对氧磷酶2对氧化低密度脂蛋白诱导的乳鼠心肌细胞损伤的影响

目的 探讨对氧磷酶2(paraoxonase 2,PON2)在氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)诱导的乳鼠心肌细胞(neonatal rat ventricular cells,NRVCs)损伤中的作用及机制.方法 分离SD大鼠(出生1～3d,雌雄不限,共90只)心肌细胞.细胞分为:①对照组(未进行任何干预)、②0.1 μmol/Lox-LDL组(ox-LDL干预24 h)、③10 μmol/Lsalubrinal+ox-LDL组(salubrinal预处理1h后加入ox-LDL干预24 h)、④20 μg/mLPON2+ox-LDL组(PON2预处理1h后加入ox-LDL干预24 h)、⑤2.5μmol/LMn(Ⅲ)TBAP+ ox-LDL组[(Mn(Ⅲ)TBAP预处理1h后加入ox-LDL干预24 h].通过MTT法和Caspase-3/7活性检测试剂盒分别检测细胞活性和细胞凋亡变化(n=6);Western blot检测心肌细胞内质网应激和氧化应激蛋白表达情况(n=4).结果 与对照组相比,ox-LDL明显降低心肌细胞活性[(0.417 ±0.047) vs(O.883 ±0.064),P ＜0.01]显著增加心肌细胞凋亡[(11 505 ±817)vs(6417 ±439),P ＜0.01].与ox-LDL组相比,PON2、salubrinal和Mn(Ⅲ)TBAP干预后,细胞活性显著升高[(0.567 ±0.066) 、0.649 ±0.082) 、0.539 ±0.049) vs0.417 ±0.047),P ＜0.01],细胞凋亡显著下降[(7 776 ±488) 、(7 545 ±547)、(7 960±480)vs (11 505 ±817),P ＜0.01).与对照组相比,ox-LDL显著增加PON2蛋白表达[(0.86 ±0.223) vs (0.561 ±0.13),P ＜0.05]、内质网应激蛋白eIF-2αphox、caspase-12和氧化应激蛋白Nox2/gp91 phox、p47 phox表达[(1.309 ±0.274) vs (0.780 ±0.186) 、(1.294±0.273) vs (0.606 ±0.064) 、(1.342 ±0.274) vs(0.788 ±0.137) 、(1.178 ±0.194) vs (0.629 ±0.125),P＜0.01].与ox-LDL组相比,PON2干预后,PON2蛋白表达明显升高[(1.190 ±0.151) vs (0.860±0.222),P＜0.05];eIF-2αphox 、caspase-12、Nox2/gp91 phox和p47 phox蛋白表达明显降低[(0.924 ±0.170) vs(1.309 ±0.274) 、(0.895 ±0.202) vs (1.294 ±0.273) 、(0.957 ±0.190) vs (1.342 ±0.274) 、(0.799±0.232) vs(1.178 ±0.194),P ＜0.05].与PON2作用类似,salubrinal干预后,eIF-2αphox和caspase-12蛋白表达明显降低[(1.010±0.096) vs(1.309±0.274)、(0.788±0.042)vs(1.294±0.273),P＜0.01],Mn(Ⅲ)TBAP干预后,Nox2/gp91 phox和p47 phox蛋白表达明显降低[(0.898 ±0.190) vs(1.342±0.274) 、(0.769±0.158) vs(1.178 ±0.194),P ＜0.01],二者均显著升高PON2蛋白表达[(1.248±0.259) 、(1.176 ±0.248) vs(0.860 ±0.222),P ＜0.05].结论 PON2可能通过代偿性升高抑制氧化应激和内质网应激而减轻ox-LDL诱导的心肌细胞损伤.     

PC12细胞应激损伤中能量限制的效应及SIRT3的表达

目的 研究H2O2诱导的PC12细胞氧化应激损伤中,能量限制(CR)及SIRT3参与的调控效应.方法 实验分4组:H2O2组,H2O2+CR组,CR组和正常对照组;MTT法检测不同浓度H2O2作用下CR对细胞生存率的影响;TUNEL染色法检测过氧化氢作用后CR对细胞的凋亡的影响;免疫荧光检测PC12细胞中SIRT3的表达定位;RT-PCR及Western印迹检测SIRT3、Caspase-3的表达变化.结果 60μmol/L H2O2作用6 h后,H2O2组细胞生存率(74.01±2.21)%可维持在70%以上,与对照组比较差异有统计学意义(P＜0.05);120μmol/L H2O2作用下,H2O2组的细胞活力显著下降(38.22±3.34)%,后续研究选取60μmol/L H2O2浓度作为应激源;60μmol/L H2O2作用6 h后H2O2组细胞生存率(74.01±2.21)%与CR+H2O2组(97.26±1.92)%间比较差异有统计学意义(P＜0.05).TUNEL凋亡检测H2O2+CR组凋亡率较H2O2组显著降低.免疫荧光双染色证实PC12细胞中SIRT3为一种线粒体蛋白.Western印迹显示与正常对照组(5256±144)比较,CR组中SIRT3表达升高(6857±157),差异有统计学意义(P＜0.05)、H2O2组中表达降低(3786±160),差异有统计学意义(P＜0.05).与H2O2组(3786±160)比较,CR+H2O2组中SIRT3表达上升(5056±121),差异有统计学意义(P＜0.05).与正常对照组比较(5342±420),H2O2组中Caspase-3表达升高(8499±426),差异有统计学意义(P＜0.001).CR+H2O2组(5750±438)中Caspase-3表达较H2O2组表达下降(8499±426),差异有统计学意义(P＜0.001).RT-PCR显示与正常对照组(6204±134)比较,CR组(7214±148)中SIRT3表达升高,差异有统计学意义(P＜0.05)、H2O2组中表达降低(4807±143),差异有统计学意义(P＜0.05).与H2O2组(4807±143)比较,CR+H2O2组中SIRT3表达上升(6195±166),差异有统计学意义(P＜0.05).结论 PC12细胞中,CR具有抗氧化应激损伤及凋亡的效应;CR可上调PC12细胞中SIRT3的表达,在H2O2诱导的PC12应激损伤中CR-SIRT3的调控具有保护效应,SIRT3是否具有延缓神经元衰老作用值得进一步研究.

