内含子源性microRNA对内皮型一氧化氮合酶表达及血管内皮细胞增殖的作用

前期工作表明,内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)第4内含子中的27碱基 (nucleotide,nt)重复序列是27-nt microRNA的来源,并对eNOS具有重要的调节作用．为进一步探讨该内含子源性27-nt microRNA参与调节eNOS表达的分子机制及其在内皮细胞增殖中的可能作用,通过构建27-nt microRNA高表达质粒,用脂质体将该质粒转染人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC),Western blot和RT-PCR检测该细胞系中eNOS蛋白和mRNA表达情况以及胞核转录因子的表达改变,并观察HUVEC增殖的变化情况．结果发现：27-nt microRNA 高表达能降低eNOSmRNA的水平和蛋白质表达;同时对转录因子Sp1、Ap1的蛋白质表达也产生了不同程度的抑制作用;转染后细胞的生长速度比未转染的细胞明显减慢,尤其转染了27-nt microRNA的双倍长度突变体(pEGP-mut-54nt-mi)质粒的HUVEC,其生长倍增时间比正常对照组明显延长达49.4%．结果表明,27-nt microRNA明显抑制eNOS蛋白及其mRNA表达,同时 HUVEC增殖受到明显抑制,转录因子Sp1 和Ap1 在27-nt microRNA对eNOS的表达调节中起重要作用．实验提示,内含子源性microRNA与转录因子共同参与对内皮细胞增殖及其相关性基因的表达调节,可能是众多真核细胞中某些疾病相关性基因表达自我调节的重要机制之一．     

人参皂苷Rb1调节脑微血管NOS/NO改善脑细胞膜通透性

目的:探究人参皂苷Rb1在脂多糖(LPS)诱导的人脑微血管细胞模型中对脑细胞膜通透性的影响。方法:采用噻唑蓝比色法(MTT)及乳酸脱氢酶(LDH)释放检测不同浓度人参皂苷Rb1对LPS刺激人脑微血管内皮细胞系(HBMEC)存活率的影响;采用EVOM法检测HBMEC细胞通透性;采用Griess法、ELISA法及DAF-FM DA荧光探针分别检测NO、ONOO~-含量及eNOS、iNOS活性;Western blot法检测p-eNOS(ser1177)、iNOS蛋白的表达水平。结果:与正常对照组比较,LPS(5μg/m L)可明显降低HBMEC细胞存活率(P0.01);与LPS组比较,随着人参皂苷Rb1浓度的增加细胞存活率明显升高,且中、高剂量组(40、80μmol/L)具有显著性差异(P0.05、P0.01);LPS导致HBMEC单层细胞电阻值明显降低(P0.01),人参皂苷Rb1高剂量组(80μmol/L)可显著抑制LPS所致的细胞膜通透性升高(P0.05);LPS可明显降低HBMEC细胞NO含量(P0.01)升高ONOO~-含量(P0.05),人参皂苷Rb1中、高剂量组(40、80μmol/L)较LPS组具有显著升高NO含量,降低ONOO~-含量的作用(P0.05);与正常对照组比较,LPS组可明显降低HBMEC细胞磷酸化eNOS (ser1177)蛋白的表达水平,而显著升高iNOS蛋白表达水平(P0.01)。人参皂苷Rb1高剂量组(80μmol/L)较LPS组可显著升高p-eNOS(ser1177)蛋白的表达水平,降低iNOS蛋白的表达水平(P0.05)。结论:人参皂苷Rb1可有效降低iNOS活性,升高eNOS活性继而减少NO水平及ONOO~-含量,显著降低LPS导致的脑细胞膜通透性升高。     

GABPα抑制鱼藤酮诱导的PC12细胞氧化应激及凋亡

[目的]明确GABPα在鱼藤酮诱导的PC12细胞氧化应激及凋亡中的作用。[方法]利用鱼藤酮制备PC12细胞氧化应激模型,通过脂质体转染的方法在PC12细胞中过表达GABPα。利用Western Blot检测GABPα的表达,通过可见分光光度法和微量法分析氧化应激标记物NO和MDA水平,抗氧化标记物GSH的水平和SOD的活性,利用流式细胞仪检测细胞凋亡。[结果]与对照组相比,鱼藤酮处理后,PC12细胞的活力明显降低(46.71%±1.7%vs 99.88%±0.649%),NO和MDA的水平明显增高(0.285±0.004 vs 0.151±0.003,0.115±0.003 vs 0.044±0.002),GSH的水平及SOD的活性显著下降(11.53±0.572 vs 22.86±1.338,0.161±0.008 vs 0.315±0.026),细胞的凋亡明显增加(30.26%±2.359%vs 3.037%±0.043%)。在PC12细胞过表达GABPα并用鱼藤酮处理后,与空载体组相比,PC12细胞的活力明细增高(62.21%±2.344%vs 47.65%±3.228%),氧化...     

