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1.
慢性乙型肝炎中CD4+ CD25+ T调节细胞水平的判定   总被引:6,自引:1,他引:5  
韩捷  陈海燕  李扬  钟惠德  沈培辰 《医学争鸣》2006,27(15):1425-1427
目的:了解慢性乙型肝炎(CHB)患者CD4 CD25 T调节细胞(Tregs)的真实水平. 方法: 采用细胞内外三色荧光标记、流式细胞仪测定、RT-PCR等方法检测CHB患者外周血中Tregs的主要标志CD4 CD25 ,CD4 CD25h,CD4 CD25 Foxp3 细胞和Foxp3 mRNA水平,并观察其与患者肝功能、乙肝病毒复制标志的相关性. 结果: CHB外周血中CD4 CD25 细胞水平与正常无明显差异,CD4 CD25h/PBMC(外周血单个核细胞)则较正常对照组升高[(0.5±0.3) vs (1.1±0.6), P<0.05],而CD25 Foxp3 /CD4 T细胞比值及Foxp3 mRNA水平则较正常明显降低[(4.5±2.7) vs (7.1±2.3)]. RT-PCR结果: △CT:13.98 vs 12.86, △△CT: 1.12 vs 0, 2-△△CT: 0.46 vs 1, P<0.05. 外周血中CD25h T细胞水平与谷丙转氨酶呈正相关(r=0.5,P<0.05),T细胞中CD25(包括高表达)阳性比例与Foxp3阳性的符合率明显低于正常人[(30.5±16.7) vs (54.6±11.1);(57.9±15.9 ) vs (87.8±13.5), P<0.01]. 结论: CHB患者存在着Tregs细胞水平的异常,CD25h的阳性率易受炎症时非调节性T细胞激活而过多表达CD25分子影响,Foxp3作为Tregs判定指标较为可靠.  相似文献
2.
目的:探究1,25二羟基维生素D3[1,25(OH)2D3]对大鼠脾CD4 CD25 调节性T细胞(Treg)及其特异性转录因子Foxp3基因表达的影响.方法:近交系雄性Lewis大鼠,随机分成对照组和3个剂量的VitD3组: 0.125 μg组,0.25 μg组和1 μg组,每组30只;灌胃给药,3次/周,共2周后对照组给予赋形剂;各组随机抽取20只大鼠,于第15 d腹腔注射脂多糖(LPS)(10 mg/kg),随机选择其中10只于6 h后切取脾,其余10只观察注射LPS后96 h大鼠的病死率,同时留取未注射LPS大鼠的脾;流式细胞仪检测脾CD4 CD25 Treg数量变化,RT-PCR检测脾Foxp3 mRNA表达.结果:1,25(OH)2D3明显上调大鼠脾CD4 CD25 Treg的数量及特异性转录因子Foxp3 mRNA的表达;1,25(OH)2D3能保护大鼠抵抗LPS的攻击.结论:1,25(OH)2D3对大鼠脾CD4 CD25 调节性T细胞及特异性转录因子Foxp3 mRNA的表达有显著影响.1,25(OH)2D3保护大鼠抵抗LPS攻击可能与其促进CD4 CD25 Treg的发育和功能有关.  相似文献
3.
 【目的】筛选高效的foxp3 RNA干扰的靶点用于阻断CD4^+CD25^+ Treg细胞的免疫抑制功能。【方法】使用oligoengine软件设计foxp3基因shRNA序列,化学合成法合成4条shRNA序列,构建含foxp3的靶序列的慢病毒载体:构建出表达FLAG融合蛋白和目的基因的工具细胞293T细胞用于筛选RNA干扰的有效靶点。【结果】成功包装了4条foxp3基因靶序列相应的慢病毒颗粒;成功构建了表达融合蛋白和目的基因的293T工具细胞:在工具细胞上筛选出了2条RNAi效率分别为91.3%和95.6%的有效靶点。【结论】使用FLAG融合蛋白和慢病毒载体能够筛选出Foxp3基因的RNAi最佳靶点。  相似文献
4.
