内皮抑素基因在其治疗的G422小鼠胶质母细胞瘤中的表达

目的观察内皮抑素（End）在携带小鼠内皮抑素基因的腺病毒（Ad-mEnd）治疗的G422荷瘤小鼠瘤体内的表达。方法用昆明种G422皮下荷瘤小鼠24只，在成瘤直径约3mm时分为空病毒保存液组、携带绿色荧光蛋白基因的腺病毒（Ad-GFP）组、Ad-mEnd单剂量组和Ad-mEnd重复给药组。各组小鼠瘤体内各注入相应试剂。用RT-PCR和western blot等方法测定End在肿瘤内的表达。结果Ad-mEnd单剂量组和重复给药组的mRNA值分别为2．73和5．14；重复给药组持续高水平表达，而单剂量组只能维持2周。结论End在Ad-mEnd治疗的小鼠G442瘤体内有良好表达，多次治疗可维持End的表达量。     

脑胶质细胞瘤病人IL-2和sIL-2R表达及细胞免疫功能变化的意义

目的 研究脑胶质细胞瘤病人外周血中自细胞介素2（IL-2）、可溶性白细胞介素2受体（sIL-2R）的表达以及红细胞免疫和T淋巴细胞亚群的变化规律．探讨它们之间的相互关系。方法 对55例脑胶质瘤病人及55例健康献血员．采用酶联免疫吸附（ELISA）法测定血清IL-2、sIL-2R含量，免疫黏附法测定红细胞免疫活性及其调节功能，链亲和素一过氧化物酶（SP）一步法测定CD3、CD4、CD8细胞数。结果 脑胶质细胞瘤组IL-2含量较对照组显著性降低（P〈0.01），sIL-2R则显著性升高（P〈0．01）；红细胞C3b受体（RBC-C3bR）、红细胞免疫调节促进因子（RFER）亦显著性降低（P〈0．01），而红细胞免疫复合物（RBC-ICR）、红细胞免疫调节抑制因子（RFm）则显著性升高（P〈0．01）；CD3、CD4细胞数显著性降低（P〈0．001），而CD8无显著性差异（P〉0．05）。结论 脑胶质细胞瘤病人存在免疫功能低下；检测血清IL-2和sIL-2R含量、红细胞免疫功能及T细胞亚群活性，对脑胶质细胞瘤病人的免疫机制研究具有重要意义。     

PHA、CD3、与IL-2协同诱导PBMC过继免疫治疗恶性脑胶质瘤的实验研究

目的 提高人脑胶质瘤过继免疫治疗的效果,探索治疗脑胶质瘤的新途径。方法 用植物血凝素（PHA）、抗CD3单抗（CD3）和IL－2共同诱导人外周血单个核细胞（PBMC）,诱导,扩增新型抗胶质瘤效应细胞PHA－CD3AK细胞,并与CD3AK细胞在某些生物学方面进行了比较。结果 两组效应细胞增殖曲线均于第6天达高峰,峰值可见,PHA－CD3AK细胞＞CD3AK（P＜0．05）;PHA－CD3AK细胞扩增倍数明显高于CD3AK细胞（P＜0．05）;两组效应细胞于培养的第6、8、10天所测得的杀伤胶质瘤细胞BT325活性可见,PHA－CD3AK细胞＞CD3AK细胞（P＜0．05）。结论 PHA、CD3和IL－2具有协同增强作用,使PHA－CD3AK细胞成为较CD3AK细胞增殖能力、杀伤活性更强的免疫效应细胞、且IL－2用量减少,为胶质瘤的过继免疫治疗打下了理论基础。     

