黄芩茎叶总黄酮对过氧化氢诱导人脐静脉内皮细胞氧化损伤的保护作用及其机制

目的探讨黄芩茎叶总黄酮(SSTF)对过氧化氢(H2O2)所致人脐静脉内皮细胞(HUVEC)氧化损伤的保护作用及其可能机制。方法在由H2O2制备血管内皮细胞氧化损伤模型的基础上,采用MTT法测定各组细胞活力;生化比色法测定各组细胞培养上清液中乳酸脱氢酶(LDH)的活性,丙二醛(MDA)的含量及超氧化物歧化酶(SOD)的活性;流式细胞术检测各组细胞的凋亡率以及western-blot法测定各组细胞凋亡抑制蛋白Bcl-2的表达情况。结果 H2O2处理HUVEC后其存活率较正常HUVEC下降,细胞培养上清液中LDH活性和MDA含量明显增加,SOD活性较正常HUVEC降低;HUVEC凋亡率明显升高(P<0.05或P<0.01);而SSTF(100~400mg/L)各剂量都能提高H2O2损伤的HUVCE的细胞活力,明显降低LDH活性,显著减少MDA的含量,提高SOD的活性,明显降低HUVEC凋亡率,明显提高Bcl-2蛋白表达水平(P<0.05或P<0.01)。结论 SSTF能减轻H2O2对HUVEC的氧化损伤作用,降低H2O2所致HUVEC的凋亡率,其机制可能与上调凋亡抑制蛋白Bcl-2的表达水平有关。     

蛋白激酶C对血管内皮功能的影响

【目的】研究血管内皮蛋白激酶C对内皮通透性及粘附功能的影响。【方法】以血管内皮细胞为研究对象,以流式分析法测定细胞间黏附分子-1（ICAM-1）,血管细胞黏附分子-1（VCAM-1）的表达;以同位素标记法测定HUVECs的粘附率;双室培养法测定HUVECs的通透率。【结果】10ng／mlPMA处理HUVECs后,HUVECs的通透性于30min时开始明显增高;VCAM-1,ICAM-1表达增强,对血小板粘附率2h达到最大值。【结论】PKC活化能其上调血管内皮细胞VCAM-1、ICAM-1的表达,并增强其粘附性。HUVECs的PKC活化与血管内皮细胞通透性增加有关。     

富马酸二甲酯对过氧化氢诱导心脏微血管内皮细胞铁死亡的影响

目的 探讨富马酸二甲酯（DMF）对过氧化氢（H2O2）诱导人心脏微血管内皮细胞（HCMEC铁死亡的影响及其机制。方法 体外培养血管内皮细胞，使用H2O2处理内皮细胞，并用不同浓度DMF干预内皮细胞，采用CCK-8法测细胞增殖；ELISA法检测细胞炎症水平；Western blot检测铁死亡蛋白GPX4和ACSL4，以及Nrf2/HO-1和JAK2/STAT1通路蛋白的改变。结果 CCK8实验结果表明，与对照组相比，H2O2可诱导HCMEC增殖能力下降，而DMF成浓度依赖性促进HCMEC增殖（P <0.05）。H2O2诱导的HCMEC细胞炎症因子TNF-α、IL-1β和IL-18水平增加，DMF处理能抑制上述炎症因子的水平（P <0.05）。Western blot结果显示，H2O2处理引起细胞内GPX4蛋白下降，而ACSL4蛋白升高；DMF能抑制H2O2诱导的HCMEC细胞铁死亡（P <0.05）。H2O2可诱导HCMEC中Nrf2和HO-1蛋白表达下降，而JAK2和STAT1的磷酸化水平的增加（P <0.05）；同H2O2组比，DMF可增加Nrf2和HO-1蛋...     

