圆偏振光与线偏振光对心肌细胞外基质组织学分析的一致性研究：Bland-Altman分析

【目的】研究圆偏振光和线偏振光结合苦味酸天狼猩红染色，对心肌细胞外基质组织学分析结果的一致性。【方法】对心肌梗死大鼠的心脏组织进行苦味酸天狼猩红染色后，固定其它光学参数，在同一视野分别使用圆偏振光和线偏振光获取图像（共随机获取40个视野）并进行图像分析，计算红色、绿色胶原纤维的面积、胶原容积分数以及胶原平均灰度等反映梗死心脏组织修复过程的组织学指标；采用Bland—Altman分析以及Pearson相关分析，对两种光学体系下测得的上述指标的一致性进行分析。【结果】通过Pearson相关分析显示两种光学系统中测得的红色纤维面积（r^2=0．9684，P〈0．01）、绿色纤维面积（r^2=0．8183，P〈0．01）、胶原容积分数（r^2=0．9595，P〈0．01）和胶原平均灰度（r^2=0．6188，P〈0．01）均有很高的相关性。然而Bland—Altman分析显示，至少30％以上的红色胶原纤维、50％绿色胶原纤维成分和30％的总细胞外基质成分在线偏振光中无法显示，并且线偏振光系统检测的偏倚随着均数的增加而增大。【结论】线偏振光结合天狼猩红染色在检测细胞外基质的沉积量、平均灰度方面，敏感性显著低于圆偏振光。在细胞外基质的相关研究中，应该尽量避免使用线偏振光。     

缺氧缺血新生大鼠海马ERK的变化及GM1的影响

目的:观察新生大鼠缺氧缺血性(HI)脑损伤后脑细胞凋亡的情况,进一步研究HI脑损伤后海马细胞外信号调节激酶(ERK)的变化,探讨单唾液酸四己糖神经节苷脂(GM1)对HI新生大鼠的脑保护作用。方法:选择新生7日龄Sprague-Dawley大鼠108只,随机分为3组:缺氧缺血组(HI组,36只),假手术对照组(Control组,36只),缺氧缺血+神经节苷脂组(GM1组,36只)。采用结扎右侧颈总动脉并吸入低氧混合气体制备HIBD模型,用TUNEL法检测细胞凋亡,免疫组化SP法检测大脑海马CA1区p-ERK的表达。结果:新生大鼠HI后脑细胞存在凋亡,GM1治疗能减少细胞凋亡。HI组、Control组和GM1组凋亡细胞数分别为(8.41±1.27)、(39.25±2.02)和(11.10±1.79),HI组阳性细胞数明显高于Control组和GMl组(P<0.05),GM1组与Control组比较阳性细胞数无明显差异(P>0.05)。HI组和GMl组p-ERK表达皆为阳性,两组比较HI组表达更强(P<0.05),Control组未见有p-ERK表达。结论:GM1可减少HI脑损伤后脑细胞凋亡,影响p-ERK表达,对缺氧缺血(HI)新生大鼠的脑具有保护作用。     

血管性痴呆小鼠海马ERK_1及ERK_2的表达特征

目的观察血管性痴呆(VD)小鼠海马神经元中细胞外信号调节激酶1、2(ERK_1、ERK_2)的表达特征,探讨其在VD发病中的作用机制。方法采用双侧颈总动脉反复缺血—再灌注法制备小鼠VD模型,设立假手术组作为对照。术后第29、30天,经跳台试验和水迷宫试验对2组小鼠进行行为学成绩测试,用免疫组化方法观察2组小鼠海马神经元中ERK的表达变化。结果VD模型小鼠学习、记忆成绩较假手术组显著下降(P<0.05)。模型组小鼠海马CA1区ERK_1、ERK_2的表达较假手术组湿著减少(P<0.05)。结论海马神经元内ERK表达减少可能参与了VD的发病机制,应用能促进ERK表达的药物可能成为治疗VD的有效药物之一。     