MBP刺激下IL-3和IL-4对树突状细胞前体定向分化的影响

目的 观察在髓鞘碱性蛋白(myelin basic protein,MBP)刺激下,不同细胞因子对树突状细胞(dentritic cell,DC)前体定向分化的影响.方法 分离实验性自身免疫性脑脊髓炎(EAE)易感性BALB/c小鼠脾脏DC.在体外不同培养条件下与MBP共培养.用流式细胞仪检测DC荧光抗体标记的CD11c和CD8α表达.结果 (1)在MBP和粒巨嗜细胞集落刺激因子(GM-CSF)培养条件下,白细胞介素-4(IL-4)能明显刺激DC CD11c表达,IL-4组其平均荧光强度为856.93±2.45,对照组为708.58±21.25,两组比较差异有统计学意义(P＜0.01);(2)在GM-CSF和MBP培养条件下,ID3能明显上调DC CD8α的表达,IL-3组平均荧光强度为7751.70±296.63,对照组为7279.17±176.77,两组比较差异有统计学意义(P＜0.01).结论 IL-4可进一步促进BALB/c小鼠脾脏DC前体向DCl分化,而IL-3则促进DC前体向DC2分化.     

腺病毒介导D2S基因联合溴隐亭治疗对GH3细胞生长的抑制作用

目的 探讨腺病毒介导多巴胺2型受体短链亚型基因(D2S)联合溴隐亭对大鼠垂体生长激素腺瘤GH3细胞株体外生长活性的影响.方法 应用携带人D2S基因及增强型绿色荧光蛋白(EGFP)报告基因的复制缺陷型腺病毒载体pAd - D2S - EGFP感染大鼠GH3细胞,RT - PCR、Western - blot法检测转染后细胞D2S mRNA及蛋白的表达,并采用CCK -8法检测转染D2S基因后联合溴隐亭药物干预治疗,对GH3细胞生长抑制及诱导凋亡作用,运用光镜及荧光显微镜检测联合作用后细胞形态变化.结果 GH3细胞转染D2S基因后检测到D2S基因的转录及蛋白表达较对照组增高.腺病毒转染D2S基因联合溴隐亭干预后第2天,体外培养的GH3细胞存活率明显下降(32.76±3.88)％,与单纯加药组的(101.78±2.05)％及空载体组的(91.79±3.80)％相比,差异有统计学意义(P＜0.01).光镜下及荧光显微镜结合Hoechst核染色观察到细胞形态出现皱缩脱落、死亡、核碎片等改变.结论 腺病毒介导D2S基因联合溴隐亭能有效抑制GH3细胞的增殖.     

左旋丁基苯酞对乳鼠脑胶质细胞氧糖剥夺/复氧后脑胶质细胞数量及IL-1β蛋白表达的影响

目的观察左旋丁基苯酞(L-3-n-butylphthalide,l-NBP)对原代培养乳鼠脑胶质细胞氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)后脑胶质细胞数量及白细胞介素-1β(IL-1β)蛋白表达的影响。方法采用DMEM培养基体外培养乳鼠脑胶质细胞,免疫细胞化学鉴定培养细胞类型,建立脑胶质细胞OGD/R损伤模型,通过测定细胞OGD/R时乳酸脱氢酶(LDH)释放量,评估脑胶质细胞OGD模型。OGD 24h/R 24 h后,采用MTT法测定脑胶质细胞数量,并观察l-NBP对OGD 24 h/R 24 h后脑胶质细胞数量的影响;采用间接酶联免疫吸附法(indirect enzyme-linked immunosorbent assay,ELISA)测定各组脑胶质细胞IL-1β蛋白表达。结果 OGD 4 h组、OGD 8 h组、OGD 12 h组、OGD 24 h组脑胶质细胞LDH释放量均比正常对照组明显增多(P<0.01);OGD不同时间组组间比较差异有统计学意义(P<0.01)。OGD 24 h/R 24 h后脑胶质细胞数量较对照组明显增加(P<0.01);l-NBP 500μmol/L组脑胶质细胞数量较OGD 24 h/R 24 h组明显减少(P<0.05)。OGD 24 h/R 24 h组IL-1β蛋白表达比对照组明显增多(P<0.05);l-NBP 100和500μmol/L组IL-1β蛋白表达较OGD 24 h/R 24 h组明显减少(P<0.05)。结论 l-NBP能减少乳鼠脑胶质细胞OGD/R后细胞数量以及IL-1β蛋白的表达。     

