首页 | 官方网站   微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 187 毫秒
1.
目的研究14-3-3蛋白过表达对1-甲基-4苯基吡啶离子(MPP )诱导的PC12细胞死亡的影响作用及其可能的机制。方法构建pcDNA3.1( )-14-3-3 真核表达质粒,用脂质体2000转染PC12细胞;Western blot技术检测PC12细胞中14-3-3蛋白、Bcl-2 蛋白, 和BAD蛋白的表达;然后分别用MTT法、酶标仪及流式细胞仪检测PC12细胞的活力、caspase的活性及PC12细胞的凋亡率。结果(1)将pcDNA3.1( )-14-3-3质粒转染PC12细胞3周后,14-3-3蛋白的表达显著增加;(2)MPP 诱导PC12细胞存活率的下降是剂量依赖性的,当MPP 的浓度达100 μmol/L时,PC12细胞的存活率丧失约50%;(3)caspase 的活性随着MPP 浓度的增加而增高,当MPP 浓度到达100 μmol/L时caspase的活性也到达最大值,而当MPP 浓度超过100 μmol/L时,caspase的活性急剧下降;(4)用100 μmol/L 的MPP 处理PC12细胞24 h后,PC12细胞的凋亡率为26.5%,14-3-3蛋白的过表达使PC12细胞的凋亡率下降到8.6%;(5)用100 μmol/L MPP 处理PC12细胞后,Bcl-2蛋白的表达趋于下调而BAD蛋白的表达上调,14-3-3蛋白的过表达能显著的增加Bcl-2蛋白的表达而使BAD蛋白的表达下调。结论14-3-3蛋白过表达通过上调Bcl-2蛋白的表达并下调BAD蛋白的表达,减少了MPP 诱导的PC12细胞的凋亡,从而发挥对PC12细胞的保护作用。这些结果可能为PD的治疗提供新的药物靶点。  相似文献   

2.
目的探讨H2O2预处理对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP^+)诱导PC12细胞毒性损伤的保护作用及其可能的机制。方法分别用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑嗅盐[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]和4,6-二氨基-2-苯基吲哚(4′,6′.diamidino-2-phenylindole,DAPI)染色技术检测PC12细胞的活力及细胞凋亡核形态改变;Westem blot检测PC12细胞中14-3-3蛋白、磷酸化p38 MAPK的水平。酶联免疫吸附实验(Enzyme—linked immunosorbent assay,ELISA)试剂盒检测ERK1/2磷酸化水平。结果MPP^+处理PC12细胞24h使细胞活力显著下降(51.6%),DAPI染色显示(51.3±6.6)%的PC12细胞呈现核固缩等细胞凋亡形态改变;H2O2预处理能显著增加PC12细胞活力(83.4%),减少凋亡细胞数[(24.9±4.3)%],说明H2O2预处理能对抗MPP^+毒性,对PC12细胞具有保护作用。Western blot结果显示,H2O2预处理在减少PC12细胞凋亡的同时伴随有14-3-3蛋白及磷酸化p38 MAPK表达上调;ELISA实验显示ERK1/2磷酸化水平显著增加。相关分析显示14-3-3蛋白表达的上调与ERK1/2磷酸化水平呈正相关(r=0.923,P〈0.01),而且PD98059抑制ERK1/2磷酸化后,H2O2预处理诱导14-3-3蛋白表达上调的作用消失。结论H2O2预处理能对抗MPP^+对PC12细胞的毒性作用,这种保护作用与细胞内14-3-3蛋白表达上调有关;而14-3-3蛋白表达上调与ERK1/2和p38MAPK信号通路的激活有关。  相似文献   