蛋白激酶C在肝细胞生长因子诱导血管内皮生长因子表达过程中的作用

目的：探讨MHCC97H细胞中蛋白激酶C（PKCs）在肝细胞生长因子（hepatocyte growth factor, HGF）诱导血管内皮生长因子（vascular endothelial growth factor ,VEGF）表达过程中的作用。方法：将MHCC97H细胞分为对照组、HGF组、calphostin C组、BIM组、R031-8220组、ξ-PS组,分别用逆转录聚合酶链反应（RT—PCR）、蛋白免疫印迹（Western Blot）法及免疫沉淀法分析各组中VEGFmRNA、蛋白质表达及磷酸化PKCξ水平变化情况。结果：广谱PKC抑制剂R031—8220能够完全抑制HGF所诱导的VEGF蛋白质升高（P〈0．01）。calphostin C、BIM能够抑制HGF诱导的PKCB和PKCs磷酸化（均P〈0．01）。BIM不能抑制HGF诱导的VEGFRNA及相应蛋白质的表达（P〉0．05）。PKCξ特异性抑制蛋白ξ-PS能够抑制HGF诱导的PKCξ磷酸化及VEGF蛋白质表达（P〈0．01）。结论：PKCs是MHCC97H细胞中HGF诱导VEGF表达路径的中间信号调节分子,HGF在MHCC97H细胞中主要通过磷酸化方式调节PKCs。HGF诱导的MHCC97H细胞的VEGF表达是通过aPKC的PKCξ调节的,cPKC、nPKC路径不参与MHCC97H中HGF诱导VEGF的表达过程。     

C-反应蛋白致内皮炎症损伤的信号转导通路筛选

目的探讨C-反应蛋白（CRP）作为急性期反应蛋白参与内皮系统炎症损伤过程涉及的信号通路及其意义。方法采用人脐静脉内皮细胞（HUVEC）进行培养、鉴定，实验用第五代细胞，给予20mg／L的CRP处理24h，设立对照，提取RNA，进行信号转导通路特异性基因芯片检测。结果CRP处理组分别有13种基因表达增加、25种基因表达降低，其中炎症生长因子相关基因WISP2表达增加37．63倍，诱导凋亡关键信号通路p53相关基因表达增加30．50倍，而抑制凋亡相关信号通路Bcl-x和Bcl-2相关基因表达降低9．61％和49．95％。CRP处理后血管细胞黏附分子（VCAM）相关基因（VCAM-1）表达增加2．75倍，而血清细胞间黏附分子（ICAM）相关基因表达两组差异无统计学意义。结论CRP可能参与了内皮系统的炎症发生，其机制可能是通过诱导细胞凋亡及激活细胞黏附相关蛋白信号通路的表达实现的。     

神经干细胞微囊泡抑制H2O2 诱导DRG 神经元氧化应激损伤机制研究

目的 探究神经干细胞微囊泡（neural stem cell microvesicles, NSC-MVs）对H2O2 诱导背根神经节（dorsal rootganglion, DRG）神经元氧化应激损伤的作用及机制。方法 超速离心提取NSC-MVs，并进行电镜和纳米颗粒示踪分析。原代培养大鼠DRG 神经元，β-tubulin Ⅲ荧光染色。建立H2O2 诱导DRG 神经元氧化应激损伤模型，确定作用浓度。经NSC-MVs 预处理，MTT( 四唑盐) 检测神经元活力，流式细胞术检测Annexin Ⅴ和PI，蛋白质印迹检测凋亡相关蛋白cleaved caspase 3，cleaved caspase 9，Bax 和Bcl-2 的表达。结果 NSC-MVs 在透射电镜下呈圆盘状，包膜完整，纳米颗粒示踪显示其粒径为50 ～ 450 nm。MTT 结果显示，与对照组相比，H2O2 组神经元活力明显抑制。当H2O2 浓度为25，50，100 和200μmol/L 时具有显著性差异，细胞活力分别为84.4 %，73.7 %，69.8 % 和49.5 %（F=127.7，P ＜ 0.01）。经100，200 和400 μg/ml 的NSC-MVs 预处理DRG 神经元，细胞活力得到明显提升，分别为51.4 %，67.4 % 和73.5 %（F=49.47，P=0.023）。流式细胞术检测结果显示，与对照组相比，H2O2 组神经元凋亡率显著上升（P ＜ 0.05），NSCMVs预处理组细胞凋亡率明显下降（P ＜ 0.05）。蛋白质印迹结果显示，与H2O2 组相比，NSC-MVs 显著抑制cleavedcaspase3，cleaved caspase 9 和Bax 蛋白表达（均P ＜ 0.05），上调Bcl-2 蛋白表达（P ＜ 0.05）。结论 NSC-MVs 能够抑制H2O2 诱导DRG 神经元氧化应激损伤，发挥神经保护作用。     

