哮喘小鼠骨髓DC细胞因子mRNA的分析

通过体外诱导和扩增小鼠骨髓来源的树突状细胞(DC),研究其中的细胞因子mRNA表达,探讨DC在哮喘中可能的作用机制.为此,利用rmGM-CSF和rmIL-4体外诱导骨髓细胞分化为DC,采用多探针模板的核酸酶保护分析检测骨髓来源的DC中细胞因子mRNA的转录.结果表明,在两种细胞因子的作用下,从骨髓中可以诱导出大量的DC;核酸酶保护分析显示骨髓来源的DC中有IL-13,IL-9和IL-3mRNA的转录,并且哮喘组表达IL-13mRNA和IL-9mRNA的相对水平与对照组相比有明显的差异性,P<0.05;而IL-3mRNA的表达在两组鼠中的表达水平没有差异性,P>0.05.可以认为DC在哮喘的形成和发展中起重要作用,提示了靶向于DC的治疗可能成为哮喘治疗的新目标.     

Transient aggregation of ubiquitinated proteins during dendritic cell maturation

Dendritic cells (DCs) are antigen-presenting cells with the unique capacity to initiate primary immune responses. Dendritic cells have a remarkable pattern of differentiation (maturation) that exhibits highly specific mechanisms to control antigen presentation restricted by major histocompatibility complex (MHC). MHC class I molecules present to CD8(+) cytotoxic T cells peptides that are derived mostly from cytosolic proteins, which are ubiquitinated and then degraded by the proteasome. Here we show that on inflammatory stimulation, DCs accumulate newly synthesized ubiquitinated proteins in large cytosolic structures. These structures are similar to, but distinct from, aggresomes and inclusion bodies observed in many amyloid diseases. Notably, these dendritic cell aggresome-like induced structures (DALIS) are transient, require continuous protein synthesis and do not affect the ubiquitin-proteasome pathway. Our observations suggest the existence of an organized prioritization of protein degradation in stimulated DCs, which is probably important for regulating MHC class I presentation during maturation.     

AF-1对内毒素诱导RAW264．7细胞IL-10表达的影响

目的探讨抗炎素-1（Antiflammin-1,AF-1）对脂多糖（LPS）诱导的RAW264．7细胞白细胞介素10（interleuki-10,IL-10）表达的影响。方法采用体外培养巨噬细胞RAW264．7细胞,实验分为正常对照组,LPS组和不同浓度的LPS＋AF-1组。应用逆转录PCR（RT—PCR）技术检测IL-10表达的变化。结果1μg／ml的LPS刺激RAW264．7细胞后IL-10的表达较正常对照组增加（P〈0．05）,LPS＋AF-1组IL-10表达较LPS组表达显著升高（P〈0．05）,并具有剂量依赖性结论AF-1可促进LPS诱导的RAW264．7细胞IL-10表达的增加。     

Notch signaling stimulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Notch signaling is one of the most important pathways mediating cell determination and differentiation.In this study, the roles of Notch signal in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) were investigated. The expression of Notch1, Jaggedl and DTXI detected by reverse transcrip-tion polymerase chain reaction (RT-PCR) suggested that Notch signal might exhibit a physiological regulatory role in the differentiation of MSCs. Constitutive expression of the intracellular domain of Notchl (ICN), the active form of Notchl protein, can activate Notch signal in cells without ligands‘ binding, hMSCs were isolated, expanded, and infected with retrovirus carrying green fluorescent protein (GFP) gene or ICN. Overexpression of ICN in hMSCs resulted in enhanced osteogenic differentiation induced by dexamethasone (Dex), which was characterized by an increase of cellular alkaline phosphatase (ALP) activity and calcium deposition. These results indicate that Notch stimulates differentiation of MSCs into osteoblasts.     