Abstract：

Objective To study the regulation effects of CR (caloric restriction) and SIRT3 in the H2O2-induced oxidative stress injury of PC12 cell. Methods The cells were divided into four groups:H2O2, H2O2+CR, CR and control (high glucose). For control and H2O2 group, cells were cultured in DMEM containing 0.45% glucose; for group in CR condition, cells were treated with the medium containing 0.1% glucose. For groups with H2O2, the H2O2 was diluted in EMEM medium to obtain the final concentration containing 60 μmol/L. Viability of PC12 cells were measured by MTT assay. The medium was refreshed with different concentration of H2O2 (from 10 to 120 μmol/L). The absorbance of the samples was measured at 492 nm using a microtiter plate reader. We detected TUNEL-positive cells using the In Situ Cell Apoptosis Detection kit pretreated with CR and H2O2. Immunofluorescence double staining detected the expression and localization of SIRT3. RT-PCR and Western-blot mehtods detected the expression of SIRT3,Caspase-3. Results After pretreating with 60 μmol/L H2O2 for 6 h, the viability of PC12 cells in H2O2 group (74.01±2.21)% retained above 70%, and have statistical significance contrasted with control group (P＜0.05); After pretreating with 120μmol/L H2O2, the viability of PC12 cells declined significantly (38.22±3.34)%. So 60μmol/L H2O2 is our experiment concentration . The viability of H2O2 group (74.01±2.21)% was much lower than CR + H2O2 group (97.26±1.92)% (P＜0.05). After pretreating with H2O2, TUNEL staining showed the apoptosis cells of CR+H2O2 group decreased significantly contrasted with H2O2 group. The immunofluorescence double staining results showed that SIRT3was a mitochondria protein. Western-blot showed the expression of SIRT3 in CR group (6857±157) (P＜0.05) increased and decreased in H2O2 group (3786±160) (P＜0.05) contrasted with control group (5256±143). The expression of SIRT3 in CR + H2O2 group(5056±121)(P＜0.05)increased contrasted with H2O2 group(3786±160). We also detected that Caspase-3 in H2O2 group(8499±426)(P＜0.001 )was much higher than control group than (5342±420), but in the CR + H2O2 group (5750±438 ) theexpression of Caspase-3 was much lower than H2O2 group(8499±426) (P＜0.001). RT-PCR also showed that the expression of SIRT3 in CR group (7214±148) increased and decreased in H2O2 group (4807±143 ) (P＜0.05) contrasted with control group (6204 ± 134 ). The expression of SIRT3 in CR + H2O2 group (6195 ± 166) increased contrasted with H2O2 group (4807 ± 143 ) ( P＜0.05). Conclusion CR causes anti-oxidative injury and has apoptotic effects in PC12 cell. It up-regulates the expression of SIRT3 and the effects of CR-SIRT3 can prevent PC12 cell from H2O2-induced apoptosis. And SIRT3 may be a novel molecule of regulating target in the delay of neuronal senescence.     

三氧化二砷对兔晶体上皮细胞增殖的影响及其机制的实验研究

目的:观察不同浓度的三氧化二砷作用不同时间对兔晶体上皮细胞影响及其作用机理。方法:应用不同浓度的三氧化二砷作用于兔晶体上皮细胞后,观察三氧化二砷对兔晶体上皮细胞生长状态的影响;用噻唑蓝(MTT)比色法、透射电镜技术观察其对兔晶体上皮细胞增殖的影响;应用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)及流式细胞技术检测三氧化二砷对兔晶体上皮细胞凋亡的诱导作用。结果:不同浓度的三氧化二砷作用于兔晶体上皮细胞有明显的时间和剂量依赖性。三氧化二砷作用后细胞生长明显受抑制,并出现明显的凋亡特征性改变;8μmol/L三氧化二砷作用24小时、48小时及72小时后TUNEL法可检测到细胞凋亡水平显著性升高,流式细胞仪分析显示,兔晶体上皮细胞在G1峰前出现明显的凋亡峰。结论:三氧化二砷可明显抑制兔晶体上皮细胞的生长,其机理主要是诱导兔晶体上皮细胞凋亡。     