3.
目的探讨促红细胞生成素(erythropoietin,EPO)对1-甲基-4-苯基吡啶离子(MPP^+)诱导的PC12细胞,变性损伤的保护作用及机制。方法用MPP^+处理PC12细胞制作帕金森病细胞模型,采用四甲基偶氮哗监泫检测暴露于不同浓度EPO后细胞的活性;流式细胞术与DNA断端原位标记法(terminal deoxynucleotidyl transferase dUTP nick end labeling,TUNEL)检测各组的细胞凋亡率;免疫印迹法检测不同处理组PC12细胞Bcl-2和Bax的表达,并采用荧光法观察不同处理组PC12细胞活性氧(reactive oxygen species,ROS)与线粒体膜电位水平以及caspase-3活性的变化。结果MPP^+可以使PC12细胞存活率下降,凋亡率增高;同时PC12细胞内ROS增多,线粒体膜电位下降。MPP^+还可以明显地提高Bax/Bcl-2比值并激活caspase-3。而EPO可以抑制这些由MPP^+引发的改变,并在1U/mL时发挥最大保护作用。结论EPO可抑制MPP^+诱导的PC12绌胞死亡,其作用机制可能其自身抗氧化和抗凋亡的特性有关。  相似文献   

4.
14-3-3蛋白过表达减轻MPP+对PC12细胞的毒性损伤   总被引:4,自引:0,他引:4  
目的 探讨14-3-3蛋白过表达对1-甲基-4-苯基吡啶离子(MPP+)诱导PC12细胞损伤的保护作用和可能的机制.方法 构建pcDNA3.1(+)-14-3-3真核表达质粒,转染PC12细胞,建立稳定过表达14-3-3蛋白细胞株;通过四甲基偶氮唑盐法(MTT法)、流式细胞术和酶标仪分别检测14-3-3蛋白过表达对MPP+诱导的PC12细胞存活力、凋亡率和SOD及谷胱甘肽过氧化物酶(GSH-Px)活性的影响.结果 14-3-3蛋白过表达显著增加PC12细胞SOD活性[质粒转染组(9.13±0.41)U/mg,MPP+组(6.45±0.52)U/mg]和GSH-Px活性[质粒转染组(89.66±3.42)μmol/mg,MPP+组(82.73±4.15)μmol/mg]、增强细胞活力[吸光度(A570):质粒转染组0.78±0.06,MPP+组0.54±0.07]、抑制细胞的凋亡(质粒转染组11.87%±3.26%,MPP+组36.30%±2.39%).结论 14-3-3蛋白过表达对MPP+的毒性有保护作用,这是通过增加SOD和GSH-Px的活性,减少氧化应激实现的.  相似文献   

5.
3-硝基丙酸多次化学预处理对多巴胺能神经元的保护作用   总被引:2,自引:0,他引:2  
目的探讨3-硝基丙酸(3-NP)多次化学预处理对多巴胺能神经元的保护作用及可能机制。方法应用MPTP(30mg/kg)在C57BL小鼠上复制帕金森病模型,以3-NP(20mg/kg)行预处理,检测小鼠中脑黑质凋亡率和转录因子c-Jun的阳性细胞数量及c-Jun的蛋白水平;应用MPP^+(0.25mmol/L)在SH—SY5Y细胞制作帕金森病模型,以3-NP(0.2mmol/L)进行预处理,并将携带显性突变体c—JuncDNA片段的真核表达载体质粒pcDNA3(HA)-Jun—dn转染SH—SY5Y细胞,检测各组细胞的c-Jun表达水平及凋亡率。结果小鼠中脑黑质凋亡率:MPTP组较对照组明显升高(P〈0.01),3-NP单次、多次预处理后均明显降低(P〈0.05,P〈0.01);c—Jun阳性细胞数:MPTP组较对照组明显增加(P〈0.05),3-NP单次预处理组与MPTP组比较无明显差异,3-NP多次预处理后明显降低(P〈0.05);c—Jun蛋白水平:与其阳性细胞数变化一致;细胞凋亡率:MPP^+组较对照组明显升高,3-NP单次、多次预处理组细胞凋亡率明显降低(P〈0.05,P〈0.01);c-Jun蛋白水平变化与中脑黑质一致;经pcDNA3(HA)-Jundn转染的细胞,其c-Jun的表达较未转染细胞明显降低(P〈0.01),其凋亡率也下降(P〈0.01)。结论3-NP单次、多次预处理对多巴胺能神经元确有保护作用,多次预处理保护效果更强,其机制与抑制转录因子c-Jun的表达,降低其蛋白水平有关。  相似文献   

