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1.
目的 观察高迁移率族蛋白B1(high mobility group box-1 protein,HMGB1)对分泌IL-10树突状细胞(dendritic cell,DC)亚群CD11clowCD45RBhigh DC功能的影响. 方法 采用磁珠分选技术获得Bab/c小鼠脾脏CD11clowCD45RBhighDC和CD4+T淋巴细胞,加不同浓度HMGB1处理(20,100,500 ng/ml,以不加HMGB1的细胞作为对照)CD11clowCD45RBhighDC细胞;流式细胞术检测CD11clow CD45RBhigh DC表面分子CD40、CD80、CD86、Ⅰ-a/e及Toll样受体(TLR)4的表达强度;应用ELISA检测CD11clowCD45RBhighDC培养液中IL-10的含量.将CD4+T淋巴细胞分为正常对照组(未作任何处理)、未刺激组(加入未经HMGB1处理的CD11clowCD45RBhighDC与CD4+T淋巴细胞混合培养)、高浓度HMGB1刺激组(加入经500 ng/ml HMGB1处理后的CD11clowCD45RBhighDC与CD4+T淋巴细胞混合培养)、高浓度HMGB1刺激+抗体1组(加入经500 ng/mlHMGB1处理后的CD11clowCD45RBhighDC、抗IL-10抗体与CD4+T淋巴细胞混合培养)和高浓度HMGB1刺激+抗体2组(加入经500 ng/ml HMGB1处理后的CD11clowCD45RBhighDC、IL-10的同型对照抗体与CD4+T淋巴细胞混合培养).流式细胞术检测CD4+T淋巴细胞培养液中IL-4和干扰素-γ(IFN-γ)的含量. 结果 与未加HMGB1刺激相比,HMGB1能显著增强CD11clowCD45RBhighDC表面分子CD86、TLR4的表达及IL-10的分泌,其中IL-10分泌呈HMGB1浓度依赖性.高浓度HMGB1刺激组IFN-γ水平为(279±17) pg/ml,显著低于未刺激组(963±11)pg/ml(P<0.05);高浓度HMGB1刺激组IL-4水平为(372±14) pg/ml,明显高于未刺激组(213±10) pg/ml(P <0.05). 结论 HMGB1可促进CD11clowCD45RBhighDC IL-10的分泌,诱导CD4+T淋巴细胞向Th2型反应分化,通过激活CD11clowCD45RBhighDC介导机体免疫抑制活性.  相似文献   
2.
Objective To observe the effect of regulatory dendritic cells (DCregs) on burn injury induced proinflammatory cytokine production and mortality rate after a single intraperitoneal injection of CD11clowCD45RBhigh DCs to injured mice. Methods DCregs were isolated and purified from spleen of 100 normal BALB/c mice to procure CD11clowCD45RBhigh DCs by MiniMACS. Mice were subjected to a 15% total body surface area (TBSA) burn injury on the back. Twenty mice were used, and splenic DCregs (1×105/ml, 5×105/ml, 10×105/ml) were given to them to investigate the protective effect of DCregs against lethality at postburn hours (PBH) 48, and to decide the optimal dosage of intervention. Another group of 70 mice were used, and they were divided into normal control group (n=7), sham burn injury group (n= 21), burn injury group (n = 21), and DCregs treatment group (n = 21). The mice in burn injury group received intraperitoncally 1 ml of Ringer solution for resuscitation. 10×105/ml of CD11clowCD45RBhigh DCs were added to lactated Ringer solution for intraperitoneal injection in DCregs treatment group. Seven animals of each group were sacrificed at PBH 12, 24 and 48, respectively, and blood samples were collected aseptically for measurement of cytokine levels in plasma and phenotype expressions on DCs by flow cytometry. Results Treatment with 10×105/ml DCregs showed a significant decrease in mortality rate compared with burn injured mice and burn injured mice given lower doses of DCregs (1×105/ml, 5×105/ml DCregs, 0% vs. 80%, 80% and 60%, all P<0. 01). A single intraperitoneal injection of 10×105/ml DCregs to burn injury mice showed a significant decrease in plasma interleukin-6 (IL-6, ng/L: 98. 76 ±10.02, 57. 83 ±6. 83, 13.29 ±1.07) compared with burn injury mice (156.32 ± 12. 85, 84. 50 ±9. 29,23.04±2. 53) at PBH 12, 24 and 48 (all P<0. 01). Similarly, in 10×105/ml DCregs treatment group,plasma macrophage chemoattractant protein-1 (MCP-1) levels (ng/L: 102.79 ±9. 88, 42. 56 ±5. 90,12. 96±1. 34) were markedly lower than those in burn injury group (168. 23±23. 85, 83. 39±8. 41, 42. 92±4. 96) at PBH 12, 24 and 48 (all P<0. 01). A single intraperitoneal injection of 10×105/ml DCregs to burn injury mice showed significant reduction in plasma tumor necrosis factor-α (TNF-α) levels (ng/L: 16. 84±1.92, 16. 62±1.28, 10. 26±1. 10) compared with burn injury mice (24. 16±4. 93, 24.25±4. 01, 17. 91±1.82) at PBH 12, 24 and 48 (all P<0. 01). Conclusion DCregs may effectively improve the outcome of mice with severe burn injury through a single intraperitoneal injection of DCregs accompanied by lowering excessive inflammatory reaction.  相似文献   
3.