甲氧滴滴涕对雄性小鼠生精细胞p34cdc2和cyclinB1的影响

【目的】探讨甲氧滴滴涕（methoxychlor，MXC）对雄性小鼠生精细胞周期调控因子细胞周期蛋白激酶Ⅰ（p34cdc2）和细胞周期蛋白B1（cyclinBl）表达水平的影响和意义。【方法】40只成年雄性昆明小鼠随机分为四组，其中三组分别喂服不同浓度MXC15天（50，100，150mg／kg body weight／day），另一组为正常对照组。应用免疫组化SABC法检测四组小鼠生精细胞中p34cdc2和cyclinB1的表达情况。【结果】小鼠生精细胞cyclinB1阳性表达部位主要在精原细胞胞核而p34cdc2主要在初级精母细胞胞浆。两种蛋白的表达均受MXC作用的影响．各实验组与对照组比较均明显降低（P〈0．001）。各实验组两两比较显示随MXC作用浓度增高两种蛋白表达降低，呈现明显差异（P〈0．01）。【结论】MXC可通过抑制生精细胞eyclinB1和p34cdc2的表达干扰细胞周期，产生生殖毒性，且作用强度随MXC浓度增强而增加。对于cyclinB1和p34cdc2的影响作用在不同细胞水平。     

维生素C保护视网膜色素上皮细胞的相关机制研究

目的研究氧化应激状态下维生素C作为抗氧化剂对视网膜色素上皮细胞的保护作用以及维生素C和SIRT1之间的调节机制。方法以人视网膜色素上皮细胞-19(ARPE-19)细胞为研究对象,分为空白对照组,无维生素C组(0μmol),低浓度维生素C组(20μmol),中等浓度维生素C组(100μmol)和高浓度维生素C组(500μmol)。培养过程中添加不同浓度维生素C之后对细胞加以H2O2(100μmol)处理12 h或24 h建立氧化应激模型。利用MTT法检测ARPE-19细胞存活率,膜联蛋白V-FITC凋亡检测试剂盒检测细胞凋亡以及活性氧(ROS)试剂盒测定细胞内活性氧的改变。使用SIRT1靶向siRNA进行SIRT1敲除,细胞使用预定浓度的维生素C进行孵育,并分为空白对照组,阴性对照组,siRNA干扰组。先用10 m M SIRT1激动剂白藜芦醇(RSV)和5 m M SIRT1抑制剂烟酰胺(NA)对细胞进行孵育,然后加入H2O2(100μmol)处理12 h或24 h,RT-PCR及Western blot方法检测SIRT1、p53和Foxo3基因表达水平。结果经过H2O212 h处理后,较高浓度的维生素C(500μmol)与较低浓度(20μmol)的维生素C无明显保护作用,而中等浓度的维生素C(100μmol)能够显著提高细胞的生存能力(P0.05),减少ARPE-19细胞的凋亡数量(P0.05),降低细胞内ROS水平(P0.05),差异有统计学意义。维生素C的作用可上调H2O2作用后SIRT1转录因子和应激反应因子(p53和Foxo3)的表达。RSV及NA可分别上调及下调维生素C对H2O2刺激的ARPE-19细胞存活力,细胞凋亡和细胞内ROS水平的影响。RT-PCR及Western blot结果表明:维生素C浓度增加时,SIRT1表达增加,p53和Foxo3基因表达水平升高。敲除或上调SIRT1表达,可相应明显地增加或减少p53和Foxo3转录和蛋白水平的表达。结论维生素C可降低细胞内ROS水平,减少ARPE-19细胞凋亡,起到抗氧化损伤保护作用,有望成为年龄相关性黄斑变性的有效治疗方法。     

