灵芪胶囊对S180荷瘤小鼠p53和bcl-2蛋白表达影响的实验研究

[目的]探讨灵芪胶囊对S180荷瘤小鼠的抑瘤作用及其抑瘤作用机制。[方法]采用体内实验法，于无菌条件下抽取传代7d、生长良好的S180荷瘤小鼠腹水，无菌生理盐水1：3稀释（约含瘤细胞2×10g／L），按0．2mL／只接种于消毒后的小鼠右前肢腋部皮下，制成肿瘤模型，经口灌胃灵芪胶囊11d后，称取小鼠体质量，脱椎处死小鼠，取脾脏及胸腺称质量并计算胸腺指数和脾脏指数。用免疫组化法检测各组凋亡相关基因突变型p53和bcl-2基因的表达。[结果]灵芪胶囊在明显抑制肿瘤生长的同时不降低体质量，在一定剂量对荷瘤动物的免疫器官指数无明显影响，明显优于化疗药物；灵芪胶囊3个剂量组突变型p53基因和bcl-2基因的表达均小于模型组。[结论]灵芪胶囊对S180荷瘤小鼠有明显抑瘤作用，其机制可能与抑制突变型p53基因和bcl-2基因表达有关。实验证明灵芪胶囊不失为一种很有前途的抗癌中药。     

17 β雌二醇通过丝裂原活化蛋白激酶抑制前列腺癌细胞系PC3增殖

目的探讨丝裂原活化蛋白激酶在17β雌二醇(E2)抑制前列腺癌PC3细胞生长中的作用。方法检测不同浓度E2作用不同时间后PC3细胞生长抑制率。流式细胞仪分析细胞周期分布,TUNEL染色检测凋亡。Western blot检测ERK1/2,JNK和p38活性。RT-PCR法检测雌激素受体(ER)α、ERβ、P21WAF1和cyclinD1的表达。结果E2抑制PC3细胞增殖,并且可以激活ERK1/2、JNK和p38。处理因素作用48h后,对照组、E2、E2 PD98059、E2 SP600125、E2 SB203580组细胞的凋亡率分别为(0.9±0.1)%;(23.0±1.4)%;(30.0±1.2)%;(10.6±0.8)%和(14.6±0.7)%,(P<0.05)。E2使细胞阻滞在G1期,PD98059、SP600125、SB203580分别预处理1h后细胞分别进一步阻滞在G1期;轻度阻滞在G1期和进入S期。RT-PCR发现PC3细胞表达ERα和ERβ,E2、E2 PD98059、E2 SP600125、E2 SB203580组中cyclinD1、P21WAF1基因表达分别为对照组的(0.42±0.03)、(3.13±0.02)倍;(0.21±0.03)、(3.08±0.05)倍;(0.43±0.01)、(1.31±0.04)倍;(2.81±0.02)、(3.14±0.02)倍(P<0.05)。结论E2激活JNK增加P21WAF1基因表达,并激活p38抑制cyclinD1表达,使细胞阻滞在G1期,JNK和p38通路还介导E2引起PC3细胞凋亡。同时E2又可激活ERK1/2,轻度拮抗上述作用。     

胰腺癌中p16基因甲基化改变及其蛋白表达分析

目的:探讨胰腺癌与癌旁组织中p16基因启动子区异常甲基化的改变及其蛋白表达的特点,以及其与胰腺癌发生发展的关系。方法:分别采用免疫组化SP法及甲基化特异PCR(MSP)检测46例人胰腺癌和癌旁组织中p16基因表达及其甲基化的水平,并结合临床资料进行分析。结果:胰腺癌中p16蛋白表达率为41.3%(19/46),而癌旁组织表达率为95.7%(44/46),两者有差异显著性(P<0.01)。p16蛋白阳性的19例胰腺癌标本中均未检出基因甲基化;p16蛋白缺失的27例标本中有18例检出基因甲基化,甲基化率为39.1%。p16基因甲基化与蛋白缺失有明显关系(P<0.05)。p16基因表达及其启动子区甲基化的发生率与胰腺癌的大小,患者性别﹑年龄的差异无显著意义(P>0.05),但与组织分化程度﹑淋巴结转移﹑PTNM分期有显著意义(P<0.05)。结论:p16基因启动子的异常甲基化可影响p16蛋白的表达,它们与胰腺癌的发生发展有关;p16甲基化和蛋白表达可能成为胰腺癌诊断及预后的候选标志物之一。     

p38丝裂原活化蛋白激酶信号级联在炎症反应中的作用

在急、慢性疾病中，细胞释放的炎症介质可以活化多种信号转导级联反应，丝裂原活化蛋白激酶(MAPK)信号转导通道在招募白细胞于炎症部位聚集起着重要的作用。同时，p38MAPK异构体的活化可以激活致炎细胞因子，而后刺激白细胞活化。然而，p38MAPK引起的白细胞招募这一系列的功能过程包括：粘附、游走和效应器的功能如氧化爆发以及p38MAPK介导的复杂细胞因子网络在炎症中的作用仍有待研究。针对减少炎症介质产生和以p38MAPK为治疗靶点的研究正在进行中，不远的将来可能会成为治疗炎症疾病的新策略。     