熊果酸对ox-LDL诱导的人脐静脉血管内皮细胞NQO1表达的影响(英文)

目的:研究熊果酸对经氧化性低密度脂蛋白(ox-LDL)干预后人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)醌还原氧化酶1表达的影响,以进一步探讨熊果酸抗动脉粥样硬化的机制。方法:体外培养人脐静脉内皮细胞,进行分组处理,每组n=5。对照组,不加任何处理;ox-LDL组,加入ox-LDL培养24h,终浓度为20mg/L;ox-LDL+低浓度熊果酸组,先加入ox-LDL(浓度20mg/L)孕育半小时,然后与熊果酸(浓度1.5μmlo/L)共同培养24h;ox-LDL+高浓度熊果酸组,先加入ox-LDL(浓度20mg/L)孕育半小时,然后与熊果酸(浓度4.5μmlo/L)共同培养24h;采用MTT试验测定细胞吸光度值,检测熊果酸对ox-LDL损伤的保护作用,采用RT-PCR法检测NQO1mRNA的表达,采用Western blot法检测NQO1蛋白的表达。结果:熊果酸减弱ox-LDL对HUVECs的损伤作用;ox-LDL组NQO1mRNA的表达量(0.624±0.009)明显高于对照组(0.521±0.007),P0.01。熊果酸呈浓度依赖性的提高NQO1mRNA的表达量(ox-LDL+低浓度熊果酸组vs ox-LDL组:0.722±0.058 vs 0.624±0.009,P0.01;ox-LDL+高浓度熊果酸组vs ox-LDL组:0.826±0.059 vs 0.624±0.009,P0.01)。ox-LDL组NQO1蛋白的表达量(0.624±0.009)明显高于对照组(0.521±0.007),P0.01。熊果酸呈浓度依赖性的提高NQO1蛋白的表达量(ox-LDL+低浓度熊果酸组vs ox-LDL组:0.710±0.058 vs 0.574±0.024,P0.01;ox-LDL+高浓度熊果酸组vs ox-LDL组:0.831±0.034 vs 0.574±0.024,P0.01)。结论:熊果酸可上调ox-LDL诱导的人脐静脉血管内皮细胞NQO1的表达,表明其可能具有抗氧化应激及抗动脉粥样硬化的作用。     

车前子多糖对氧化型低密度脂蛋白致人脐静脉内皮细胞损伤的保护作用

利用ox-LDL对培养的人脐静脉内皮细胞(HUVECs)造成脂质过氧化损伤模型,探讨车前子多糖(PSP)对HUVECs的保护作用及其可能机制。用MTT和化学方法,从细胞水平观察ox-LDL对细胞增殖活性、细胞上清液中一氧化氮(NO)含量、胞内一氧化氮合酶(NOS)活性、丙二醛(MDA)含量、SOD活力;用ELISA和RT-PCR技术,从分子水平观察ICAM-1和凋亡相关基因mRNA的表达,探讨不同浓度的PSP(25 mg/L、50 mg/L、100 mg/L)对上述指标的影响。结果表明ox-LDL导致HUVECs损伤,而伴随PSP浓度的增加,HUVECs增殖活性呈明显升高趋势(P0.05);而PSP使HUVECsMDA含量显著下降(P0.05),SOD、NO和NOS水平明显升高(P0.05),ICAM-1、c-myc mRNA和p53 mRNA表达降低,表明PSP对受损的内皮细胞具有保护作用,其机制可能与抑制ox-LDL诱导的ICAM-1、c-myc mRNA和p53 mRNA的表达有关。     