6.
九节龙-Ⅲ经BAD凋亡途径诱导胶质瘤U251细胞凋亡   总被引:1,自引:1,他引:0  
目的研究九节龙-Ⅲ诱导胶质瘤U251细胞凋亡及其内在机制。方法四甲基偶氨唑蓝(MTT)分析药物对U251细胞及胶质细胞增殖的影响;膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V.FITC/PI)染色、Hoechst33342染色和透射电镜检测细胞凋亡;Western blotting分析Bcl-xL/Bcl-2相关死亡启动子(BAD)蛋白表达和磷酸化水平。结果九节龙-Ⅲ以剂量和时间依赖方式抑制U251细胞活性(P〈0.05,IC50=8.2μg/ml),诱导染色质浓聚边集、凋亡小体形成等凋亡改变,且浓度低于40μg/ml时不抑制胶质细胞活性(P〉0.05)。Western blotting发现U251凋亡细胞内BAD表达明显增加、去磷酸化并被裂解。结论九节龙-Ⅲ经BAD去磷酸化和裂解的凋亡途径诱导了胶质瘤U251细胞的显著凋亡。  相似文献   

7.
目的探讨雷沙吉兰(Rasagiline)对β-淀粉样蛋白(Ap)诱导PCI2细胞损伤阿尔茨海默病(AD)模型的保护作用及其机制。方法不同浓度的Aβ25-35(1μmol/L,10μmol/L,20μmol/L)作用于PCI2细胞48h,MTT法检测细胞存活率.选用使细胞存活率降低到64%的Aβ浓度20μmol/L。用不同浓度的雷沙吉兰(0.1μmol/L,1μmol/L,10μmol/L)预孵育PC12细胞1h,再加入20μmol/l,的Aβ共孵育48h,再测MTT活性,并用荧光染料丫啶橙和溴化乙啶染色,在荧光显微镜下计数凋亡细胞检测凋亡细胞百分率。结果 Aβ在20μmol/L时使PC12细胞存活率降低至64%,与对照组差异显著,1μmol/L的雷沙吉兰可显著提高细胞存活率至85%。对照组细胞凋亡率为2%.20μmol/L,Aβ作用48h后,PC12细胞凋亡率达13%,1μmol/L的雷沙吉兰使20μmol/L。Aβ诱导的PC12细胞凋亡率下降到5%。结论 雷沙吉兰对Aβ引起的PC12细胞损伤具有明显的保护作用.其机制可能与抑制Aβ诱导的凋亡有关。  相似文献   

8.
目的 探讨灯盏细辛对缺氧复氧(HR)损伤的PC12细胞保护机制。方法 将体外培养的PC12细胞分为正常对照(NC)组、HR组、灯盏细辛10μmol/L(E1)组、20μmol/L(E2)组、30μmol/L(E3)组。将HR组及E1~E3组细胞经HR处理后,测定各组细胞的存活率;通过Western Blot方法观察各组细胞Bcl-2和Bax蛋白含量的变化。结果 (1)与NC组比较,HR组的细胞存活率明显下降(P〈0.01);与HR组比较,E1-E3组细胞存活率明显升高(均P〈0.01),且呈剂量依赖性(P〈0.01)。(2)与NC组比较,HR组PC12细胞Bcl-2蛋白表达明显下降,Bax蛋白表达明显增加(均P〈0.01);与HR组比较,E1-E3组PC12细胞Bcl-2表达明显增加,Bax表达明显下降,并呈剂量依赖性(均P〈0.01)。结论 灯盏细辛对HR损伤的PC12细胞保护作用是通过增加Bcl-2表达、抑制Bax表达的抗凋亡作用实现的。  相似文献   

