δT in preventing hyperoxia-induced cell death. Addition of exogenous αT to charcoal-treated medium prevented lactate dehydrogenase (LDH) leakage from PC12 cells and also inhibited the apoptosis, which was accompanied by DNA fragmentation. Additional αT was rapidly concentrated in PC12 cells, suggesting that it exerts antioxidant effects. Our data show that PC12 cell death under high-oxygen conditions is due to apoptosis and that, among the vitamin E homologs, αT most effectively prevents hyperoxic apoptosis. J. Neurosci. Res. 52:184–191, 1998. © 1998 Wiley-Liss, Inc.     

P~(38)激酶在PC12细胞缺氧/再给氧损伤中的作用

目的:探讨PC12细胞缺氧/再给氧损伤的信号转导机理。方法:培养的PC12细胞先缺氧(95％N2/5％CO2)6h,然后重新给氧,观测不同时间点细胞的存活率和caspase-3的活性;用MTT法测存活率,caspase-3检测试剂盒测caspase-3活性。用p38拮抗剂SB203580孵育细胞2h,之后缺氧/再给氧,观察SB203580对细胞存活率和caspase-3活性的影响。结果:PC12细胞缺氧/再给氧后caspase-3活性明显增加并使细胞存活率下降,SB203580明显降低缺氧/复氧后caspase-3的活性并使细胞死亡减少。结论:PC12细胞缺氧/再给氧后至少可以通过激活p38、caspase-3信号分子诱导PC12细胞死亡。     

3aicalin inhibits colistin sulfate-induced apoptosis of PC12 cells

Baicalin, a type of flavonoid extracted from the dried root of Scutellaria baicalensis georgi, has been shown to effectively inhibit cell apoptosis. Therefore, we assumed that baicalin would suppress colistin sulfate-induced neuronal apoptosis. PC12 cells exposed to colistin sulfate （62.5-500 μg/mL） for 24 hours resulted in PCl2 cell apoptosis. In addition, caspase-3 activity, lactate dehydrogenase level and free radical content increased in a dose-dependent manner. Subsequently, PC12 cells were pretreated with baicalin （25, 50 and 100 pg/mL）, and exposed to 125 pg/mL colistin sulfate. Cell morphology markedly changed, and cell viability increased. Moreover, caspase-3 activity, lac- tate dehydrogenase level and free radical content decreased. Results indicated that baicalin inhib- ited colistin sulfate-induced PC12 cell apoptosis by suppressing free radical injury, and reducing caspase-3 activity and lactate dehydrogenase activity.