Changes of Regulatory T Cells in Graves' Disease   总被引:2,自引:0,他引:2  
The immune mechanism of Graves' diseases (GD) and the roles of regulator T cells were investigated. In 32 patients with GD (GD group) and 20 healthy volunteers (control group), flow cytometry was used to detect the proportion of CD4^+CD25^+ cells, MACS to isolate CD4^+ CD25^+ cells, RT-PCR to assay the expression of FOXP3, and ELISA to test the leyel of IL-10, respectively. It was found that there was no significant change in the proportion of CD4^+CD25^+ T cells between GD group and control group (P〉0.05), while secretion of IL-10 and expression of FOXP3 in GD group were lower than control group (P〈0.01 and P〈0.05, respectively). In conclusion, though the proportion of regulatory T cells of peripheral blood lymphocytes in the patients with GD, the functions of them were significantly weakened, which might be a pathogenic factor in GD.  相似文献
5.
左归丸对小鼠CD4+ CD25+调节性T细胞的影响   总被引:2,自引:0,他引:2  
目的:观察左归丸制剂对小鼠脾细胞中Treg亚群及其相关细胞因子表达的影响。方法:Balb/c小鼠给予不同剂量左归丸处理后,利用流式细胞术检测小鼠脾细胞中Treg亚群(CD4^+/CD25^+)的变化;采用RT-PCR法检测小鼠脾细胞中IL-10、TGF-β、IFN-γ及Foxp3的表达水平;采用ELISA法检测小鼠外周血中IFN-γ的分泌水平。结果:小剂量左归丸对小鼠脾脏Treg亚群及相关细胞因子的表达没有显著影响;中剂量左归丸可明显上调小鼠脾脏Treg亚群比例(P〈0.05),提高Treg特异性胞内信号Foxp3及相关细胞因子IL-10、TGF-β的转录水平,同时抑制,IFN-γ的表达;大剂量左归丸可显著降低Treg细胞亚群比例,对Foxp3、IL-10、TGF-β、IFN-γ的表达均有明显的抑制作用(P〈0.05)。随着中、大剂量左归丸处理小鼠IFN-γ的转录下调,血清中IFN-γ的水平也明显下降(P〈0.05)。结论:左归丸可上调Treg细胞及相关细胞因子的表达水平,抑制IFN-γ的表达,但这种免疫效应有剂量限制性,大剂量应用时显示抑制作用,提示左归丸对Treg亚群有剂量依赖性的双向调节作用。  相似文献
6.
PTD-Foxp3融合蛋白的表达、纯化及其生物学功能初步研究   总被引:2,自引:2,他引:0  
目的:构建带HIV-1 TAT转录因子的穿膜序列(Protein transduction domain,PTD)的pET28a-PTD-Foxp3原核表达载体,表达纯化PTD-Foxp3融合蛋白,并研究其生物学作用.方法:利用基因重组技术构建pET28a-PTD-Foxp3融合蛋白表达载体,并在大肠埃希菌Rosetta(DE3)中表达融合蛋白,经Ni2 分离柱纯化pET28a-PTD-Foxp3融合蛋白.流式细胞术检测融合蛋白穿越细胞膜进入小鼠T淋巴细胞瘤株EL-4细胞中的能力,并通过混合淋巴细胞反应初步分析融合蛋白抑制T细胞活化增殖的生物学作用.结果:成功地构建了pET28a-PTD-Foxp3融合蛋白表达载体,表达并纯化了pET28a-PTD-Foxp3融合蛋白.通过流式细胞术分析证实pET28a-PTD-Foxp3融合蛋白能有效地进入细胞内;同时经混合淋巴细胞反应证明该融合蛋白能明显抑制T细胞的活化增殖能力.结论:成功表达具有生物学活性的PTD-Foxp3融合蛋白,为进一步研究PTD-Foxp3融合蛋白免疫抑制功能和构建表达人PTD-Foxp3融合蛋白,最终应用于临床疾病的治疗奠定了基础.  相似文献
7.