碱性纤维母细胞生长因子对AD细胞模型PKCγ活性影响

目的 研究碱性纤维母细胞生长因子(bFGF)对β-淀粉样蛋白25-35(Aβ25-35)诱导大鼠肾上腺嗜铬细胞瘤(PC12)细胞存活率、蛋白激酶Cγ(PKCγ)蛋白表达的影响.方法 经体外培养的PC12细胞分正常对照组(常规培养液)、Aβ损伤组(加入培养液及老化Aβ25-35)、bFGF组(加入培养液、bFGF及老化Aβ25-35).MTT法检测不同处理组PC12细胞存活率,Western Blot 免疫印迹法分析PKCγ蛋白表达变化.结果 Aβ损伤组PC12细胞存活率低于正常对照组(P ＜0.05),bFGF组细胞存活率高于Aβ损伤组(P＜0.05).Western Blot 结果 显示,Aβ损伤组PKCγ(0.68±0.07)较对照组PKCγ(0.8±0.08)表达显著下降;Aβ损伤组在加入不同浓度的bFGF后PKCγ的值分别为:1.04±0.10,1.20±0.12,1.39±0.03,1.69±0.16,较 Aβ损伤组 PKCγ(0.65±0.06)蛋白表达显著增强,并与 bFGF浓度呈正相关.结论 bFGF对Aβ诱导的PC12细胞损伤具有一定保护作用,可能是通过增强PC12细胞PKCγ蛋白表达而实现.     

天麻素干预对过氧化氢诱导的PC12细胞凋亡、活力及PI3K／AKT信号通路蛋白的影响

【目的】探讨磷脂酰肌醇3激酶（PI3K）蛋白表达量及蛋白激酶 B（AKT ）信号通路在过氧化氢（H2 O2）诱导的 PC12细胞中的作用及天麻素的干预对其通路的影响。【方法】PC12细胞随机分为正常对照组,模型组（400μmol／L H2 O2）,天麻素低、中、高浓度组（0．1、1、10μmol／L 天麻素＋400μmol／L H2 O2）。咪唑蓝法检测各组细胞活力,Hoechst 染色观察各组细胞凋亡情况,比色法检测天冬氨酸蛋白水解酶（Caspase 3）、Caspase 8及 Caspase 9活性,western blot 分析 Bcl‐2、Bax 及 PI3K 蛋白表达量与 AKT 磷酸化水平。【结果】与正常对照组比较,模型组中细胞活力下降,细胞凋亡率提高,Caspase 3、Caspase 8及 Caspase 9活性提高,Bcl‐2及 PI3K 表达量下降,Bax 表达量上升,AKT 磷酸化水平降低,差异均具有统计学意义（ P ＜0．01）;与模型组比较,天麻素低、中、高浓度组细胞活力提高,细胞凋亡率降低,Caspase 3、Caspase 8及 Caspase 9活性降低,Bcl‐2、PI3K 蛋白表达量及 AKT 磷酸化水平提高,天麻素中、高浓度组 Bax 表达量降低,差异均具有统计学意义（ P ＜0．01）。【结论】天麻素可通过激活 PI3K／AKT 信号通路,从而抑制 H2 O2诱导的 PC12细胞凋亡。     

Hydrogen peroxide-induced extracellular signal-regulated kinase activation in cultured feline ileal smooth muscle cells