三氧化二砷联合阿霉素对人淋巴瘤细胞增殖及凋亡的影响

目的：探讨三氧化二砷（As2O3）联合阿霉素（adriamycin, ADM）对淋巴瘤细胞增殖及凋亡的影响。方法：将Raji细胞分为实验组与对照组,实验组分为1 μmol/L As2O3组、2 μmol/L As2O3组、ADM组、1 μmol/L As2O3与ADM联合组、2 μmol/L As2O3与ADM联合组。用As2O3和ADM干预不同时间的人淋巴瘤细胞株Raji,采用Wright-Giemsa染色法观察细胞凋亡的形态学变化;采用噻唑蓝比色法检测As2O3联合ADM对淋巴瘤细胞的增殖抑制作用;流式细胞术检测As2O3联合ADM对淋巴瘤细胞凋亡率及淋巴瘤细胞内ADM荧光强度的影响;半定量聚合酶链反应检测As2O3和ADM对淋巴瘤细胞p53表达的影响。结果：As2O3联合ADM作用细胞24h后,可观察到细胞出现凋亡形态学改变。与As2O3和ADM单药组相比,As2O3联合ADM可增强对Raji细胞的抑制率,差异有统计学意义（P＜0.05）,其作用呈浓度和时间依赖性;As2O3与ADM联合组与单药组相比,Raji细胞的凋亡率差异具有统计学意义（P＜0.05）;As2O3和ADM单药组及联合组p53的表达水平均低于对照组,差异有统计学意义（P＜0.05）;另外,加入1 μmol/L和2 μmol/L As2O3后细胞内ADM的荧光强度分别为18.53和18.12,分别增加0.056倍和0.023倍,差异无统计学意义（P＞0.05）。结论：As2O3和ADM单药及两种药物联合体外均可抑制淋巴瘤细胞增殖,诱导凋亡和下调突变型p53;As2O3和ADM联合体外有协同抗淋巴瘤作用;As2O3对Raji细胞内ADM浓度无显著影响;As2O3可增强淋巴瘤细胞的化疗敏感性,其下调突变型p53的表达可能是其分子水平的作用机制之一。     

Survivin反义寡核苷酸逆转顺铂诱导的人肺腺癌细胞凋亡耐受作用

目的 探讨survivin反义寡核苷酸逆转人肺腺癌细胞对顺铂诱导的细胞凋亡耐受的效应.方法 常规体外培养A549/CDDP细胞,以脂质体包裹的survivin反义寡核苷酸(ASODN)体外转染细胞.采用二苯胺反应法(DPA)测定DNA片段化、激酶法检测Caspase-3酶活性及流式细胞术检测细胞凋亡率(AI),评价细胞凋亡程度.采用MTT法测定细胞存活率和生长抑制率,计算半效抑制浓度(IC50)及耐药倍数(RI).结果 单用ASODN转梁组DNA片段化比率、细胞凋亡率和Caspase3相对活性分别增高至(43.55±6.07)%、(34.03±2.25)%和(1.1298±0.2502),和各对照组相比较,均有显著性变化(P＜0.05);而ASODN和CDDP联用组上述凋亡指标增高更为明显,分别达(76.73±2.04)%、(65.85±5.47)%和(1.6805±0.2758)(P＜0.05).转染组A549/CDDP细胞生长抑制显著,单用转染组和联合CDDP组生长抑制率分别增高达59.3%和83.7%(P＜0.05).3.ASODN转染后CDDP对细胞IC50由(225.03±10.59)μmol/L减低至(158.84±4.256)μmol/L,其RI由11.9减为8.39.结论 Survivin反义寡核苷酸体外转染可降低耐顺铂人肺腺癌细胞的凋亡阈值,从而较大程度上逆转其对顺铂诱导的细胞凋亡耐受.     

基于小鼠黑色素瘤肺转移模型探讨丙泊酚上调DR5表达发挥抑瘤作用的研究

  目的  恶性黑色素瘤是一种恶性化程度高、易转移的由黑色素细胞恶性增殖引起的皮肤癌。本研究旨在探讨丙泊酚对小鼠B16F10黑色素瘤肺转移的抑制作用，以期为丙泊酚是否更适用于黑色素瘤相关手术麻醉提供一定的理论基础。  方法  体外细胞实验采用CCK-8方法考察不同浓度的丙泊酚对B16F10细胞增殖的抑制作用；流式细胞术检测不同实验组B16F10细胞凋亡率；Western blotting法检测各实验组DR5蛋白表达的变化。体内实验采用尾静脉注射B16F10细胞方法建立模型组，将C57BL/6J雄性小鼠24只, 采用随机数字表法分为4组, 即正常对照组, 黑色素瘤肺转移模型组, 丙泊酚50 mg/kg、10 mg/kg剂量组, 每组6只，观察腹腔注射丙泊酚对小鼠肺组织肿瘤转移结节的影响。  结果  体外实验结果显示，对照组与10 μmol/L、50 μmol/L、100 μmol/L 3个浓度实验组于72 h时的细胞相对增殖能力(490 nm OD值)分别为0.91±0.03、0.81±0.02、0.71±0.02和0.61±0.02；100 μmol/L丙泊酚处理B16F10细胞在24 h、48 h和72 h三个时间点的细胞凋亡率分别为(8.09±1.01)%、(26.04±1.10)%、(32.94±1.30)%，100 μmol/L丙泊酚处理B16F10细胞72 h，DR5蛋白表达显著上调。体内实验证实，50 mg/kg剂量的丙泊酚腹腔注射可以显著抑制肺转移黑色素瘤的结节数，且免疫组化结果显示，丙泊酚组肺组织中DR5的表达显著增加。  结论  丙泊酚能够抑制小鼠肺转移黑色素瘤，其机制可能与上调DR5蛋白表达相关。      