lncRNA NEAT1沉默通过IL-10/STAT3信号通路调节小胶质细胞极化对脑缺血再灌注损伤大鼠的影响

目的 探讨长链非编码RNA核富集转录体1(lncRNA NEAT1)沉默通过白细胞介素(IL)-10/信号传导与转录激活因子3(STAT3)信号通路调节小胶质细胞极化对脑缺血再灌注损伤(CIRI)大鼠的影响。方法 SD大鼠随机分为假手术组(Sham组)、CIRI组、CIRI+si-NC组、CIRI+si-NEAT1组,每组24只。除Sham组外,其余各组大鼠采用线栓法构建CIRI模型。建模结束后,各组大鼠给予对应处理7 d。对大鼠神经功能缺损进行评分;观察脑组织病理学变化;检测脑梗死体积百分比,脑组织中离子钙接头蛋白分子1阳性(Iba1+)细胞中M1型、M2型极化标志物阳性(Iba1+CD86+、Iba1+CD206+)细胞的数量,IL-1β、肿瘤坏死因子-α(TNF-α)、IL-4、IL-10,lncRNA NEAT1、诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg-1)、IL-10 mRNA,IL-10、STAT3、磷酸化STAT3(p-STAT3)蛋白水平。结果 与Sham组相比,CIRI组大鼠脑组织病理损伤严重,神经功能缺损评分、脑梗死体积百分比、脑组织中Iba1+CD8...     

逆转录病毒介导人肿瘤坏死因子基因治疗胶质瘤的实验研究

运用小鼠白血病病毒MoMLV为载体,将TNFa基因转导致人胶质瘤细胞SHG-44,14dG418筛选出细胞克隆.检测TNFa的表达.观察细胞生长.结果:转基因细胞培养上清液中有高浓度TNFa表达分别为(5 198.7±3 757.4)pg/ml和(3 217.4±1 180.6)pg/ml(P<0.05),生物活性达320.0U/ml.“RT-PCR”检测转基因细胞有特异mRNA表达,转基因细胞生长减慢.转染TNFa基因可诱导胶质瘤细胞的生长抑制.     

p53基因增强恶性胶质瘤HSV-TK/ACV系统疗效的体外研究

目的 :体外研究p5 3基因提高恶性胶质瘤HSV TK/ACV系统自杀基因治疗疗效的可行性。方法 :以腺病毒为载体转导p5 3和HSV TK基因入C6鼠胶质瘤细胞 ,给以不同浓度的ACV ,MTT法监测细胞生存率 ,TUNEL法检测凋亡。评价p5 3基因提高HSV TK/ACV系统治疗胶质瘤的疗效。结果 :p5 3明显提高鼠胶质瘤细胞对HSV TK/ACV的敏感性 ,p5 3联合HSV TK转染C6的ACVID50 (1μg·mL-1)和ID10 0 (10 μg·mL-1)较单独HSV TK转染C6的ACVID50 和ID10 0 提高 10倍 ,并可使C6细胞凋亡增加。结论 :p5 3基因可明显增强胶质瘤HSV TK/ACV系统的体外抑瘤作用     

人参皂甙、CD3与IL-2协同诱导PBMC治疗恶性脑胶质瘤的实验研究

目的　提高人脑胶质瘤过继免疫治疗的效果 ,探索治疗脑胶质瘤的新途径。方法　用人参皂甙 (GS)、抗CD3单抗 (CD3 )和 IL-2共同诱导人外周血单个核细胞 (PBMC) ,诱导、扩增新型抗胶质瘤效应细胞 GS-CD3 AK细胞 ,并与 CD3 AK细胞在某些生物学方面进行了比较。结果　两组效应细胞增殖曲线均于第 6天达高峰 ,峰值可见 GS-CD3 AK细胞 >CD3 AK细胞 (P <0 .0 5 ) ;GS-CD3 AK细胞扩增倍数明显高于 CD3 AK细胞 (P <0 .0 5 ) ;两组效应细胞于培养的第 6天所测得的杀伤恶性脑胶质瘤细胞 BT3 2 5活性可见 GS-CD3 AK细胞 >CD3 AK细胞 (P <0 .0 5 )。结论　GS、CD3和 IL-2具有协同增强作用 ,使 GS-CD3 AK细胞成为较 CD3 AK细胞增殖能力、杀伤活性更强的免疫效应细胞 ,且 IL-2用量减少 ,为胶质瘤的过继免疫治疗打下了理论基础。     