HIV susceptibility conferred to human fibroblasts by cytomegalovirus-induced Fc receptor

The main receptor for the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) on T and B lymphocytes, monocytes and macrophages is the CD4 antigen 1-3. Infection of these cells is blocked by monoclonal antibodies to CD4(1,2) and by recombinant soluble CD4(4-9). Expression of transfected CD4 on the surface of HeLa and other human cells renders them susceptible to HIV infection 10. HIV-antibody complexes can also infect monocytes and macrophages by means of receptors for the Fc portion of immunoglobulins (FcR)11-13), or complement receptors 14,15. The expression of IgG FcRs can be induced in cells infected with human herpes viruses such as herpes simplex virus type 1 (HSV-1)16,17 and human cytomegalovirus (CMV)18-21. Here we demonstrate that FcRs induced by CMV allow immune complexes of HIV to infect fibroblasts otherwise not permissive to HIV infection. Infection was inhibited by prior incubation with human IgG, but not by anti-CD4 antibody or by recombinant soluble CD4. Once HIV had entered CMV-infected cells by means of the FcR, its replication could be enhanced by CMV transactivating factors. Synergism between HIV and herpes viruses could also operate in vivo, enhancing immunosuppression and permitting the spread of HIV to cells not expressing CD4.     

乳糖诱导mIL-9蛋白高效表达研究

研究了乳糖诱导重组工程菌pET(32a+)-rmIL-9/BL21(DE3)高效表达rmIL-9融合蛋白的实验条件,探讨规模化生产rmIL-9的可行性.分别考察乳糖诱导时机、诱导浓度、诱导时间等参数对rmIL-9融合蛋白表达的影响,并通过正交实验,筛选最佳的乳糖诱导条件.结果显示,对于rmIL-9融合蛋白,乳糖作为诱导剂可达到良好的诱导效果,诱导产物的表达量可优于IPTG诱导产物.最优诱导表达条件为:当菌液OD600值为1时,添加终浓度为5g/L的乳糖,诱导9 h可以高效表达目的蛋白.实验表明,采用乳糖可以诱导rmIL-9融合蛋白基因的高效表达,此为动物实验评价rmIL-9治疗黑色素瘤提供了前期基础.     

熊果酸对H22荷瘤小鼠抗肿瘤及免疫调节作用

目的 探讨熊果酸（UA）对H22荷瘤小鼠抗肿瘤作用及免疫功能的影响.方法 皮下移植建立H22荷瘤小鼠模型,腹腔注射不同剂量UA,检测抑瘤率和免疫器官指数,MTT法检测脾脏T、B淋巴细胞增殖能力,流式细胞术检测CD4＋、CD8＋T细胞亚群含量及比例,ELISA法检测血清细胞因子IL-2、IL-4和TNF-α的表达量.结果 UA对小鼠皮下移植性肿瘤H22有显著的抑制作用,可降低免疫器官中异常增大的脾指数,增强脾脏中T、B淋巴细胞增殖能力,提高淋巴细胞亚群CD4＋T细胞表达及CD4＋/CD8＋T细胞亚群比例,促进血清IL-2、TNF-α表达,降低IL-4表达.结论 UA可抑制小鼠肝癌H22肿瘤生长,体内可以提高荷瘤小鼠的免疫能力,其抗肿瘤作用可能与机体的免疫调节作用相关.     

Characterization of a common precursor population for dendritic cells

Dendritic cells (DCs) are essential for the establishment of immune responses against pathogens and tumour cells, and thus have great potential as tools for vaccination and cancer immunotherapy trials. Experimental evidence has led to a dual DC differentiation model, which involves the existence of both myeloid- and lymphoid-derived DCs. But this concept has been challenged by recent reports demonstrating that both CD8- and CD8+ DCs, considered in mice as archetypes of myeloid and lymphoid DCs respectively, can be generated from either lymphoid or myeloid progenitors. The issue of DC physiological derivation therefore remains an open question. Here we report the characterization of a DC-committed precursor population, which has the capacity to generate all the DC subpopulations present in mouse lymphoid organs---including CD8- and CD8+ DCs, as well as the B220+ DC subset---but which is devoid of myeloid or lymphoid differentiation potential. These data support an alternative model of DC development, in which there is an independent, common DC differentiation pathway.     