二苯乙烯苷对抗MPP^＋诱导的PC12细胞凋亡的作用

目的：探讨不同浓度的二苯乙烯苷（TSG）对1-甲基-4苯基吡啶离子（MPP＋）诱导的PC12细胞损伤的保护作用.方法：MTT比色试验检测细胞活性; Hoechst33258染色观察凋亡细胞核形态; 流式细胞仪（FCM）检测细胞凋亡比例; TUNEL酶标记法检测凋亡细胞断裂的DNA片段.结果：1,5,10μmol/LTSG对MPP＋诱导的PC12细胞损伤具有一定的保护作用.与MPP＋组相比,1,5,10μmol/LTSG预处理组细胞活力分别上升为（57.8±1.2）%（P〈0.05）,（74.3±2.7）%（P〈0.05）,（86.8±2.0）%（P〈0.05）.Hoechest33258染色发现MPP＋组较多胞核出现固缩凝集,而TSG处理组预处理组则分别减少为（31.6±2.3）%,（22.4±1.8）%,（13.4±1.1）%（P〈0.05）.FCM检测也提示TSG预处理可降低细胞凋亡百分率.TUNEL染色显示MPP＋组TUNEL阳性细胞（35.3±1.2）%增加,而TSG预处理组TUNEL阳性细胞显著降低为（25.3±1.2）%,（17.3±0.9）%,（11.7±0.9）%.结论：TSG可减轻MPP＋诱导的PC12细胞损伤和凋亡.     

激活视黄醇类X受体抑制轻度氧化低密度脂蛋白诱导的小鼠巨噬细胞系RAW264.7增殖

目的 探讨激活视黄醇类X受体(RXR)对轻度氧化低密度脂蛋白(LoxLDL)诱导巨噬细胞增殖的影响及其作用机制.方法 饥饿处理后的小鼠巨噬细胞系RAW264.7经LoxLDL(20μg/ml)刺激24 h诱导细胞增殖,干预组同时给予不同浓度的RXR特异性激动剂顺式维甲酸9(9-cisRA),四甲基偶氮唑蓝方法检测细胞活力,脱氧尿嘧啶核苷免疫组化方法检测细胞DNA合成,流式细胞仪PI单染法检测细胞凋亡,Western印迹方法检测Nur77和RARB蛋白表达.结果 LoxLDL诱导24 h后,细胞活力和DNA合成(A值)分别升高0.79±0.12和1.81±0.31,RXR的特异性配体9-cisRA能够显著抑制LoxLDL的作用,在10-8mol/L和10-7mol/L浓度分别使细胞活力下降到0.43±0.10和0.26±0.07(P＜0.05),DNA合成升高为1.14±0.43和0.72±0.06(P＜0.05),且对细胞凋亡没有影响.Western印迹检测表明,孤儿核受体Nur77蛋白表达在LoxLDL刺激后显著升高5倍以上,9-cisRA显著下调Nur77表达,同时上调下游靶基因RARB蛋白表达.结论 激活核受体RXR能够显著抑制LoxLDL刺激引起的巨噬细胞增殖,其机制可能与调控RXR/Nur77异源二聚体及其下游靶基因RARβ有关.     

丁苯酞对Aβ25-35诱导的阿尔茨海默病细胞模型的保护作用

目的 研究丁苯酞对Aβ25-35诱导的AD细胞模型的保护作用及其作用机制.方法 取对数生长期的N2a细胞,用20 μmol/LAβ25-35建立阿尔茨海默病细胞模型;实验分为AD组、丁苯酞组和对照组,其中丁苯酞组设立4个药物浓度梯度的亚组,分别为0.1、1、10、100μmol/L丁苯酞.MTT法检测细胞存活率;流式细胞仪检测细胞凋亡率;倒置相差显微镜下观察细胞形态学变化;qRT-PCR检测细胞TNF-β和IL-1β的mRNA含量.结果 与对照组比较:AD组细胞存活率显著下降,细胞凋亡率明显增加,贴壁细胞数量明显减少,突起结构减少,TNF-α和 IL-1β mRNA表达增高(P＜0.05);与AD组比较:丁苯酞组的细胞存活率提高,细胞凋亡率下降,贴壁细胞数量增多且结构较正常,TNF-α和IL-1β的mRNA表达减少(P＜0.05).结论 丁苯酞对Aβ25-35诱导的AD细胞模型具有保护作用,可能与抑制TNF-α和IL-1β等炎性因子的表达有关.     