NSAIDS inhibit the IL-1β-induced IL-6 release from human post-mortem astrocytes: the involvement of prostaglandin E2

Epidemiological studies have shown that steroidal as well as non-steroidal anti-inflammatory drugs lower the risk of developing Alzheimer's Disease (AD). A suppressive effect of these anti-inflammatory drugs on local inflammatory events in AD brains has been suggested, however the mechanisms responsible are still unknown. In this study we investigated at cellular level the influence of two anti-inflammatory drugs—dexamethasone and indomethacin—and an experimental specific cyclooxygenase-2 inhibitor, BF389, on the production of the pro-inflammatory cytokine IL-6 and the inflammatory mediator PGE₂ by human astrocytes. Two human post-mortem astrocyte cultures (A157 and A295) and astroglioma cell lines (U251 and U373 MG) were found to secrete considerable amounts of IL-6 upon stimulation with IL-1β. The glucocorticoid dexamethasone inhibited the IL-1β-activated release of IL-6 from the postmortem astrocyte cultures A157 and A295 and from the astroglioma cell lines. The non-specific cyclooxygenase inhibitor indomethacin and BF389 only suppressed the IL-6 release by post-mortem astrocyte culture A157. This post-mortem astrocyte culture was found to produce large amounts of PGE₂ upon stimulation with IL-1β, whereas in the supernatants of the postmortem astrocyte culture A295 and the astroglioma cell lines, low PGE₂ concentrations were detected. Addition of exogenous PGE₂ prevented the inhibitory effect of indomethacin and BF389 on the IL-1β-activated IL-6 release from A157 astrocytes and largely potentiated the IL-1-induced release of IL-6 from all astrocytes/astroglioma cells tested. Dexamethasone also inhibited the PGE₂ release from the astrocytes and astroglioma cells, however the inhibitory effect of dexamethasone on the IL-1β-activated IL-6 release could not be prevented by the addition of PGE₂. The observed reduction of IL-6 and/or PGE₂ from astrocytes may be involved in the mechanism underlying the beneficial effects of these drugs in AD.     

侧脑室注射质粒pLXSN介导bcl-2基因对大鼠脑梗死及BDNF影响的研究

目的 研究大鼠短暂大脑中动脉闭塞（MCAO）模型经侧脑室注射质粒pLXSN介导bcl-2基因后，该基因对脑梗死及脑源性神经营养因子（brain-derived neurotrophic factor,BDNF）表达的影响。方法取135只Wistar大鼠随机分配为3组,每组45只．对照组为脑室注射生理盐水和空质粒两组．治疗组为脑室注射Bcl-质粒pLXSN介导bcl-2基因。以改良线栓法制备短暂缺血2h脑梗死模型。每组分别于缺血后3、6、24、48、72h为时间点。分别测量梗死体积变化以及BDNF蛋白的表达。结果MCAO后24、48、72h．梗死体积治疗组显著小于对照组（P〈0．05）；3、6h时间点无显著变化（P〉0．05）。MCAO后24、48、72h。治疗组Bcl-2和BDNF蛋白的表达较对照组显著增加（P〈0．01）．而3、6h时间点无显著改变（P〉0．05）。结论脑室注射质粒pLXSN介导bcl-2基因对短暂脑缺血有治疗作用．并可增加BDNF表达。上调BDNF表达所起的神经保护作用可能是短暂脑缺血后bcl-2基因的治疗作用机制之一。     