miR-608靶向PCDHGA9调控胃癌细胞凋亡的机制

[目的]探讨miR-608调控胃癌细胞SGC-7901凋亡的潜在机制。[方法]将胃癌中表达失调的miRNA以mimics的形式在胃癌细胞SGC-7901中过表达,检测细胞的凋亡水平。将能够调节凋亡的miRNA在多种胃癌细胞中敲低或过表达,检测这些胃癌细胞的凋亡水平。miRDB和荧光素酶报告实验验证miRNA的潜在底物。胃癌细胞SGC-7901中过表达或敲低底物后,检测凋亡水平。[结果] miR-608能够抑制胃癌细胞SGC-7901的凋亡水平(26.31±6.54 vs 7.04±2.19,P<0.05)。在胃癌细胞株MKN-1、 MNK-28、MNK-45、HGC-27、SNU-1、SNU-5、 Hs-746T、SGC-7901、SNU-719、KATO 3中过表达miR-608后,这些胃癌细胞株的凋亡水平均显著下降(P<0.05)。胃癌细胞SGC-7901中过表达miR-608后,PCDHGA9的表达水平显著下降(1.42±0.31 vs 0.42±0.10,P<0.05)。此外,miR-608靶向PCDHGA9的3′端非编码区(100±7 vs 23±5,P&l...     

重组福安泰-03抑制人脐静脉血管内皮细胞的迁移和增殖

研究重组福安泰-03(recombinant Fuantai-03,rFAT-03)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)迁移和增殖的影响.聚碳酸酯膜小室趋化运动模型(transwell model)检测rFAT-03对HUVECs迁移能力的影响;MTT法检测rFAT-03对HUVECs和多种人癌细胞生长的影响;荧光显微镜观察rFAT-03作用下HUVECs的形态变化;膜联蛋白V-异硫氰酸荧光素(annexin V-fluorescein isothiocyanate,annexinV-FITC)双染检测rFAT-03对HUVECs早期凋亡的影响;流式细胞术分析rFAT-03对HUVECs周期及凋亡的影响;Western印迹检查rFAT-03对HUVECs血管内皮细胞生长因子(VEGF)、Bax和Bcl-2表达的影响.结果显示,rFAT-03明显抑制HUVECs细胞的迁移和增殖,其抑制效果与剂量和作用时间相关.0.20mg/mL恩度(endostar),0.10、0.20mg/mLrFAT-03作用HUVECs24h,对细胞迁移的抑制率分别为32.0%、32.6%、57.1%(P0.01).0.20mg/mL恩度,0.05、0.10、0.20mg/mLrFAT-03作用HUVECs72h,其对细胞生长的抑制率分别为40.9%、63.7%、69.3%、87.0%.但rFAT-03对多种人癌细胞株的生长却无明显影响.rFAT-03处理HUVECs24h,细胞的早期凋亡率增加(P0.05).rFAT-03阻滞HUVECs于G0/G1期,并呈现典型的凋亡峰.终浓度为0.05、0.10、0.20mg/mLrFAT-03作用于HUVECs24h,G0/G1期细胞指数分别为63.4%、67.5%和75.7%(对照组为62.1%),凋亡指数分别为4.2%、8.5%和10.3%.rFAT-03下调HUVECs的VEGF和抑调亡基因Bcl-2的表达,上调促凋亡基因Bax的表达,其效果与剂量相关.结果提示,rFAT-03明显抑制HUVECs细胞的迁移和增殖,诱导其凋亡,它的这些作用与其下调VEGF、Bcl-2表达,上调Bax表达密切相关.     

莪术油对Hela细胞迁移及侵袭的初步实验研究

目的:探讨莪术油对宫颈癌细胞系Hela细胞迁移和侵袭的影响。方法:宫颈癌细胞系Hela细胞培养后分实验组及对照组,实验组以125μg/m L浓度的莪术油作用48 h,对照组细胞不进行任何处置。细胞划痕实验检测莪术油对Hela细胞迁移的影响;Transwell小室实验检测莪术油对Hela细胞侵袭的影响。结果:0 h处理后实验组、对照组划痕宽度无统计学差异(P0.05);48 h后实验组划痕宽度明显大于对照组的划痕宽度(4.33±0.58 m vs 2.17±0.29 m,P0.05)。实验组48 h后的穿透细胞数明显少于对照组(26.2±1.3个vs 62.2±2.3个,P0.05)。结论:莪术油可抑制宫颈癌细胞的迁移与侵袭。     