丁苯酞对Aβ25-35诱导的PC12细胞凋亡的作用及其机制

目的 探讨丁苯酞(NBP)对β淀粉样蛋白(Aβ)诱导的PC12细胞凋亡的保护作用及其机制.方法 不同浓度的NBP(0.1、1.0、10、100 μmol/L)作用到经Aβ诱导或未经诱导的PC12细胞上,然后分别采用细胞存活率检测(MTT)及PI单染、透射电镜方法检测细胞凋亡,采用western印迹法检测Bcl-2和Cyt-C蛋白的表达,进而观察NBP对Aβ25-35诱导的PC12细胞凋亡的保护作用.结果 MTT显示不同浓度(0.1、1.0、10、100 μmol/L)NBP组细胞存活率分别为76.5%±1.1%、84.2%±1.3%、89.5%±1.3%、81.9%±1.9%,明显高于模型组(71.7%±1.4%),其中10 μmol/LNBP组最明显,差异有统计学意义(P＜0.05);流式细胞学结果显示10 μmol/L NBP组细胞凋亡率明显低于模型组(P＜0.05);电镜下模型组细胞凋亡特征明显,10 μtmol/L NBP组细胞形态接近正常;Western印迹显示:10 μmol/L NBP组Bcl-2蛋白表达增加,而模型组Bcl-2表达明显减少(P＜0.05);模型组胞质Cyt-C表达最强,而10μmol/L NBP组有较弱的Cyt-C表达(P＜0.05).结论 NBP对Aβ诱导的PC12细胞凋亡有保护作用,其机制可能和NBP增加Bcl-2蛋白水平,抑制Cyt-C从线粒体的释放有关.     

人参皂苷Rg1对β淀粉样蛋白诱导细胞凋亡的影响

目的 探讨人参皂苷Rg1对β淀粉样蛋白(Aβ)诱导细胞凋亡的影响及其机制.方法 利用中国仓鼠卵巢瘤细胞系(CHO)采用MTY方法筛选人参皂苷Rgl抗Aβ细胞毒性的有效浓度.通过转染突变型(M146L)PS1基因的CHO细胞(PS1M146L),利用免疫荧光染色、蛋白质免疫印迹方法,探讨人参皂苷Rg1对PS1M146L细胞中Aβ42和凋亡效应基因半胱氨酸蛋白水解酶(caspase-3)活性蛋白表达的影响,通过脱氧核苷酸转移酶介导的dUTP缺口末端标记法和Annexin V-FITC/PI染色流式细胞仪检测,观察人参皂苷Rg1对Aβ促细胞凋亡的影响.结果 加用25、50、100 μmol/L人参皂苷Rg1的CHO细胞活性明显高于未加用组(均P＜0.05).在转染突变型PS1M146L的CHO细胞中,加用人参皂苷Rg1作用24 h后早期细胞凋亡数(10.11.11±0.76)较未加用组(15.01±1.46)少,差异有统计学意义(P＜0.05);同时Aβ42和caspase-3活性片段的蛋白质表达量较未加用组细胞亦不同程度降低(均P＜0.05).结论 人参皂苷Rg1可能通过降低Aβ42生成和降低caspase-3蛋白质表达抑制Aβ诱导的细胞凋亡.     

三氧化二砷联合维A酸对恶性黑色素瘤细胞株A375的增殖、迁移能力影响及其可能分子机制的探讨简

目的 研究三氧化二砷（As2O3）联合维A酸对恶性黑色素瘤细胞株A375的增殖、迁移能力影响及其可能的分子机制.方法 常规培养黑色素瘤细胞株A375,分为空白对照组（不含药物的DMEM处理）、As2O3组（10μmol/LAs2O3）、全反式维A酸（at-RA）组（10 μmol/L at-RA）、As2O3＋at-RA组（10.μmol/LAs2O3联合10 μmol/L at-RA）.MTT法检测细胞活力,划痕试验检测细胞迁移能力,Western-blot法检测MMP-2、Caspase-2的蛋白水平.结果 ①As2O3＋at-RA组的细胞活力为（35.8±7.3）％,低于As2O3组的（62.4±10.3）％和at-RA组的（59.3±11.3）％,差异有统计学意义（P＜ 0.05）;②As2O3＋at-RA组的迁移能力相对值为（21.3±6.3）％,低于As2O3组的（55.4±9.5）％和at-RA组的（57.1±10.4）％,差异有统计学意义（P＜ 0.05）;③As2O3＋at-RA组的Caspase-2蛋白含量为（274.1±42.4）％,高于As2O3组（175.2±29.4）％和at-RA组（181.4±23.8）％;As2O3＋at-RA组的MMP-2蛋白含量为（36.2±8.2）％,低于As2O3组（64.2±10.4）％和at-RA组（62.7±9.4）％.结论 As2O3和at-RA联合应用能够抑制黑色素瘤细胞株A375的增殖和迁移能力,这一作用可能是通过上调Caspase-2、下调MMP-2来发挥的.     