H(2)O(2) has been shown to act as a signaling molecule involved in many cellular functions such as apoptosis and proliferation. In the present study, we characterized the effects of H(2)O(2) on the activation of mitogen-activated protein (MAP) kinases and examined the factors involved in the process of extracellular signal-regulated kinase (ERK) activation by H(2)O(2) in ileal smooth muscle cells (ISMC). ISMC were cultured and exposed to H(2)O(2). Western blot analysis was performed with phosphospecific MAP kinase antibodies. Potent activation of ERK and moderate activation of stress-activated protein kinase/c-Jun NH(2)-terminal kinase occurred within 30 min of 1 mM H(2)O(2) treatment. However, p38 MAP kinase was not activated by H(2)O(2). The activation of ERK by H(2)O(2) was reduced by the mitogen-activated/ERK-activating kinase inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one], Ras inhibitor S-farnesylthiosalicylic acid, removal of extracellular Ca(2+), depletion of the intracellular Ca(2+) pool by thapsigargin, or pretreatment of ISMC with the calmodulin antagonist W-7. Also, H(2)O(2)-induced ERK activation was attenuated by a receptor tyrosine kinase inhibitor, tyrphostin 51, but not by down-regulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or by a PKC inhibitor, GF109203X [3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride]. Growth factor receptor antagonist suramin pretreatment inhibited H(2)O(2)-induced ERK activation, highlighting a role for growth factor receptors in this activation. Furthermore, the ERK activation by H(2)O(2) was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol indicating that metal-catalyzed free radical formation may mediate the initiation of signal transduction by H(2)O(2). These data suggest that short-term stimulation with H(2)O(2) activates the signaling pathways of cell mitogenic effects which are thought to be a protective response against intestinal oxidative stress.     

Enhanced thromboxane receptor-mediated responses and impaired endothelium-dependent relaxation in human corpus cavernosum from diabetic impotent men: role of protein kinase C activity

We have evaluated the influence of protein kinase C (PKC) activity on penile smooth muscle tone in tissues from diabetic and nondiabetic men with erectile dysfunction. Human corpus cavernosum (HCC) strips were obtained from impotent diabetic and nondiabetic men at the time of penile prosthesis implantation and studied in organ chambers. Contractility responses to a prostanoid precursor, to prostanoids, and to the endothelium-dependent vasodilator acetylcholine were studied. Arachidonic acid (AA; 100 microM) caused cyclooxygenase-dependent relaxation of HCC. This relaxation was impaired in diabetic tissues and normalized by blocking thromboxane (TP) receptors with 20 nM [1S-[1alpha,2alpha(Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ29548). Diabetes did not affect prostaglandin (PG)E(1)-induced relaxation, but it reduced relaxation induced by the PGE(1) metabolite PGE(0). This effect was related to an interaction of PGE(0) with TP receptors. Diabetic tissues had reduced endothelium-dependent relaxation, which was partially improved by SQ29548 and completely normalized by the PKC inhibitor 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X; 1 microM). In HCC from nondiabetic patients, treatment with the PKC activator phorbol-12,13-dibutyrate (0.3 microM) significantly attenuated endothelium-dependent relaxation, an effect prevented by coadministration of GF109203X. Tissues from diabetic patients had enhanced sensitivity to the contractile effects of the TP receptor agonist 9,11-dideoxy-9alpha,11alpha-epoxymethano PGF(2alpha) (U46619) (EC(50) = 0.65 +/- 0.42 and 6.01 +/- 2.28 nM in diabetic and nondiabetic patients, respectively). Inhibition of PKC with 1 microM GF109203X, prevented diabetes-induced hypersensitivity to U46619-induced contractions (EC(50) = 8.55 +/- 3.12 microM). Overactivity of PKC in diabetes is responsible for enhanced contraction and reduced endothelium-dependent relaxation of HCC smooth muscle. Such alterations can result in erectile dysfunction.     

甘露糖结合凝集素对甲状腺癌细胞p53及Bcl-2蛋白表达的影响

目的探讨甘露糖结合凝集素（MBL）对甲状腺癌细胞株p53及凋亡基因Bcl-2基因表达的影响。方法将0、1mg／L的重组人甘露糖结合凝集素（RhMBL）（对照组、实验组）分别作用于两种甲状腺癌细胞株（甲状腺乳头状癌细胞和甲状腺滤泡状癌细胞）,利用蛋白印记（WesternBlot）检测p53、Bcl-2的表达。结果重组人甘露糖结合凝集素作用于甲状腺癌细胞株后,可使p53及Bcl-2蛋白相对表达量下调。（1）甲状腺乳头状癌细胞：p53蛋白对照组的表达量为1．51±0．29、实验组为0．74±O．07,两组比较差异有统计学意义（t=7．63,P=0．040）;Bcl-2蛋白对照组的表达量为1．20±0．07,实验组为0．59±0．04,两组比较差异有统计学意义（t=6．28,P=0．001）。（2）甲状腺滤泡状癌细胞：p53蛋白对照组的表达量为0．88±O．14,实验组为0．35±0．08,两组比较差异有统计学意义（t=3．29,P=0．003）;Bcl-2蛋白对照组的表达量为1．07±0．11,实验组为0．33±0．06,两组比较差异有统计学意义（t=5．73,P=0．000）。结论MBL在其诱导甲状腺癌细胞凋亡、抑制其增殖的的过程中,Bcl-2和p53可能发挥着一定的作用。     