基因芯片检测难治性癫脑组织中sh3gl2的表达

目的：运用基因芯片和RT-PCR技术检测难治性癫脑组织中sh3gl2 mRNA表达,从分子水平探讨难治性癫可能的发病机制。方法：在应用基因芯片对难治性癫患者手术切除的颞叶组织与对照组行基因表达谱分析研究的基础上,筛选出目的基因后用RT-PCR对芯片扫描结果进行验证。结果：候选基因sh3gl2在难治性癫患者脑部颞叶组织中出现高表达,与对照组相比差异有显著统计学意义（P〈0.01）;RT-PCR结果与芯片结果一致。结论：sh3gl2在难治性癫颞叶皮质中的表达增加,提示了其可能是难治性癫发生发展中的一个重要因素。     

Changes in expressions of proinflammatory cytokines IL-1β, TNF-α and IL-6 in the brain of senescence accelerated mouse (SAM) P8

The senescence-accelerated mouse (SAM) is known to be a murine model for accelerated aging. The SAMP8 strain shows age-related deterioration of learning and memory at an earlier age than control mice (SAMR1). In the present study, we investigated the changes in expressions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the brain of SAMP8. In the hippocampus of 10 months old SAMP8, the expression of IL-1 mRNA was significantly elevated in comparison with that of SAMR1. In both strains of SAMs, increases in IL-1β protein in the brain were observed at 10 months of age compared with 2 and 5 months. The only differences found between the strain in protein levels were at 10 months and were elevations in IL-1β in the hippocampus and hypothalamus, and in TNF-α and IL-6 in the cerebral cortex and the hippocampus in SAMP8 as compared with SAMR1. However, lipopolysaccharide-induced increases in the expression of these cytokines in brain did not differ between SAMP8 and SAMR1. Increases in expression of proinflammatory cytokines in the brain may be involved in the age-related neural dysfunction and/or learning deficiency in SAMP8.     

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice

The pro-inflammatory cytokine interleukin-1β (IL-1β) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC₅₀ = 20–150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1β release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED₅₀ = 100 μmol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED₅₀ = 40 μmol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1αβ knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1β.     

Inhibition of interleukin-1β and prostaglandin E2 thermogenesis by glycyl-glutamine, a pro-opiomelanocortin-derived peptide

Interleukin-1β (IL-1β) and other cytokines produce fever by stimulating prostaglandin E₂ (PGE₂) synthesis in thermoregulatory regions of the preoptic area and anterior hypothalamus (POA/AH). Prostaglandin E₂ is thought to raise body temperature, at least in part, by stimulating β-endorphin release from pro-opiomelanocortin neurons that innervate the POA/AH. In this study, we investigated whether glycyl-glutamine (β-endorphin_30–31), an inhibitory dipeptide synthesized from β-endorphin post-translationally, inhibits IL-1β and PGE₂-induced hyperthermia. Hyperthermic sites were identified by microinjecting PGE₂ (3 fmol/1 μl) into the medial preoptic area (mPOA) of conscious, unrestrained rats. Interleukin-1β (1 U) injection into the same PGE₂ responsive thermogenic sites in the mPOA elicited a prolonged rise in colonic temperature (T_c) (+1.02±0.06°C) that persisted for at least 2 h. Glycyl-glutamine (3 nmol) co-injection into the mPOA inhibited IL-1β thermogenesis completely (T_c=−0.18±0.22°C). Glycyl-glutamine had no effect on body temperature when given alone to normothermic rats. Co-injection of individual amino acids, glycine and glutamine (3 nmol each amino acid), failed to influence IL-1β-induced thermogenesis, which indicates that Gly-Gln hydrolysis does not explain its inhibitory activity. Glycyl-glutamine (3 nmol) also prevented the rise in body temperature produced by PGE₂ (PGE₂=0.89±0.05°C; PGE₂ plus Gly-Gln=−0.16±0.14°C), consistent with evidence that PGE₂ mediates IL-1β-induced fever. These findings demonstrate that Gly-Gln inhibits the thermogenic response to endogenous pyrogens.