整合素αvβ3对MDA-MB-231BO乳腺癌细胞AKT信号通路的调控

旨在探究整合素αvβ3的单克隆抗体LM609在BSP不同表达水平的乳腺癌细胞中对AKT(蛋白激酶B)信号通路的影响。利用免疫细胞化学法检测BSP不同表达水平的乳腺癌细胞中整合素αvβ3的表达量。BSP基因沉默乳腺癌MDA-MB-231BO细胞,Western blotting在蛋白水平检测磷酸化AKT的表达,MTT试验和细胞划痕试验分别检测细胞增殖、迁移能力的变化。结果显示,与231BO-Scrambled细胞相比,231BO-BSP27细胞中BSP蛋白水平明显降低,抑制率达到(59.43±1.71)%;LM609分别处理两株细胞后,与对照组231BO-Scrambled细胞相比,BSP基因沉默组21BO-BSP27细胞中AKT磷酸化水平下调明显,为(33.78±1.51)%(P<0.01);231BO-BSP27细胞和对照组231BO-Scrambled中细胞的增殖和迁移能力均有不同程度的下降(P<0.05)。LM609能够抑制胞内整合素αvβ3功能的表达,进而对AKT信号通路进行调控,并影响细胞增殖和迁移的发生。     

miR-181a通过调控上皮间质转化过程影响卵巢癌细胞的迁移和侵袭

目的:探讨mi R-181a在卵巢癌细胞中的表达及对卵巢癌细胞的迁移和侵袭的影响和可能机制。方法:采用细胞免疫荧光检测卵巢癌细胞中抗波形蛋白和E-钙粘蛋白的表达,Western blotting检测mi R-181a对抗波形蛋白和E-钙粘蛋白的表达情况的调控;划痕愈合实验检测mi R-181a对卵巢癌细胞迁移能力的影响;Transwell侵袭实验检测mi R-181a对卵巢癌细胞侵袭能力的影响。结果:增加mi R-181a的表达后,EMT过程中的相关蛋白激活水平下调,mi R-181a一定程度上可以抑制卵巢癌细胞的EMT过程;过表达mi R-181a后可以明显影响Vimentin[D1(4.58±0.85)vs(0.29±0.02),P0.05;D5(4.16±0.79)vs(0.29±0.02),P0.05]和E-Cadherin[D1(4.75±0.41) vs.(4.56±0.38),P0.05;D5(2.19±0.18) vs.(4.56±0.38),P0.05]蛋白的表达;COC1细胞的迁移能力随mi R-181a的表达的增高而降低[(19.24±4.31)%vs.(25.95±6.02)%,P0.05;(51.25±8.75)%vs.(73.49±12.54)%,P0.05];过表达mi R-181a后可以一定程度上减弱卵巢癌COC1细胞的侵袭能力[(74.64±8.21)vs(231.98±21.72),P0.05]。结论:mi R-181a通过调控上皮间质转化过程影响卵巢癌细胞的迁移和侵袭行为。     

Biogenesis of short intronic repeat 27-nucleotide small RNA from endothelial nitric-oxide synthase gene

Endothelial nitric-oxide synthase (eNOS) is a constitutively expressed gene in endothelium that produces NO and is critical for vascular integrity. Previously, we reported that the 27-nucleotide (nt) repeat polymorphism in eNOS intron 4, a source of 27-nt small RNA, which inhibits eNOS expression, were associated with cardiovascular risk and expression of the eNOS gene. In the current study, we investigated the biogenesis of the intron 4-derived 27-nt small RNA. Using Northern blot, we showed that the eNOS-derived 27-nt short intronic repeat RNA (sir-RNA) expressed only in the eNOS expressing endothelial cells. Cells containing 10 x 27- or 5 x 27-nt repeats produced higher levels of 27nt sir-RNA and lower levels of eNOS mRNA than the cells with 4 x 27-nt repeats. The 27nt sir-RNA was mostly present within the endothelial nuclei. When the splicing junctions of the 27-nt repeat containing intron 4 in the full-length eNOS cDNA vector were mutated, 27nt sir-RNA biogenesis was abolished. Suppression of Drosha or Dicer diminished the biogenesis of the 27nt sir-RNA. Our study suggests that the 27nt sir-RNA derived through eNOS pre-mRNA splicing may represent a new class of small RNA. The more eNOS is transcribed or higher number of the 27-nt repeats, the more 27nt sir-RNA is produced, which functions as a negative feedback self-regulator by specifically inhibiting the host gene eNOS expression. This novel molecular model may be responsible for quantitative differences between individuals carrying different numbers of the polymorphic repeats hence the cardiovascular risk.     

Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity

Plasma adiponectin level is significantly reduced in patients with metabolic syndrome, and vascular dysfunction is an important pathological event in these patients. However, whether adiponectin may protect endothelial cells and attenuate endothelial dysfunction caused by metabolic disorders remains largely unknown. Adult rats were fed with a regular or a high-fat diet for 14 wk. The aorta was isolated, and vascular segments were incubated with vehicle or the globular domain of adiponectin (gAd; 2 mug/ml) for 4 h. The effect of gAd on endothelial function, nitric oxide (NO) and superoxide production, nitrotyrosine formation, gp91(phox) expression, and endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) activity/expression was determined. Severe endothelial dysfunction (maximal vasorelaxation in response to ACh: 70.3 +/- 3.3 vs. 95.2 +/- 2.5% in control, P < 0.01) was observed in hyperlipidemic aortic segments, and treatment with gAd significantly improved endothelial function (P < 0.01). Paradoxically, total NO production was significantly increased in hyperlipidemic vessels, and treatment with gAd slightly reduced, rather than increased, total NO production in these vessels. Treatment with gAd reduced (-78%, P < 0.01) superoxide production and peroxynitrite formation in hyperlipidemic vascular segments. Moreover, a moderate attenuation (-30%, P < 0.05) in gp91(phox) and iNOS overexpression in hyperlipidemic vessels was observed after gAd incubation. Treatment with gAd had no effect on eNOS expression but significantly increased eNOS phosphorylation (P < 0.01). Most noticeably, treatment with gAd significantly enhanced eNOS (+83%) but reduced iNOS (-70%, P < 0.01) activity in hyperlipidemic vessels. Collectively, these results demonstrated that adiponectin protects the endothelium against hyperlipidemic injury by multiple mechanisms, including promoting eNOS activity, inhibiting iNOS activity, preserving bioactive NO, and attenuating oxidative/nitrative stress.     

miR-125a-5p转染对肝癌细胞增殖、侵袭、迁移的影响及相关机制研究

摘要 目的：探究miR-125a-5p转染对肝癌细胞增殖、侵袭、迁移的影响及相关机制。方法：将肝癌细胞分为对照组、下调组和上调组,并通过细胞转染建立稳定转染的下调组和上调组。MMT法检测细胞增殖能力,流式细胞仪检测细胞凋亡能力,Transwell小室实验检测细胞侵袭能力,细胞划痕实验检测细胞迁移能力,Western blot法检测P13K/Akt通路中AKT、Bax、Bcl-2、P13K、P-AKT蛋白表达量。结果：与上调组相比,下调组24、48、72 h细胞增殖率,细胞侵袭、迁移细胞数,AKT、Bcl-2、P13K、P-AKT蛋白表达量显著降低,具有统计学差异（29.67±9.87 vs 17.34±5.71,t=5.192,P＜0.05、34.75±11.56 vs 15.17±5.04,t=7.365,P＜0.05、38.48±12.81 vs 12.51 ±4.13,t=9.153,P＜0.05,72.53±24.17 vs 36.28±12.07,t=6.365,P＜0.05、86.51±28.75 vs 46.28±15.32,t=5.858,P＜0.05,1.26±0.41 vs 0.81±0.26,t=4.397,P＜0.05、1.35±0.44 vs 0.76±0.24,t=5.584,P＜0.05、1.48±0.46 vs 0.79±0.26,t=6.194,P＜0.05、1.22±0.39 vs 0.73±0.24,t=5.584,P＜0.05）;与上调组相比,下调组24、48、72h细胞凋亡率,Bax蛋白表达量显著升高,具有统计学差异（17.62±5.84 vs 29.31±9.75,t=4.879,P＜0.05、14.97±4.65 vs 34.19±11.36,t=7.427,P＜0.05、11.26±3.74 vs 38.62±12.86,t=9.690,P＜0.05,0.75±0.24 vs 1.33±0.43,t=5.587,P＜0.05）。结论：下调miR-125a-5p的表达,可通过作用于P13K/Akt通路,调控AKT、Bax、Bcl-2、P13K、P-AKT蛋白表达量,进而起到抑制肝癌细胞增殖、促进肝癌细胞凋亡以及抑制肝癌细胞的侵袭、迁移能力。     

Physcion,a tetra-substituted 9,10-anthraquinone,prevents homocysteine-induced endothelial dysfunction by activating Ca2+- and Akt-eNOS-NO signaling pathways