Mechanism of the vascular angiotensin II/alpha2-adrenoceptor interaction

alpha(2)-Adrenoceptors potentiate vascular responses to angiotensin II. The goal of this study was to test the hypothesis that the phospholipase C (PLC)/protein kinase C (PKC)/c-src/phosphatidylinositol 3-kinase (PI3K) pathway contributes to the vascular angiotensin II/alpha(2)-adrenoceptor interaction. In rats in vivo, intrarenal infusions of angiotensin II (10 ng/kg/min) increased renal vascular resistance by 5.8 +/- 0.5 units, and this response was enhanced (p < 0.05) to 9.1 +/- 1.2 units by UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; 3 microg/kg/min; alpha(2)-adrenoceptor agonist]. Intrarenal infusions of U-73122 [1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione; 3 microg/min; PLC inhibitor], GF109203X [bisindolylmaleimide I; 10 microg/min; PKC inhibitor], CGP77675 [1-(2-{4-[4-amino-5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]phenyl}ethyl)piperidin-4-ol; 5 microg/min; c-src inhibitor], and wortmannin (1 microg/min; PI3K inhibitor) abolished the angiotensin II/alpha(2)-adrenoceptor interaction. In isolated perfused rat kidneys, angiotensin II (0.3, 1, and 3 nM) increased perfusion pressure (by 15 +/- 8, 39 +/- 4, and 93 +/- 9 mm Hg, respectively), and UK-14,304 (1 microM) potentiated these responses (to 36 +/- 4, 67 +/- 7, and 135 +/- 17 mm Hg, respectively). This angiotensin II/alpha(2)-adrenoceptor interaction was abolished by U-73122 (10 microM), GF109203X (3 microM), CGP77675 (5 microM), and wortmannin (0.2 microM). Preglomerular microvascular smooth muscle cells expressed phospholipase (PLC)-beta(2), PLC-beta(3), c-src, phospho(tyrosine 416)-c-src, and PI3K. In these cells, angiotensin II (0.1 microM) and UK-14,304 (1 microM) per se did not increase phospho-c-src; however, the combination of angiotensin II plus UK-14,304 doubled phospho-c-src, and this interaction was abolished by U-73122 (10 microM) and GF109203X (3 microM). In conclusion, the PLC/PKC/c-src/PI3K pathway may contribute importantly to the interaction between alpha(2)-adrenoceptors and angiotensin II on renal vascular resistance.     

葡萄籽提取物对高尿酸诱导内皮细胞功能紊乱的保护作用

目的探讨葡萄籽提取物（Grape seed extract,GSE）对尿酸（UA）诱导内皮细胞功能紊乱的保护作用和机制。方法常规培养的人脐静脉内皮细胞（HUVEC）分为对照组、UA处理组和UA＋GSE（不同浓度）组。RT-PCR和Western blot法检测各组细胞eNOS mRNA和蛋白表达水平,硝酸还原酶法检测各组细胞上清NO的含量,化学发光法检测细胞过氧化物丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。结果 （1）人脐静脉内皮细胞经UA处理后,eNOS mRNA、蛋白质和NO释放量明显下降,细胞MDA含量明显增加,SOD活性下降（P〈0.05）;（2）经GSE干预后,细胞eNOS mR-NA、蛋白质和NO释放水平上调,细胞MDA含量明显降低、SOD活性部分恢复（P〈0.05）。结论 GSE可通过抑制氧化应激水平,抑制高尿酸诱导的皮细胞功能紊乱。     