Verapamil modulating arsenic trioxide-induced QT interval rolongation in guinea pig

Objective To investigate therapeutic action of verapamil on QT prolongation induced by arsenic trioxide (As2O3) in guinea pig and to further explore its possible mechanism. Methods Different doses of As2 O3 was infused intravenously to observe the changes of QT interval on the electrocardiogram (ECG) at different times in guinea pig. Patchclamp technique and laser scanning confocal microscopy were utilized to study the action of As2 O3 on action potential duration (APD),L-type calcium current (ICa-L ), rapid delayed rectifier potassium current (IKr) and intracellular calcium concentration ([Ca2+]i) of guinea pig myocytes. At the same time, verapamil was applied preliminarily to evaluate effects of verapamil on changes of the above index induced by As2O3. Results Intravenous administration of As2 O3 at the dose of 1.6mg/kg and 0.8mg/kg prolonged QT interval on ECG obviously in guinea pig hearts dose dependently and time dependently. QTc (corrected QT interval) was progressively prolonged in the 2-hour period of intravenous infusion of 1.6mg/kg As2O3 from (328.5 ± 30.9)ms of control to (388.4 ± 31.3)ms at 2h following As2O3 ( P ＜ 0.01) .When verapamil was pretreated for 5min, then 1.6mg/kg As2 O3 was added, the results showed that QTe was shorter in verapamil-treatment group (357.3±21.4)ms than that in As2O3 group (388.4±31.3)ms (P＜0.05) at 2h. Confocal experiments showed that in normal Tyrode solution, As2 O3 (1μmol/L and 10μmol/L) had no obvious effects on resting [Ca2+]i (P＞0.05) in guinea pig cardiomyocytes, however, 10μmol/L As2 O3 could markedly enhance [Ca2+]i increase induced by KCl 60mmol/L and the peak value increased from 903.4±369.4 to 1674.6±563.2 ( P＜0.05). The action of elevating [Ca2+]icould be blocked by 10μmol/L verapamil incompletely. The patch-clamp studies indicated that As2 O3 at concentration of 10μmol/L prolonged APD50 from (263.6 -±75.2)ms to (523.9±47.8)ms (P＜0.01) and APD90 from (277.5 ± 77.5)ms to (536.3 ±49.6)ms (P ＜0.01) ,and increased ICa-L from (- 6.0±1.5)pA/pF to (-8.7±2.0)pA/pF (P＜0.01) at 0mV and also reduced IKr from (6.7±1.8)pA/pF to (4.5±1.8)pA/pF (P＜0.05). However, 10μmol/L verapamil could modulate prolonging APD50 from (523.9 ± 47.8) ms to (340.4±83.8) ms ( P＜0.01) and APD90 from (536.3 ±49.6)ms to (348.9 ± 85.5)ms (P＜0.01) and correct increasing Ica-L induced by 10μmol/L As2O3 from (-8.7±2.0)pA/pF to ( - 6.6±1.4)pA/pF (P＜0.05) at 0mV. Conclusion As2O3 could induce prolongation of the QT interval on the ECG in guinea pig hearts and the ionic mechanism is associated with increasing Ica-L and inhibiting IKr/HERG. Verapamil may be useful in normalizing QT prolongation during As2 O3 therapy by decreasing Ica-L and [Ca2+]iof ventricular myocytes in guinea pig.     

Apoptosis of Human Trabecular Meshwork Cells Induced by Transforming Growth Factor-p2 in vitro

Summary: Whether transforming growth factor-β2 (TGF-β2) induces apoptosis of human trabecular meshwork cells was investigated in vitro. Cultured 3-5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF-β2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy.DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44) %, (4.43±1.17) % and (9. 60±2.05) % respectively with different concentrations [1 ng/ml (P＜0. 05), 3.2 ng/ml (P＜0.01)] of TGF-β2 with the difference being significant between experimental group and control group[(1. 41±0.34) %]. It was concluded that TGF-β2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.     

Nuclear matrix associated protein PML: an arsenic trioxide apoptosis therapeutic target protein in HepG2 cells

Objective To investigate arsenic trioxide(As2O3)-induced apoptosis and the effects on cell nuclear matrix related protein promyelocytic leukaemia(MPL).Methods HepG2 cells were cultured in MEM medium and treated with 0.5,2.5 and 10μmol/L As2O3 for either 24 h or 96 h at each concentration.In situ terminal deoxynucleotidyl trangferase(TdT) labeling (TUNEL)and DNA ladders were used to detect apoptosis.Confocal microscopy and Westem blotting were used to observe the expression of PML.Results The growth rates of HepG2 cells were slower in the As2O3 treated than the untreated control group.DNA ladder and TUNEL positive apoptotic cells could be detectedin As2O3 treated groups.The expression of PML decreased in HepG2 cells with 2 μmol/L As2O3 treatment,Confocal images demonstrated that the expression of PML protein in HepG2 cell nuclei decreased after treatment with 2μmol/L As2O3,and micropunctates characteristic of PML protein in HepG2 cell nuclei disappeared after treatment with 5μmol/L As2O3.Conclusions Our results show that arsenic trioxide can significantly inhlbit the growth of HepG2 cellsin vitro.As2O3 induces apoptosis in HepG2 tumor cells in a time and concentration dependent manner.As2O3 may degrade the PML protein in HepG2 cell nuclel.The decreased expression of PML in As2O3 treated tumor cells is most likely to be caused by apoptosis.Nuclear matrix associated protein PML could be the target of As2O3 therapy.     