Background FOXP3 was thought to express in the T-cell lineage exclusively until recently when FOXP3 was shown to be expressed by cancer cells. It was indicated that FOXP3 may play a wider role in biology by endowing tumor cells with immune suppressive activity. However, researches between FOXP3 and lymph node metastasis of gastric cancer were relatively infrequent, so the present work was aimed to investigate the relationship between FOXP3 expression and lymph node metastasis in human gastric cancer.Methods A total of 122 gastric cancer patients were enrolled in this study, and gastric tumor specimens and lymph nodes were acquired. Thirty patients who had chronic superficial gastritis diagnosed by gastroscopy contemporaneously in the Peking University People's Hospital were chosen randomly as the control group. Immunohistochemistry was performed to evaluate FOXP3 expression. A survival analysis on the 122 patients was then performed. Then, NCI-N87cell lines were used to confirm FOXP3 expression in gastric carcinoma cells. Finally, evaluation of FOXP3 expression in gastric tumor and peritumor tissues in 12 patients were conducted using immunohistochemistry and Western blotting. A X2 test or Fisher's exact test (bilateral) was conducted to compare the percentage of positive percentage staining between groups. Kaplan-Meier analysis was performed for survival analysis.Results FOXP3 was expressed by gastric cancer cells and peritumor epithelial cells. FOXP3 expression was increased in primary tumors (58.2%) than that in control group (26.7%). In the lymph-node metastasis group, the incidence of lymph node metastasis which was less than 60% had a significant upregulation of FOXP3 in primary tumors and lymph nodes.However, the frequency of FOXP3 expression had no relationship with survival.Conclusion FOXP3 probably has a relationship with lymph node metastasis of gastric cancer.  相似文献
8.
Background Rapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells. Methods Peripheral CD4+CD25- naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4+CD25- T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4+CD25+CD127- subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4+CD25+ T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1×105 carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4+CD25- T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25×105/well) in 96-well round-bottom plates for 6 days. Then 1×105 or 0.25×105 converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4+CD25- T cells using flow cytometry. Data were analyzed using CellQuest software.Results We found that RAPA can convert peripheral CD4+CD25- naive T Cells to CD4+Foxp3+ Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity. Conclusion Our findings provide evidence that RAPA induces Treg cell conversion from Teff cells and uncovers an additional mechanism for tolerance induction by RAPA.  相似文献
9.
哮喘患儿CD4+CD25+调节性T细胞及FOXP3的表达   总被引:2,自引:0,他引:2  
目的:研究CD4+CD25+调节性T细胞在哮喘儿童外周血中的比例改变,并探讨其临床意义.方法:采用细胞内染色的流式细胞术检测急性发作期哮喘患儿外周血CD4+CD25+Treg及其调控基因FOXP3的表达,并与健康对照组进行比较.结果:急性发作期哮喘患儿外周血CD4和CD25双阳性细胞所占比例与健康对照组无明显差异(P>0.05),而CD4+CD25+FOXP3调节性T细胞在外周血中的比例明显低于健康对照组(P<0.01).结论:哮喘患儿外周血CD4+CD25+FOXP3调节性T细胞在外周血中的比例明显减少,可能与哮喘的发病机制有关.  相似文献
10.
携带鼠Foxp3基因的自身失活型慢病毒载体的构建及表达   总被引:2,自引:0,他引:2  
目的 构建携带鼠Foxp3基因的自身失活型慢病毒载体,并研究其在CD4+CD25-T细胞中的表达.方法 构建含鼠Foxp3基因及内部核糖体进入位点序列(IRES)和绿色荧光蛋白基因(GFP)的双顺反子慢病毒载体.采用脂染法将慢病毒载体三质粒转染293T细胞,经共培养法感染免疫磁珠分离的小鼠CD4+CD25-T细胞,流式细胞术(FCM)检测基因转导效率,通过RT-PCR及Western Blotting法分别从mRNA和蛋白水平检测Foxp3的表达.结果 成功构建了慢病毒表达质粒pXZ208-IRES-GFP/pXZ208-Foxp3-IRES-GFP,包装的重组慢病毒滴度达106 IU/mL.以pXZ208-IRES-GFP为阴性对照组,共培养法感染CD4+CD25-T细胞,实验组细胞基因转导效率达55.95%,并且存在Foxp3 mRNA和蛋白水平的高表达.结论 成功构建了携带鼠Foxp3基因的自身失活型慢病毒载体;该系统可有效的介导Foxp3基因在CD4+CD25-T细胞中表达.  相似文献
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