氧化应激对大鼠肾小管上皮细胞生物学行为的影响及L-EGCG的保护作用

[目的]探讨氧化应激对大鼠肾小管上皮细胞（NRK）生物学行为的影响及L -表没食子儿茶素没食子酸酯（L -EGCG）对NRK细胞氧化性损伤的保护作用.[方法]利用H2O2刺激离体培养的NRK细胞建立氧化损伤的模型,筛选H2O2对NRK细胞损伤研究的最适剂量与最佳时点.然后将培养细胞分EGCG正常对照组、H2O2组、不同浓度儿茶素组.MTT法检测细胞活力,流式细胞仪测定细胞凋亡,荧光探针JC-1测定线粒体膜电位.利用生物化学法检测各组NRK细胞培养上清中丙二醛（MDA）含量及乳酸脱氢酶（LDH）漏出量.[结果]①250 μmol/L H2O2作用6 h剂量组即可引起NRK细胞损伤;②L-EGCG能明显改善H2O2导致的细胞损伤,可使细胞存活率升高,LDH释放量降低,MDA生成减少,稳定了线粒体膜电位,减轻了细胞凋亡.[结论]氧化应激能导致大鼠肾小管上皮细胞活力下降,凋亡增加,L-EGCG可能是通过提高NRK细胞的抗氧化能力而提高其对H2O2损伤的保护及损伤修复能力.     

Contributions of the mitogen-activated protein kinase and protein kinase C cascades in spatial learning and memory mediated by the nucleus accumbens

Several studies have reported a role for the nucleus accumbens (NAcc) in learning and memory. Specifically, NAcc seems to function as a neural bridge for the translation of corticolimbic information to the motor system mediating locomotor learning, but the signaling mechanisms involved in this striatal learning await further investigation. The present experiments investigated the role of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) cascades within the NAcc of Long-Evans rats in a food-search spatial learning task (FSSLT). First, we used immunoblotting to examine changes in MAPK p42/p44 phosphorylation within the NAcc in the acquisition phase of the FSSLT. Second, we examined the effect on the acquisition and retention phases in the FSSLT of pretraining intra-accumbal microinjections of the MAPK [U0126; 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene, 1 microg/side] or PKC [GF109203X; bisindolylmaleimide or 1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol-3-yl) maleimide, 0.5 ng/side] inhibitors (four training sessions; one session/day). Third, the potential coupling of PKC and MAPK signaling pathways in the NAcc in spatial learning was studied using microinjections of GF109203X, radioactive activity assays, and immunoblotting. Results showed that 1) MAPK p42/p44 phosphorylation is augmented within the NAcc after spatial learning, 2) MAPK and PKC inhibition caused differential deficits in the acquisition and formation of spatial memories, and 3) inhibition of PKC activity by GF109203X caused a reduction in MAPKs phosphorylation in the NAcc in an early stage of the acquisition phase. Overall, these findings suggest that NAcc-PKC and -MAPK play important roles in spatial learning and that MAPKs phosphorylation seems to be mediated through the activation of the PKC signaling pathway.     

Signal transduction pathways involved in the mechanical responses to protease-activated receptors in rat colon