Objective To investigate immunomodulatory effect of Astragalus polysaccharides (APS) on IL-12-secreting dendritic cell (DC) subset CD11chigh CD45RBlow DC. Methods Spleen CD11chighCD45RBlow DC and CD4 +T lymphocytes in BALB/c mice were purified by magnetic beads sorting,and were treated with 0 (as control), 50, 100, 200 μg/mL APS. Immunofluorescence staining and flow cytometry were used to determine expressions of CD11chighCD45RBlow DC surface molecules, including CD40,CD80, CD86, I-A/E, and Toll-like receptor (TLR) 4. IL-12 level in CD11chighCD45RBlow DC culture supernatant was determined by ELISA. The CD4+ T lymphocytes were divided into: normal control group,non-stimulation group ( CD4 + T lymphocytes cocultured with APS-unstimulated CD11 chigh CD45RBlow DC ) ,high-dose APS stimulation group (CD4+T lymphocytes cocultured with 200 μg/mL APS-stimulated CD11ch'ghCD45RBlow DC) , high-dose APS stimulation + antibody 1 group ( CD4 + T lymphocytes cocultured with 200 μg/mL APS-stimulated CD11chighCD45RBlow DC and IL-12 antibody), high-dose APS stimulation +antibody 2 group (CD4 +T lymphocytes cocultured with 200 μg/mL APS-stimulated CD11chigh CD45RBlow DC and IL-12 antibody isotype). Proliferation ability of CD4 + T lymphocytes was determined with MTT method.IL-4 level as well as IFN-γ level in CD4 + T lymphocyte culture supernatant was determined by flow cytometry. Data were processed with one-way analysis of variance. Results Compared with those in control, the expressions of CD 11 chigh CD45 RBlow DC surface molecules ( except for CD86 ) on CD 11 chigh CD45RBlow DC surface, as well as IL-12-secreting level with dose-dependence were increased in cells stimulated with 50,100, 200 μg/mL APS. Proliferation ability of CD4 +T lymphocytes in high-dose APS stimulation group was higher as compared with that in non-stimulation group ( F = 13. 438, P <0.05). IFN-γlevel in high-dose APS stimulation group [(2784 ± 137 ) pg/mL] was higher than that in non-stimulation group [(1952 ±101 ) pg/mL, F = 12. 177, P <0.05]. IL-4 level in high-dose APS stimulation group was (172 t 20) pg/mL,which was lower than that in non-stimulation group [( 193 ± 19) pg/mL, F = 11.963, P <0.05]. Proliferation ability of CD4+ T lymphocytes, IFN-γ level, and IL-4 level in high-dose APS stimulation + antibody 1 group were all ameliorated when compared with those in non-stimulation group; while levels of the 3 indexes in high-dose APS stimulation + antibody 2 group were similar to those in high-dose APS stimulation group.Conclusions APS can activate IL-12-producing CD11 chighCD45RBlowDC, and further induce the activation of immune function of T lymphocyte with shifting of Th2 to Th1 in vitro. APS can enhance the immune response via promoting the phenotypic and functional maturation of CD11 chighCD45RBlow DC.  相似文献   
4.
白细胞介素-12是由p35和p40亚单位组成的致炎细胞因子异二聚体。为确定其在腹腔脓毒症中的作用,挪威研究人员将大肠埃希菌(E.coli)分别注入基因剔除小鼠和野生小鼠的腹腔。发现腹膜炎的严重程度与细菌浓度呈剂量依赖关系,表现为腹腔液和血浆中的IL-12p40和IL-12p35的浓度显著升高。而在感染后6h其含量在两组小鼠中差异无显著性,但20h后,p35基因剔除小鼠肝组织匀浆检测发现细菌明显增加,  相似文献   
5.
越来越多的证据表明,氧化还原酶趋化抑制因子(MIF)是调节内毒素以及核转录因子产生的重要因子,因此在严重脓毒症患者免疫治疗中有可能成为一种新的靶因子。而迄今为止,MIF主要采用酶联免疫吸附法检测,对不同白细胞亚群MIF的表达则无法进行分析。最近,德国学者报道了高灵敏的流式细胞术用于MIF细胞内水平检测的方法。这种方法既能应用于经体外培养全血白细胞内MIF的测定,也能用于未经培养的人全血白细胞亚群分析,对T淋巴细胞、B淋巴细胞、  相似文献   
6.