绿茶提取物EGCG对视网膜神经节细胞氧化应激损伤的防护作用

目的 研究绿茶提取物表没食子儿茶素没食子酸酯(EGCG)对视网膜神经节细胞(RGC-5)氧化应激损伤的防护作用.方法 利用外源性活性氧(H2O2)造成RGC-5细胞的氧化应激损伤,同时应用EGCG进行保护.通过末端脱氧核苷酸转移酶介导的脱氧尿嘧啶核苷三磷酸缺口末端标记(TUNEL)验证RGC-5细胞的死亡方式,2',7'-二氢乙啡啶(OHE)染色实验检测细胞内活性氧簇(ROS)生成情况,蛋白质印迹法定量分析细胞核酶(PARP-1)的激活,噻唑蓝(MTT)法分析比较EGCG、维生素E水溶性衍生物(Trolox)和PARP-1抑制剂NU1025对RGC-5细胞的保护作用.结果 H2O2以浓度和时间依赖的方式降低RGC-5细胞活性,500μmol/L H2O2在24 h可以导致RGC-5细胞活性下降50%.H2O2可以导致RGC-5细胞凋亡,并显著增加细胞内ROS的生成,上调细胞核酶PARP-1的表达;而预先给予EGCG可以明显减少RGC-5细胞凋亡和细胞内ROS生成.MTT实验显示EGCG可以使氧化应激损伤中RGC-5细胞活性恢复到87%,而Trolox和NU1025可以分别提高细胞活性到62%和71%.结论 EGCG能够有效地防护氧化应激对视网膜神经节细胞的损伤,是一种极具开发潜力的神经保护性药物.     

3-溴丙酮酸增强肝癌细胞对顺铂敏感性的作用

目的探讨3-溴丙酮酸（3-BP）增强肝癌细胞对顺铂敏感性的作用及其可能机制。方法MTT法检测3-BP、顺铂对HepG2、
SMMC7721细胞的增殖抑制作用。选择低于半数抑制浓度（IC50）的100 μmol/L的3-BP和8 μmol/L的顺铂单独或联合处理细
胞,PI 单染流式细胞术检测细胞凋亡,caspase 3 活性检测试剂盒测定caspase-3 的活性变化,ATP检测试剂盒测定细胞内ATP
水平,Western blot 检测XIAP、PARP蛋白的表达。结果3-BP 在50~400 μmol/L 浓度范围内对HepG2、SMMC7721 细胞具有
明显的增殖抑制作用（P<0.01）,作用48 h 的IC50分别为238.9±13.9 μmol/L、278.7±11.7 μmol/L;顺铂在2~32 μmol/L 浓度范围
内对HepG2、SMMC7721细胞具有明显的增殖抑制作用（P<0.01）,作用48 h的IC50分别为16.4±0.9 μmol/L、20.9±1.8 μmol/L。
100 μmol/L 3-BP与8 μmol/L顺铂联合作用于HepG2、SMMC7721细胞48 h 的增殖抑制率分别为（60.6±2.2）%、（56.8±2.3）%,明
显高于对照组和单独用药组（P<0.01）。100 μmol/L 3-BP与8 μmol/L顺铂联合作用于HepG2、SMMC7721细胞48 h 的凋亡率
分别为（51.1±4.3）%、（46.5±3.9）%,较单用3-BP、顺铂的凋亡率明显提高（P<0.01）。结论3-BP 能增强肝癌细胞HepG2、
SMMC7721对顺铂诱导的凋亡的敏感性,其机制可能是通过引起细胞内ATP缺乏、下调XIAP蛋白的表达以及增加caspase-3的
活性。     

姜黄素对过氧化氢诱导内皮细胞凋亡及衰老的影响

目的 探讨姜黄素对过氧化氢(H2O2)诱导人脐静脉内皮细胞(HUVECs)自噬、凋亡及衰老的影响.方法 体外培养HUVECs,分为对照组(只给予培养基),模型组(给予60 μmol/L H2O2),姜黄素组(给予60μmol/L H2O2+ 10 μmol/L姜黄素),3-MA组[(给予60 μmol/L H2O2+ 10μmol/L姜黄素+1 mmol/L3-甲基腺嘌呤(3-Methyladenine)],培养4d后,DCFH-DA法检测细胞培养液中活性氧(ROS)的含量,流式细胞仪检测细胞凋亡水平,衰老相关β-半乳糖苷酶(SA-β-gal)染色检测细胞衰老程度,Western blot技术检测自噬相关蛋白LC3和p62的表达水平.结果 与对照组相比,模型组ROS含量、凋亡率、SA-β-gal阳性率及p62水平升高(均P＜0.05),而LC3-Ⅱ/Ⅰ比值差异无统计学意义(P＞0.05).与模型组比,姜黄素组ROS含量、凋亡率及SA-β-gal阳性率及p62水平降低(均P＜0.05),但LC3-Ⅱ/Ⅰ比值升高(P＜0.05).与姜黄素组比较,3-MA组ROS含量、凋亡率、SA-β-gal阳性率及p62水平升高(均P＜0.05),但LC3-Ⅱ/Ⅰ比值降低(P＜0.05).结论 姜黄素可减轻H2O2诱导内皮细胞凋亡及衰老,其机制与提高内皮细胞自噬有关.     