9.
目的探讨肺腺癌转移相关转录本1(MALAT1)对β-淀粉样蛋白1-42片段(Aβ1-42)诱导肾上腺嗜铬细胞瘤PC12细胞损伤及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)通路的影响。方法体外培养PC12细胞,并随机分为正常对照组(正常生长PC细胞)、模型组(Aβ1-42诱导PC细胞损伤)、sh-MALAT1组(Aβ1-42组基础上转染MALAT1干扰序列)、sh-对照组(Aβ1-42组基础上转染MALAT1对照序列)。采用实时定量PCR(RT-qPCR)检测各组PC12细胞MALAT1表达水平,采用四甲基偶氮唑蓝(MTT)法检测PC12细胞增殖,采用流式细胞仪及AnnexinV-FITC/PI法体外检测PC12细胞凋亡,采用免疫印迹(WB)检测PC12细胞PI3K/Akt通路相关p-PI3K/PI3K、p-Akt/Akt蛋白及其下游凋亡相关蛋白Cleaved-caspase3/caspase3、B细胞淋巴瘤-2基因相关X蛋白(Bax)、B细胞淋巴瘤-2(Bcl-2)蛋白表达。结果与正常对照组比较,模型组、sh-对照组组PC12细胞存活率、p-PI3K/PI3K、p-Akt/Akt、Bcl-2蛋白水平低(均P0.05),MALAT1表达水平、PC12细胞凋亡率、Bax、Cleaved-caspase3/caspase3蛋白表达水平高(均P0.05);与模型组、sh-对照组比较,sh-MALAT1组MALAT1表达水平、细胞存活率及p-PI3K/PI3K、p-Akt/Akt、Bcl-2蛋白水平高(均P0.05),Bax、Cleaved-caspase3/caspase3蛋白表达水平低(均P0.05)。结论下调MALAT1表达可能通过激活PI3K/Akt通路,上调Bcl-2蛋白表达,下调Bax、Cleaved-caspase3/caspase3蛋白表达抑制Aβ1-42诱导的PC12损伤,发挥保护作用。  相似文献   

10.
目的:评价尿酸减轻6.羟基多巴胺(6-OHDA)对PC12细胞的毒性作用。方法:应用PCI2细胞制作帕金森细胞模型,分为对照组、尿酸组、6-OHDA组、尿酸+6-OHDA组。采用MTT测定各组PC12细胞活性,免疫荧光法观察各组PCI2细胞caspase-3激活情况,流式细胞术检测各组PC12细胞凋亡率。尿酸100~400μmol·L^-1不影响PCI2细胞生存率,尿酸100~400μmol·L-1可显著提高6-OHDA50gmol-L。作用6、12和24h造成的PCI2细胞生存率的下降(P〈0.01);尿酸能减少6-OHDA导致的PCI2细胞caspase-3激活,降低6-OHDA导致的凋亡率(P〈0.05)。结论:尿酸具有减轻6-OHDA对PC12细胞的毒性作用。  相似文献   

11.
OBJECTIVE: To investigate the protective effects of hydrogen peroxide preconditioning (HPP) on the pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium (MPP(+)) and to explore the potential mechanisms. METHODS: The viability and apoptosis of PC12 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4',6'-diamidino-2-phenylindole (DAPI) staining, respectively. The expressions of 14-3-3 protein and phosphorylated p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. Enzyme-linked immunosorbent assay (ELISA) was used to measure the activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2). RESULTS: The cell viability decreased and the number of apoptotic cells increased dramatically in MPP(+) group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP(+)-treated PC12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC12 cells induced by HPP. CONCLUSION: HPP protects PC12 cells against MPP(+) toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.  相似文献   