兔骨髓间充质干细胞的分离、培养与鉴定   总被引:1,自引:1,他引:0  
背景:目前对骨髓间充质干细胞常用的分离方法有密度梯度离心法、贴壁筛选分离法。目的:联合应用密度梯度离心法和贴壁筛选分离法体外分离培养、扩增兔骨髓间充质干细胞,并对其进行鉴定。设计、时间及地点:对比观察的细胞学实验,于2007-10/2008-03在上海市第六人民医院中心实验室完成。材料:2月龄新西兰纯种大耳白兔6只用于骨髓间充质干细胞取材与原代培养,1.073kg/L的Percoll分离液。方法:实验采用Percol分离液利用密度梯度离心法及结合贴壁分离筛选法来分离、纯化骨髓间充质干细胞,在采用密度梯度离心法得到骨髓间充质干细胞后,经贴壁培养及反复换液纯化骨髓间充质干细胞。分别取第3,5,7,9代骨髓间充质干细胞,行细胞计数,绘制细胞生长曲线。主要观察指标:倒置显微镜下观察原代及传代细胞的形态、生长情况。采用CD44及CD34抗体进行间接免疫荧光标记鉴定培养的干细胞。CD44染色呈阳性,CD34染色呈阴性,说明所提取、纯化的细胞是骨髓间充质干细胞。结果:增殖传代的骨髓间充质干细胞呈长梭形均匀分布生长,形态比原代培养的细胞更均匀,细胞生长旺盛、增殖迅速,胞核明显,核仁清晰,核浆比例大,细胞形态均匀,平行排列呈螺旋状或漩涡状,传代至第5代时无明显变化。随传代次数的增加,细胞增殖能力逐渐下降,第3~5代细胞增殖能力强。所分离培养的细胞均表达CD44,不表达CD34。结论:在体外采用密度梯度离心及贴壁培养法可获得高纯度的兔骨髓间充质干细胞。  相似文献   
7.
"全厚皮"法根治腋臭   总被引:9,自引:8,他引:1  
目的:探讨采用手工剪除大汗腺至腋部成近“全厚皮”方法治疗腋臭的疗效。方法:从2005年7月~2007年10月共对108例患者采用沿腋窝皱襞1cm切口,手工剪除腋部皮肤与皮下组织之间包含大汗腺在内的一定厚度的皮下组织,直至腋部毛发区域皮肤形成近全厚皮片厚度。术后采用引流、打包包扎、胶布及绷带固定等多种措施至7天后切口拆线愈合。结果:215个腋部中206个气味消除效果为好,9个为一般,0个为差。所有患者术后瘢痕不显,效果满意。结论:采用手工剪除大汗腺至腋部成近“全厚皮”法治疗腋臭是一种可行性强,操作简便,疗效可靠持久,并发症少,外观完美且容易推广的有效方法。  相似文献   
8.
目的为组织工程动脉提供种子细胞 ,从而构建出组织工程化动脉. 方法实验于 2004- 03/05在首都医科大学宣武医院外科实验室完成.采用组织贴块法培养成年狗主动脉平滑肌细胞,并进行细胞传代、鉴定及形态学观察. 结果通过严格取材即可获得高纯度培养的平滑肌细胞,其形态学特征明显,但随培养代次的增加,其增殖能力及形态出现改变. 结论成年狗主动脉平滑肌细胞可作为动脉组织工程的种子细胞,其增殖能力有待于进一步加强.  相似文献   
9.
犬内皮与平滑肌细胞的短期静态联合培养   总被引:2,自引:2,他引:0  
目的:为获得组织工程化自体血管,观察体外静态培养条件下犬内皮细胞与平滑肌细胞联合培养组织学及形态学的特征。方法:实验于2004-07/2005-06在首都医科大学宣武医院外科院级实验室完成。①实验材料:雄性杂种犬,3个月龄,体质量8~12kg。②实验方法:贴块法及酶解法对犬内皮细胞及平滑肌细胞进行原代分离培养及扩增,将第Ⅱ代平滑肌细胞以1×109L-1的密度种植于胶原膜上培养13d,再将第Ⅱ代内皮细胞接种于生长平滑肌细胞的胶原膜上2d。③实验评估:行苏木精-伊红染色同时扫描电镜和透射电镜观察平滑肌细胞和内皮细胞在胶原载体上联合培养后的形态。结果:苏木精-伊红染色见平滑肌细胞较均匀的分布于支架材料表面及内部;扫描电镜下,平滑肌细胞可以在胶原载体材料上生长,增殖明显并在短期内形成多层细胞。内皮细胞与平滑肌细胞联合培养2d就可在平滑肌细胞层表面获得连续的单层内皮细胞层。结论:在短期静态培养条件下犬的血管平滑肌细胞和内皮细胞可以在胶原载体材料上形成具有两层细胞结构的组织工程化动脉血管组织片。  相似文献   
10.
美国新泽西州医学院的外科专家在研究两组出血性休克动物模型时发现,缺氧诱导因子一1(HIF-1)参与了休克后胃肠缺血引起的SIRS和远隔器官损伤。HIF-1作为转录因子,由对氧稳定的HIF-1α和HIF-1β两个亚单位组成,参与调节低氧血症和局部缺血时的多种病理生理反应。研究者观察了两组胃肠道缺血-再灌注大鼠模型中HIF-1的变化。他们发现,大鼠在创伤(剖腹手术)复合出血性休克打击90 min后,回肠黏膜细胞核中HIF-1α含量升高,其肠黏膜细胞HIF-1α蛋白则持续  相似文献   
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