依维莫司和LBH589对耐药急性髓系白血病细胞增殖、凋亡及多药耐药相关蛋白表达的影响

目的 探讨雷帕霉素衍生物依维莫司(RAD001)或联合组蛋白去乙酰化酶抑制剂LBH589对耐药急性髓系白血病细胞株HL-60/ADM细胞增殖、凋亡和逆转耐药的影响.方法 采用不同浓度RAD001或联合LBH589处理HL-60/ADM细胞,四甲基偶氮唑盐(MTT)法检测细胞增殖活力,Hoechst33342染色法和AnnexinV-FITC/PI染色流式细胞仪检测细胞凋亡、阿霉素摄取率和多药耐药相关蛋白1(MRP1)表达评估逆转耐药效应.进一步检测处理前后细胞信号通路蛋白变化,探讨其机制.结果 10～50 μmol/L RAD001均能够抑制HL-60/ADM细胞增殖、诱导凋亡,在作用48和72 h时30 μmol/L药物浓度达到最大抑制效果,进一步增加药物浓度细胞增殖抑制率并没有明显增加(P＜0.05).不同浓度RAD001和LBH589联合处理HL-60/ADM细胞,没有明显的协同抑制增殖作用[协同指数(CI)≥1.0].10 μmol/LRAD001处理HL-60/ADM细胞可以明显下调细胞表面MRP1表达(93.90%±4.20%比79.10%±3.28%,P＜0.05),提高HL-60/ADM细胞阿霉索蓄积率(8.53%±0.68%比15.37%±1.46%,P＜0.01).深入机制研究表明,RAD001可以阻断PI3K/AKT/mTOR信号通路活性,通过上调P53抑制MRP1蛋白的活性.结论 RAD001与LBH589联合没有协同抑制HL-60/ADM细胞增殖作用.但RAD001单药能够有效抑制HL-60/ADM细胞增殖,诱导凋亡,通过阻断PI3K/AKT/mTOR信号通路抑制细胞MRP1蛋白的表达,提高细胞阿霉素摄取率而逆转耐药.

Abstract：

Objective To investigate the effects of everolimus ( RAD001 ) or plus panobinostat (LBH589) on the proliferation, apoptosis and drug resistance in chemoresistant acute myeloid leukemic cells.Methods HL-60/ADM cells were treated with RAD001 alone or with LBH589.Proliferation and apoptosis were evaluated by 3-( 4, 5 ) -dimethylthiahiazo ( -z-y1 )-3, 5-di-phenytetrazoliumromide ( MTT )assay, Hoechst33342 and AnnexinV-FTTC/PI stain.The altered expressions of multidrug resistance-associated protein 1 ( MRP1 ) and intercellular adriamycin accumulation were analyzed by flow cytometry.The change in protein level was analyzed by Western blot.Results Effective proliferative inhibition and apoptotic induction in HL60/ADM cells were observed in the treatment of 10 -50 μmol/L RAD001.The maximal effect was shown for the concentration of 30 μmol/L RAD001 at 48 and 72 hours.The inhibition ratio remained unchanged with the adjustment of drug doses (P ＜0.05).Moreover, there was no synergistic effects in the treatment with different concentration of RAD001 and LBH589 (CI ≥ 1.0).A down-regulation of MRP1 (93.9% ±4.2% vs 79.10% ± 3.28% ) and an up-regulation of adriamycin ( 8.53% ± 0.68% vs 15.37% ± 1.46% ) were induced by the treatment with 10 μmol/L RAD001 ( both P ＜ 0.01 ).RAD001 inhibited the p53-dependent expression of MRP1 via an inhibition of phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway.Conclusion The combined treatment of RAD001 and LBH589 has no synergistically inhibitory effect on HL60/ADM cells.But the sole treatment of RAD001 may inhibit proliferation, induce apoptosis and accumulate intercellular adriamycin through a down-regulated expression of MRP1 in HL60/ADM cells via an inhibition of PI3K/AKT/mTOR signaling pathway.     

氧化砷诱导食管癌细胞系细胞凋亡的研究

目的:研究食管癌细胞系SHEEC1以三氧化二砷（AS2O3）诱导细胞凋亡光镜和电镜的形态改变。方法：SHEEC1细胞常规培养在199培养基,在5umoL/LAs2O3作用72h收获细胞以苏木精－伊红染色,TUNEL标记,透射电镜和流式细胞仪检查。结果：光镜下凋亡细胞核致密,染色质片段化,胞浆红染,TUNEL标记阳性,流式细胞仪检查出现凋亡峰（亚G1／G 0峰）。电镜下可见两种死亡细胞：一种是核固缩,有染色质凝集靠近核膜等典型凋亡改变;另一种细胞浆退行性变及细胞坏死改变。结论：食管癌细胞系SHEEC1可以用As2O3引起凋亡,提示其可以作为治疗食管癌的辅助药物。