12.
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。  相似文献   

13.
Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The effect of econazole against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells was assessed in relation to the mitochondrial membrane permeability changes. Treatment of PC12 cells with MPP(+) resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. Econazole (0.25-2.5 microM) inhibited the cytotoxicity of MPP(+) or rotenone. The addition of econazole (0.5 microM) significantly attenuated the MPP(+)-induced mitochondrial damage, elevation of intracellular Ca(2+) level and cell death. However, because of the cytotoxicity, econazole at 5 microM did not attenuate the toxicity of MPP(+). The results show that econazole at the low concentrations may reduce the MPP(+)-induced viability loss in PC12 cells by suppressing the mitochondrial permeability transition, leading to activation of caspase-3 and the elevation of intracellular Ca(2+) levels, which are associated with the increased formation of ROS and depletion of GSH.  相似文献   

14.
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。  相似文献   

15.
16.
17.
18.
目的 探讨亲环素A(CyPA)对Aβ25-35诱导的PC12细胞凋亡影响及可能的作用机制.方法 将PC12细胞分为正常对照组(0 μmol/L Aβ25-35)、Aβ25-35诱导组(10 μmol/L Aβ25-35)和药物保护组(0.1、1、10、100 nmol/L CyPA+10 μmoi/Lβ25-35),药物保护组采用相应浓度CyPA预处理PC12细胞30 min,再加入Aβ25-35继续培养.MTT法分析细胞存活率,Hoechst33258染色法检测细胞凋亡,RT-PCR检测Bcl-2和Bax mRNA表达,Western blotting检测Bcl-2和Bax蛋白表达.结果 1、10和100 nmol/L CyPA可提高细胞的存活率,减少Aβ25-35引起的细胞凋亡,增加Bcl-2mRNA表达以及Bcl-2蛋白表达,降低Bax mRNA表达以及Bax蛋白表达,与Aβ25-35诱导组比较差异有统计学意义(P<0.05),且CyPA剂量越高效果越明显.结论 CyPA可剂量依赖性对抗Aβ25-35对PC12细胞的毒性作用,减少细胞凋亡,其机制与上调抗凋亡基因Bcl-2表达和下调凋亡基因Bax表达有关.
Abstract:
Objective To explore the effect of cyclophilin A (CyPA) on apoptosis of PC 12 cells induced by Aβ25-35 and its potential mechanism. Methods PC 12 cells were divided into normal control group (0 μmol/L Aβ25-35), Aβ25-35 inducement group (10 μmol/L Aβ25-35) and drug protection groups (0.1, 1,10 and 100 nmol/L CyPA+10 μmol/L Aβ25-35). Cells in the drug protection groups were pretreated by CyPA of different concentrations for 30 min, and then co-cultured with Aβ25-35 We evaluated the survival rate of PC12 cells with MTT assay, analyzed the apoptosis of PC12 cells with Hoechst33258 staining, and detected the mRNA expressions of Bcl-2 and Bax with PT-PCR and the protein levels of Bcl-2 and Bax with Western blotting. Results Cells pretreated wth CyPA of 1, 10 and 100 nmol/L enjoyed an obvious elevation of survival rate of PC 12 cells, a significant reduction of apoptosis induced by Aβ25-35,an obvious increase of mRNA expression of Bcl-2 and protein level of Bcl-2, and a statistical decrease of mRNA expression of Bax and protein level of Bax as compared with those cells of the Aβ25-35 inducement group (P<0.05);and these effects were dose-dependent. Conclusion CyPA could resist the toxic role of Aβ25-35 on PC 12 cells and reduce the apoptosis in a dose-dependent manner by up-regulation of anti-apoptosis gene Bcl-2 and down-regulation of apoptosis gene Bax.  相似文献   