脑心康片对脑缺血大鼠细胞凋亡相关蛋白p53表达的影响

目的：观察脑心康片对局灶性脑缺血大鼠的神经保护作用,并进一步探讨其作用机制。方法：采用线栓法阻断大鼠一侧大脑中动脉（MCAO）制备局灶性脑缺血模型,观察脑心康片对MCAO模型大鼠脑缺血后24、48、72h神经功能缺损导致的行为学变化,并以免疫组化法检测其对上述三个时间点大脑皮质细胞凋亡相关蛋白p53表达的影响,以此探讨脑心康片抗细胞凋亡及对脑缺血的神经保护作用。结果：脑心康片能抑制MCAO模型大鼠大脑皮质p53蛋白表达。结论：脑心康片对大鼠局灶性脑缺血具有保护作用,其机理可能与脑心康片影响凋亡相关蛋白p53的表达有关。     

Effect of Inflammation on Costimulation Blockade-Resistant Allograft Rejection

Previously, we reported that allogeneic skin grafts were rapidly rejected by CD28 and CD40 ligand double deficient mice mediated by CD8⁺ T cells. These results indicated that some elements in addition to CD28- and CD40-mediated costimulation provide stimulatory signals for the activation of donor-specific CD8⁺ T cells. In this report, we investigated the role of inflammation associated with transplantation on costimulation-independent priming of CD8⁺ T cell during graft rejection. B6 RAG1 KO mice were transplanted with BALB/c-skin and adoptively transferred with syngeneic CD8⁺ T cells the same day or 50 days after transplantation. When blockade of CD28- and CD40-mediated costimulation failed to prevent acute rejection of freshly transplanted skin grafts, it efficiently delayed rejection of well-healed skin grafts. These results showed that factors associated with transplantation have essential roles in inducing costimulation blockade-resistant allograft rejection. Costimulation blockade failed to prevent acute graft-infiltration of NK cells and increasing expression of intragraft IL-12 and IL-15. These factors may trigger the graft-infiltration and priming of CD8⁺ T cells to induce costimulation blockade-resistant allograft rejection.     

Autotransfusion after open heart surgery: the oxygen delivery capacity of shed mediastinal blood is maintained

Autotransfusion of mediastinal shed blood after open heart surgery has become a common and accepted procedure in reducing the need for homologous transfusion during the last 15 years. The objectives of the present study were to investigate the oxygen delivery capacity of autotransfused shed mediastinal blood, compared to patient-blood, during cardiopulmonary bypass and in the postoperative period.
Ten consecutive patients undergoing elective cardiac surgery were studied. Mediastinal shed blood was collected in the cardiotomy reservoir and retransfused during the first 18 postoperative hours. The oxygen delivery capacity of the blood to the tissues was calculated by use of the oxygen status algorithm (OSA 2.0) programme and measurement of the 2,3-diphosphoglycerate (2,3-DPG) concentration.
Autotransfusion volume ranged from 450–1530 ml per patient (median 824 ml). Shed blood had a mean haemoglobin level of 8.8 g/dl and 7.4 g/dl at 1 h and 6 h of autotransfusion, respectively. There were no significant changes of 2,3-DPG concentration in the patient-blood during cardiopulmonary bypass or after autotransfusion compared to preoperative values. P50 for oxygen (3.6 and 3.6 kPa) and 2,3-DPG concentrations (5.3 and 5.1 mikromol/ml erythrocyte) in shed mediastinal blood (1h and 6h postoperatively) were not significantly different compared to patient-blood.
The results demonstrate that the oxygen delivery capacity of shed mediastinal blood is maintained and that the oxygen affinity of patient-blood is not influenced by autotransfusion.     

Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag? octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH₂ (peptide l), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH₂ (peptide 2), which closely corresponds to the original Flag? sequence. Enrichment of the peptides with ¹⁵N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes. © Munksgaard 1995.     

Genomic Localization and Nucleotide Sequence of a lef-8 Gene of the Spodoptera Littoralis Nucleopolyhedrovirus

A gene similar to lef-8 of the Autographa californica (Ac) nucleopolyhedrovirus (MNPV) was identified in the Spodoptera littoralis (Spli) MNPV. The SpliMNPV lef-8-like gene was localized on the genomic map between 26.9 and 29 map units and is flanked by a chitinase gene and p47 gene. This gene arrangement differs from that of similar genes in the AcMNPV genome, where the lef-8 gene is located about 62 kbp from the chitinase gene and about 7 kbp from the p47 gene. Sequence analyses of the lef-8 gene revealed an ORF of 2730 nucleotides, predicted to encode a protein with M _r 104876. The putative protein is 60.9% identical to the AcMNPV LEF-8 and contains an identical sequence of a conserved motif of DNA-dependent RNA polymerases. Sequences downstream of the lef-8 gene contain two sequence repeats which resemble a repeated motif of the SpliMNPV enhancer element and other repetitive sequences from the viral genome. This revised version was published online in July 2006 with corrections to the Cover Date.