Recording simultaneously in vitro the changes of endoluminal pressure (index of circular muscle activity) and isometric tension (index of longitudinal muscle activity), we examined the mechanisms responsible for the apamin-sensitive relaxant and contractile responses induced by protease-activated receptor (PAR)-1 and PAR-2 activating peptides, SFLLRN-NH2 and SLIGRL-NH2, respectively, in rat colon. In the circular muscle, the inhibitory effects of SFLLRN-NH2 and SLIGRL-NH2 were significantly reduced by ryanodine, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, but unaffected by 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X), a protein kinase C (PKC) inhibitor, or genistein, a tyrosine kinase inhibitor. In the longitudinal muscle, the contractile responses to SFLLRN-NH2 and SLIGRL-NH2 were significantly reduced by nifedipine, an L-type calcium channel blocker, ryanodine, GF109203X, genistein, and abolished by U73122. The effects of genistein were additive with GF109203X but not with nifedipine. In the longitudinal muscle, the relaxant responses to the highest concentrations of SFLLRN-NH2 and SLIGRL-NH2 were abolished by nifedipine, reduced by genistein, and unaffected by ryanodine or GF109203X. In conclusion, influx of extracellular Ca2+ through L-type voltage-dependent channels or release of Ca2+ from intracellular stores are determining for the opening of the apamin-sensitive K+ channels responsible for longitudinal muscle relaxation or circular muscle inhibitory response, respectively, in rat colon. The longitudinal muscle contraction is mediated by activation of PLC; PKC and tyrosine kinase are involved in the cascade process, playing a parallel role. Indeed, tyrosine kinase and L-type Ca2+ channels would act sequentially. The influx of Ca2+ in turn would cause release of Ca2+ from sarcoplasmic reticulum.     

表皮生长因子对卵巢激活素基因表达的调节及其信号通路

背景：激活素作为卵巢内调控分子,对卵巢卵泡发育起着重要作用。目的：探索表皮生长因子在激活素基因表达过程中所起的重要作用以及可能参与调节的信号通路。方法：分离斑马鱼卵巢卵泡,体外培养6d,消化后传代培养24h。表皮生长因子单独或与其他分子抑制剂（AG1478、H89、GF109203X）或激动剂（FK、PMA）联合处理细胞,提取细胞RNA,反转录PCR检测细胞激活素表达量。结果与结论：表皮生长因子可快速提高激活素表达量,其作用可能是通过磷酸化信号分子丝裂原活化蛋白激酶实现,而蛋白激酶C特异性抑制剂或激动剂可减弱或加强表皮生长因子对丝裂原活化蛋白激酶信号分子的激活,显示卵巢内激活素表达受表皮生长因子调节,蛋白激酶C/丝裂原活化蛋白激酶信号通路参与了这种调节作用。蛋白激酶A抑制剂也能抑制表皮生长因子对激活素表达的促进作用。     

TGF-β1和(或)三氧化二砷作用于HL-60细胞后P27~(Kip1)表达及凋亡情况的研究

本研究观察三氧化二砷(As2O3)和/或TGF-β1对HL-60细胞凋亡及细胞周期的影响,以及作用前后P27Kip1、内源性TGF-β1、cyclinE和BCL-2的变化情况。As2O3和/或TGF-β1作用于HL-60细胞,用细胞形态学观察及流式细胞术检测细胞凋亡和细胞周期改变情况,应用免疫组织化学检测药物处理前后P27Kip1、内源性TGF-β1、cyclinE以及BCL-2表达的变化情况。结果表明:As2O3和TGF-β1单用均可以诱导HL-60细胞发生凋亡,联合处理对HL-60细胞生长抑制作用强于单药处理,其中5μmol/LAs2O3作用最强,5ng/mlTGF-β1处理可引起HL-60细胞的细胞周期阻滞于G1期(p0.05)。5ng/mlTGF-β1和联合处理组P27Kip1的表达较对照组增强(p0.05);5μmol/LAs2O3处理组P27Kip1的表达与对照组比较无明显差异(p0.05)。5ng/mlTGF-β1和联合处理组均可见cyclinE蛋白表达下调(p0.05)。5μmol/LAs2O3和联合处理组可见内源性TGF-β1表达上调(p0.05)。结论:As2O3、外源性TGF-β1均可诱导HL-60细胞凋亡,联合处理组诱导凋亡作用强于对照组和单药处理组,TGF-β1处理可引起HL-60细胞周期阻滞于G1期。TGF-β1诱导HL-60细胞凋亡过程中P27Kip1表达增强,拮抗cyclinE和BCL-2的作用,细胞周期阻滞,诱导细胞发生凋亡,这表明P27Kip1是TGF-β引起细胞周期阻滞的关键效应物,外源性TGF-β1又通过上调内源性TGF-β1的表达增强As2O3诱导原代APL细胞凋亡的作用。