细胞外信号调节蛋白激酶通路在胎盘生长因子1诱导脐静脉内皮细胞释放一氧化氮中的作用

目的 探讨细胞外信号调节蛋白激酶(ERK)通路在胎盘生长因子1(PLGF-1)诱导脐静脉内皮细胞(HUVEC)释放一氧化氮(NO)中的作用.方法 选择因头盆不称、胎儿窘迫或胎位异常行剖宫产分娩的新生儿50例,采用胰蛋白酶消化法进行脐带HUVEC原代培养.(1)培养成功后.用免疫组化法通过Ⅷ因子鉴定细胞形态;(2)用蛋白印迹法和RT-PCR技术检测HUVEC中酪氨酸激酶受体1(Fit-1)蛋白和mRNA表达;(3)用浓度为10 ng/ml的PLGF-1培养细胞(观察A组),并在培养前及培养后2.5、5、10、20 min收集细胞,提取蛋白,用蛋白印迹法检测ERK蛋白的表达;(4)用浓度为10 ng/ml的PLGF-1培养细胞,收集培养后20、40、160、360、480、720、1440 min的细胞培养液,用硝酸盐还原酶法检测培养液中NO的含量;(5)先用含ERK特异性抑制剂--PD98059(100μmo/L)的培养基培养细胞60 min后,换含PLGF-1的培养基继续培养(观察B组),收集培养后20、40、160、360、480、720、1440 min的细胞培养液,用硝酸盐还原酶法检测培养液中NO的含量.以无血清培养基培养的细胞为对照组,细胞处理时间、培养条件和收集细胞液时间同观察组.实验重复3次.结果 (1)免疫组化鉴定培养的细胞为HUVEC.(2)HUVEC中有Fit-1 mRNA和蛋白的表达.(3)观察A组PLGF-l培养HUVEC后2.5 min即有ERK蛋白表达强度的升高,5 min时表达强度达到高峰,10 min时表达强度降低.(4)PLGF-l培养20 min后HUVEC培养液中NO含量开始升高,为(6.96±0.34)μmol/L,培养40 min时NO含量为(9.45 4-0.59)μmol/L,培养360 min时NO达(15.82±0.69)μmo/L,各时间点NO含量比较,差异均有统计学意义(P<0.05).(5)观察B组NO释放显著受到抑制,从培养160 min到1440 min,NO含量下降达50%以上.结论 ERK通路可能是PLGF诱导HUVEC释放NO的重要信号通路之一.     

鱼藤酮对PC12细胞凋亡及ERK1／2蛋白表达的影响

目的 观察鱼藤酮对PC12细胞凋亡以及细胞外信号调节酶1和2(ERK1/2)蛋白表达的影响,以深入探讨ERK1/2蛋白表达与鱼藤酮诱导PC12细胞凋亡之间的关系.方法 通过建立PC12细胞鱼藤酮染毒模型,光镜下观察鱼藤酮对PC12细胞形态的影响;流式细胞仪检测PC12细胞凋亡率的改变;Western蛋白印迹法和免疫组化法检测PC12细胞ERK1/2蛋白表达及定位情况.结果 鱼藤酮处理24 h后PC12细胞形态明显改变且凋亡率增加,20 μmol/LMEK抑制剂PD98059预处理1 h与单纯鱼藤酮染毒比较凋亡率没有显著性改变;磷酸化ERK1/2蛋白表达量随着鱼藤酮剂量的加大而增高,且活化的ERK1/2蛋白主要集中在胞浆内表达.结论 鱼藤酮体外作用可以诱导PC12细胞凋亡以及ERK1/2蛋白磷酸化表达增强.     

ERK信号通路在紫杉醇诱导大肠癌细胞凋亡中的作用

目的 观察紫杉醇对大肠癌细胞系CX1细胞的杀伤作用,并探讨MAPKs/ERK信号传导通路抑制剂PD98059对其的影响.方法 通过MTT法比较紫杉醇和紫杉醇+PD98059分别作用于CX1细胞后的药物敏感度,并应用流式细胞术对二者作用下的细胞进行细胞解析,观察细胞周期变化.结果 大肠癌CX1细胞在紫杉醇作用72 h后,IC50为2.14 × 10-7mol/L,而在紫杉醇联合PD98059作用72 h后,IC50为6.91×10-9mol/L.通过细胞的周期解析发现紫杉醇和PD98059共同作用的细胞其凋亡比例为31.41%,远远高于紫杉醇单独作用引起的凋亡(15.60%),二者之间差异有统计学意义(P＜0.05).而未加药及单加PD98059的CX1细胞其凋亡细胞只占5.27%及5.61%,二者之间差异无统计学意义(P＞0.05).结论 紫杉醇可以诱导大肠癌细胞凋亡,而针对MAPK/ERK信号通路的抑制剂PD98059可增强紫杉醇的凋亡作用.     

Adenosine triggers preconditioning through MEK/ERK1/2 signalling pathway during hypoxia/reoxygenation in neonatal rat cardiomyocytes