BackgroundHomocysteine (Hcy) induced vascular endothelial dysfunction is known to be closely associated with oxidative stress and impaired NO system. 1,8-Dihydroxy-3-methoxy-6-methylanthracene-9,10-dione (physcion) has been known to has antioxidative and anti-inflammatory properties.PurposeThe purpose of the present study was to define the protective effect of physcion on Hcy-induced endothelial dysfunction and its mechanisms involved.Study Design and MethodsHyperhomocysteinemia (HHcy) rat model was induced by feeding 3% methionine. A rat thoracic aortic ring model was used to investigate the effects of physcion on Hcy-induced impairment of endothelium-dependent relaxation. Two doses, low (L, 30 mg/kg/day) and high (H, 50 mg/kg/day) of physcion were used in the present study. To construct Hcy-injured human umbilical vein endothelial cells (HUVECs) model, the cells treated with 3 mM Hcy. The effects of physcion on Hcy-induced HUVECs cytotoxicity and apoptosis were studied using MTT and flow cytometry. Confocal analysis was used to determine the levels of intracellular Ca²⁺. The levels of protein expression of the apoptosis-related markers Bcl-2, Bax, caspase-9/3, and Akt and endothelial nitric oxide synthase (eNOS) were evaluated by western blot.ResultsIn the HHcy rat model, plasma levels of Hcy and malondialdehyde (MDA) were elevated (20.45 ± 2.42 vs. 4.67 ± 1.94 μM, 9.42 ± 0.48 vs. 3.47 ± 0.59 nM, p < 0.001 for both), whereas superoxide dismutase (SOD) and nitric oxide (NO) levels were decreased (77.11 ± 4.78 vs. 115.02 ± 5.63 U/ml, 44.51 ± 4.45 vs. 64.18 ± 5.34 μM, p < 0.001 and p < 0.01, respectively). However, treatment with physcion significantly reversed these changes (11.82 ± 2.02 vs. 20.45 ± 2.42 μM, 5.97 ± 0.72 vs. 9.42 ± 0.48 nM, 108.75 ± 5.65 vs. 77.11 ± 4.78 U/ml, 58.14 ± 6.02 vs. 44.51 ± 4.45 μM, p < 0.01 for all). Physcion also prevented Hcy-induced impairment of endothelium-dependent relaxation in HHcy rats (1.56 ± 0.06 vs. 15.44 ± 2.53 nM EC₅₀ for ACh vasorelaxation, p < 0.05 vs. HHcy). In Hcy-injured HUVECs, physcion inhibited the impaired viability, apoptosis and reactive oxygen species. Hcy treatment significantly increased the protein phosphorylation levels of p38 (2.26 ± 0.20 vs. 1.00 ± 0.12, p <0.01), ERK (2.11 ± 0.21 vs. 1.00 ± 0.11, p <0.01) and JNK. Moreover, physcion reversed the Hcy-induced apoptosis related parameter changes such as decreased mitochondrial membrane potential (MMP) and Bcl-2/Bax protein ratio, and increased protein expression of caspase-9/3 in HUVECs. Furthermore, the downregulation of Ca²⁺, Akt, eNOS and NO caused by Hcy were recovered with physcion treatment in HUVECs.ConclusionPhyscion prevents Hcy-induced endothelial dysfunction by activating Ca²⁺- and Akt-eNOS-NO signaling pathways. This study provides the first evidence that physcion might be a candidate agent for the prevention of cardiovascular disease induced by Hcy.     

Endothelium-dependent vasorelaxation is inhibited by in vivo depletion of vascular thiol levels: Role of endothelial nitric oxide synthase

Thiols like glutathione may serve as reducing co-factors in the production of nitric oxide (NO) and protect NO from inactivation by radical oxygen species. Depletion of thiol compounds reduces NO-mediated vascular effects in vitro and in vivo. The mechanisms underlying these actions are not clear, but may involve decreased synthesis of NO and/or increased degradation of NO. This study investigates the effect of glutathione depletion on the response to NO-mediated vasodilation induced by acetylcholine (Ach, 10 μg/kg), endothelial NO synthase (eNOS) activity and potential markers of vascular superoxide anion (O^·-₂) production in conscious chronically catheterized rats. Thiol depletion induced by buthionine sulfoximine (BSO, 1 g ip within 24 h) decreased the hypotensive effect of Ach by 30% (MAP reduction before BSO 27 ± 3 mmHg, 19 ± 3 mmHg after BSO, (mean ± SEM), p < .05n = 8). The impaired effect of Ach was associated with a significant reduction in eNOS activity (control: 7.7 ± 0.8, BSO: 3.9 ± 0.4 pmol/min/mg protein (p < .05), n = 6). In contrast, neither NADH/NADPH driven membrane-associated oxidases nor lucigenin reductase activity were significantly (p < .05) affected by BSO (BSO: 4415 ± 123, control: 4105 ± 455 counts/mg, n = 6) in rat aorta. It is concluded that in vivo thiol depletion results in endothelial dysfunction and a reduced receptor-mediated vascular relaxation. This effect is caused by reduced endothelial NO formation.     