19.
To further characterize MPP(+)-induced cell death and to explore the role of Bcl-2-related proteins in this death paradigm, we utilized a mesencephalon-derived dopaminergic neuronal cell line (MN9D) stably transfected with human bcl-2 (MN9D/Bcl-2), its C-terminal deletion mutant (MN9D/Bcl-2Delta22), murine bax (MN9D/Bax), or a control vector (MN9D/Neo). As determined by electron microscopy and TUNEL assay, MN9D/Neo cells exposed to MPP(+) underwent a cell death that was characterized by mitochondrial swelling and irregularly scattered heterochromatin without accompanying DNA fragmentation. However, cell swelling typically seen in necrosis did not appear. To examine the biochemical events associated with MPP(+)-induced cell death, various analyses were conducted. Addition of a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (50-400 microM) or Boc-aspartyl(OMe)-fluoromethylketone (50-200 microM) did not attenuate MPP(+)-induced cell death while the same treatment protected MN9D/Neo cells against staurosporine-induced apoptotic cell death. Concurrent treatment with an inhibitor of macromolecule synthesis such as cycloheximide, emetine, or actinomycin D blocked MPP(+)-induced cell death, suggesting that new protein synthesis is required as demonstrated in many apoptotic cell death. The level of cytosolic calcium in MN9D/Neo cells was unchanged over 24 h following MPP(+) treatment, as monitored by means of the fluorescent probe Fura-2. Western blot analysis indicated that expression level of proapoptotic protein, Bax was not significantly altered after MPP(+) treatment. In this death paradigm, overexpression of Bcl-2 but not its C-terminal deletion mutant attenuated MPP(+)-induced cell death whereas overexpression of Bax had no effect. Taken together, these data indicate that (i) MPP(+) induces a distinct form of cell death which resembles both apoptosis and necrosis; and (ii) full-length Bcl-2 counters MPP(+)-induced morphological changes and cell death via a mechanism that is dependent on de novo protein synthesis but independent of cytosolic calcium changes, Bax expression, and/or activation of caspase(s) in MN9D cells.  相似文献   

20.
目的 探讨BDNF对凝聚态β-淀粉样蛋白25-35片断(Aβ25-35)诱导细胞损伤特别是凋亡的影响.方法 MTT法观察细胞活力,Annexin V-PI双染色法观察细胞凋亡,Western blotting法检测Bax及Bcl-2表达,应用Trk B受体抑制剂K252a(200 nmol/L)观察50 ng/mL BDNF对20 μmol/L的Aβ25-35诱导细胞损伤的抑制作用机制.结果 MTT检测结果及Annexin V-PI双染色法发现50ng/mL的BDNF预处理可显著地抑制20μmol/L的Aβ25-35诱导的PC12细胞活力的下降及细胞凋亡;Western blotting检测发现50 ng/mL的BDNF对20μmol/L的Aβ25-35诱导的PC12细胞Bax表达升高、Bcl-2表达降低及Bax/Bcl-2比值上调均有明显的抑制作用,该作用可被Trk B受体抑制剂K252a(200 nmol/L)抑制.结论 BDNF对Aβ25-35诱导的细胞损伤特别是细胞凋亡具有保护作用,该保护作用是通过与其特异性受体Trk B结合实现的.
Abstract:
Objective To investigate the effect of BDNF on cell injuries, cell apoptosis especially, induced by 25-35 segment of β-amyloid protein (Aβ25-35) at condensed state in PC12 cells.Methods The viability of PC12 cells, the apoptosis of PC12 cells and the expressions of Bax and Bcl-2 were detected by MTT, Annexin V-PI staining and Western blotting, respectively. Trk B receptor inhibitor K252a (200 nmol/l) was employed to observe the mechanism of 50 ng/ml BDNF on Aβ25-35 (20 μmol/L)-induced cell injury. Results BDNF (50 ng/ml) could significantly prevent the decrease of cell viability and cell apoptosis, and the increase of Bax expression and the decrease of Bcl-2 expression induced by 20 μmol/L Aβ25-35, and prevent the increase of up-regulation of Bax/Bcl-2 ratio; and these effects were blocked by K252a (200 nmol/1). Conclusion BDNF can prevent Aβ25-35-induced cell injury, cell apoptosis especially, by binding to its specific receptor Trk B.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司    京ICP备09084417号-23

京公网安备 11010802026262号