Three subtypes of adenosine receptors (A(1), A(2A) and A(3) ARs) are functionally expressed in cardiomyocytes. Adenosine released during ischemia and ischemia/reperfusion plays a major role in cardioprotection. Phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (PKB) and MEK/ERK1/2 pathways are involved in cell survival. Since the role of these pathways in AR-mediated preconditioning is poorly understood, we have investigated whether PI-3K/PKB and/or MEK1/ERK1/2 pathways are involved in AR-induced cardioprotection in neonatal rat cardiomyocytes. Cells were pre-treated (15 min) with adenosine (non-selective), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)) before 4 h hypoxia (0.5% O(2)) and 18 h reoxygenation (HX4/R). HX4/R-induced increase in LDH release was significantly reduced by adenosine (70%), CPA (59%) and Cl-IB-MECA (46%). The MEK1 inhibitor PD 98059 suppressed the effects of adenosine, CPA, and Cl-IB-MECA on LDH release, whereas the PI-3K inhibitor wortmannin did not reverse this cardioprotection. Western blotting of phosphorylated ERK1/2 and PKB during HX4/R supported the involvement of ERK1/2 and not PKB in A(1) and A(3) agonist-mediated cardioprotection. In addition, adenosine, CPA and Cl-IB-MECA inhibited HX4/R-induced caspase 3 activity by 75%, 70% and 59%, respectively, and this inhibition was abolished by PD 98059. Interestingly, wortmannin inhibited by 66% the anti-apoptotic response triggered by Cl-IB-MECA but had no effect on adenosine or CPA-induced inhibition of caspase 3. CGS 21680 did not modify cell survival or caspase 3 activity. In conclusion, these data show that the preconditioning effect of adenosine requires A(1) and A(3) but not A(2A) ARs and involves an anti-apoptotic effect via MEK1/ERK1/2 pathway in neonatal rat cardiomyocytes. In addition, A(3)AR-induced preconditioning also involves a PI-3K dependent pathway.     

Bile modulates intestinal epithelial barrier function via an extracellular signal related kinase 1/2 dependent mechanism

Objective Obstructive jaundice is frequently complicated by infections and has been associated with increased bacterial translocation and gut mucosal hyperpermeability in animal models. Proper expression of the tight junction (TJ) proteins ZO-1 and occludin is important for normal gut barrier function. We tested whether bile modulates intestinal epithelial ZO-1 and occludin expression.Animals (a) Male C57BL/6 mice; (b) male Sprague-Dawley rats.Interventions (a) Mice were subjected to common bile duct ligation (CBDL) or a sham procedure, and 96 h later all surviving animals were killed for measurement of ileal mucosal permeability to FITC-labeled dextran (everted gut sac technique), bacterial translocation to mesenteric lymph nodes, and ileal epithelial ZO-1 and occludin expression (western blots). (b) Rat IEC-6 enterocytic monolayers were incubated in the presence or absence of graded concentrations of rat bile and/or U0126, an inhibitor of extracellular signal related kinase (ERK) 1/2 activation.Results (a) Compared to sham-treated controls, CBDL significantly increased gut mucosal permeability and bacterial translocation and markedly decreased ileal epithelial expression of ZO-1 and occludin. In a follow-up in vivo experiment, gavaging mice with fresh rat bile twice daily significantly ameliorated the deleterious effects of CBDL on gut barrier function. (b) Addition of 1% (v/v) bile to media enhanced phosphorylation of ERK1/2, increased the expression of ZO-1 and occludin and decreased permeability to FITC-dextran. All of these bile-mediated effects were blocked by 10 µM U0126.Conclusions These data support the view that the presence of bile in the intestinal lumen is essential for normal gut barrier function, possibly because compounds present in bile initiate ERK1/2-dependent signaling that is essential for normal expression of key TJ proteins.This revised version was published online in April 2005 with a corrected section title.Grant support: 5R01 GM 37631-18 from the National Institutes of Health     

Significance of extracellular potassium in central respiratory control studied in the isolated brainstem-spinal cord preparation of the neonatal rat

The significance of extracellular potassium in central respiratory control was investigated using the isolated brainstem-spinal cord preparation of the neonatal rat. Depth profiles of extracellular potassium activity ([K+])ECF in the medulla were measured with ion-sensitive microelectrodes. Although [K+]ECF increased with depth in medullary tissue during control (4 mM) and low (1 mM) potassium concentration ([K+])CSF superfusion, this gradient disappeared with higher [K+]CSF. With low [K+]CSF (1 mM), respiratory CO2 responsiveness was abolished, and increased with high [K+]CSF (8 mM). Respiratory frequency (fR) was diminished at low [K+]CSF (1 mM), and increased with elevated [K+]CSF (8 and 16 mM); with yet higher [K+]CSF (32 mM) apnea occurred after a transient increase in fR. Perforated patch recording revealed that high [K+]ECF decreased membrane resistance, depolarized membrane potential, and increased firing frequency in most of the recorded medullary neurons. High [K+]ECF also increased excitatory and inhibitory post-synaptic potentials of medullary neurons and augmented the functional connectivity among neurons. It is concluded that [K+]ECF is of importance in the maintenance of respiratory rhythm and central chemosensitivity.     

Get a grip: integrins in cell-biomaterial interactions

Integrin adhesion receptors have emerged as central regulators of cell–biomaterial interactions. This opinion paper discusses how integrins control cellular and host responses to biomaterials and new strategies to manipulate these adhesive interactions in order to elicit specific cellular responses.