microRNA-21对人舌鳞癌细胞的增殖和凋亡的影响

目的:探讨micro RNA-21(mi R-21)对人舌鳞癌细胞增殖和凋亡的影响。方法:选取8例舌鳞癌组织和4例癌旁组织为研究材料,采用实时荧光定量聚合酶链式反应(q RT-PCR)法对舌鳞癌及癌旁组织中的mi R-21相对表达量进行检测,利用人工合成的mi R-21mimic对人舌鳞癌Tca8113细胞进行瞬时转染,采用q RT-PCR法对Tca8113细胞中mi R-21相对表达量进行检测,采用四唑盐比色法(MTT)法对Tca8113细胞增殖情况进行检测,采用流式细胞术对Tca8113细胞周期与凋亡情况进行检测。结果:舌鳞癌组织中mi R-21的相对表达量(3.502±0.674),高于癌旁组织(0.998±0.192),差异有统计学意义(P0.05)。mi R-21mimic导致了Tca8113细胞中的mi R-21相对表达量上调(6.864±1.324),明显高于对照scramble组[(0.997±0.187),P0.05],对Tca8113细胞的增殖发挥了促进作用(P0.05)。经mi R-21mimic转染之后,Tca8113细胞进入S期的细胞出现了明显的增加[(27.4±5.1)%vs(48.6±8.7)%,P0.05],处于G1期的细胞出现了显著的减少[(56.3±9.6)%vs(36.2±7.2)%,P0.05],细胞凋亡数量出现了显著减少[(9.4±2.3)%vs(18.6±3.9)%,P0.05]。结论:mi R-21在舌鳞癌组织中高表达,过表达mi R-21有效促进了Tca8113细胞的增殖,抑制细胞的凋亡,mi R-21在舌鳞癌诊断和治疗可能具有一定的新型靶点价值。     

n-6/n-3 多不饱和脂肪酸营养失衡对小鼠精子发生的影响

目的:探讨n-6/n-3多不饱和脂肪酸营养失衡对小鼠精子发生的影响。方法:健康的30只C57/B6雄鼠随机分为对照组(CON)、高脂组(HF)、花生四烯酸组(HF+AA)。喂食16周,做合笼实验并记录致孕率,通过精子动力分析仪检测小鼠精子活力和数量的变化,用Elisa试剂盒测血清中睾酮和甘油三酯水平。通过病理组织染色观察小鼠睾丸组织的形态学变化。利用Realtime-PCR的方法检测小鼠睾丸中Dazl基因表达水平的变化。结果:高脂组、花生四烯酸组与对照组相比精子活力[(16±0.01;12.33±2.83 vs72.2±12.73)%,P0.001],精子数量[(7.5±1.13;6±0.14 vs 13.87±0.35)million/m L,P0.001],致孕率[(28.57;14.29 vs 78.57)%,P0.001]及血清睾酮含量[(0.35±0.14;0.27±0.07 vs 3.51±0.7)ng/m L,P0.001]均显著性降低。高脂组、花生四烯酸组与对照组相比,血清中甘油三酯的含量显著增高[(0.74±0.04;0.74±0.04 vs 0.45±0.04)mmol/L,P0.001]。病理组织染色观察到花生四烯酸组小鼠睾丸组织出现了明显异形,曲细精管内部的初级精母细胞明显缺失,管腔中从初级精母细胞到精子的发生过程出现了变异,精子的数量也显著性下降。参与精子生成过程中的减数分裂前的有丝分裂增殖期、精原细胞的发育等过程的Dazl基因在高脂组和花生四烯酸组小鼠睾丸中的表达量与对照组相比显著降低(0.87±0.05;0.65±0.03 vs 1.07±0.04,P0.05)。结论:膳食中n-3/n-6多不饱和脂肪酸失衡会导致雄鼠精子发生发育的障碍