卡介苗加脂多糖建立大鼠急性免疫性肝损伤模型的研究

目的　构建接近临床病毒性肝炎发病机制的肝损伤动物模型 ,以便对病毒性暴发性肝炎作进一步的研究。方法　用不同剂量的卡介苗 (BCG)及不同剂量的脂多糖 (LPS)诱导SD大鼠建立急性免疫性肝损伤模型 ,以大鼠血清转氨酶水平变化和肝脏病理学检查等指标作为肝损伤判断标准 ,并以流式细胞仪对该模型的血清中CD3+ 、CD4 + 、CD8+ 细胞百分比计数 ,以ELISA法对TNFα IgG、白介素 (IL 6和IL 10 )水平进行了检测。结果　“BCG +LPS”所造免疫性急性肝损伤模型当以BCG剂量 5× 10 7个活菌 /只及LPS剂量 30 μg kg时ALT升高明显 ,达正常对照 10倍以上 ,AST也较正常对照升高两倍 ;肝脏组织病理损伤中“Ⅲ”级和“Ⅳ”级大于 70 % ,而且其细胞因子水平较正常大鼠升高。对血清中CD3+ 、CD4 + 、CD8+ 细胞百分比也有影响。结论　以“BCG +LPS”所造模型比较成功 ,该模型能充分造成大鼠急性肝损伤 ,且能影响免疫系统 ,与病毒性肝炎的发病机制相似 ,可以用其对病毒性暴发性肝炎进行研究     

体外诱导骨肉瘤特异性抗肿瘤的免疫实验

采用分离骨肉瘤患者外周血单核细胞体外诱导DC细胞,Trizol法提取患者骨肉瘤细胞总RNA,用总RNA转染DC并诱导特异性CTL的扩增,用MTT法检测淋巴细胞的增殖和CTL的杀伤活性。探讨骨肉瘤细胞总RNA转染的DC疫苗体外诱导特异性抗肿瘤免疫的能力。经骨肉瘤细胞总RNA转染的DC特异性表面标志及功能相关分子表达均上调,转染后的DC可显著刺激自体T淋巴细胞增殖,诱导的特异性CTL对靶细胞的杀伤率显著高于单纯淋巴细胞和未经转染的DC。说明骨肉瘤细胞总RNA转染的DC疫苗可在体外诱导出特异性抗肿瘤免疫。     

自体肝癌细胞裂解物致敏的树突状细胞诱导抗肝癌免疫的体外研究

从术后肝癌病人的外周血中诱导树突状细胞（DC），并经自体肝癌细胞裂解物致敏DC，用流式细胞仪、^3H-TdR掺入法及MTT法检测了DC表面分子的表达、DC刺激T细胞的增殖效应及DC诱导的T细胞对肝癌细胞的杀伤作用，进而比较经自体肝癌细胞裂解物致敏的DC与其它条件下的DC功能的差异．结果显示：肝癌细胞裂解物致敏DC的功能较未致敏DC显著提高，其可诱导自体混合淋巴细胞强的增殖效应，同时诱导的T细胞对自体肝癌细胞有较强的杀伤率．     

Skewed maturation of memory HIV-specific CD8 T lymphocytes

Understanding the lineage differentiation of memory T cells is a central question in immunology. We investigated this issue by analysing the expression of the chemokine receptor CCR7, which defines distinct subsets of naive and memory T lymphocytes with different homing and effector capacities and antiviral immune responses to HIV and cytomegalovirus. Ex vivo analysis of the expression of CD45RA and CCR7 antigens, together with in vitro analysis of the cell-division capacity of different memory CD8+ T-cell populations, identified four subsets of HIV- and CMV-specific CD8+ T lymphocytes, and indicated the following lineage differentiation pattern: CD45RA+ CCR7+ --> CD45RA- CCR7+ --> CD45RA- CCR7- --> CD45RA+ CCR7-. Here we demonstrate through analysis of cell division (predominantly restricted to the CCR7+ CD8+ T-cell subsets) that the differentiation of antigen-specific CD8+ T cells is a two-step process characterized initially by a phase of proliferation largely restricted to the CCR7+ CD8+ cell subsets, followed by a phase of functional maturation encompassing the CCR7- CD8+ cell subsets. The distribution of these populations in HIV- and CMV-specific CD8+ T cells showed that the HIV-specific cell pool was predominantly (70%) composed of pre-terminally differentiated CD45RA- CCR7- cells, whereas the CMV-specific cell pool consisted mainly (50%) of the terminally differentiated CD45RA+ CCR7- cells. These results demonstrate a skewed maturation of HIV-specific memory CD8+ T cells during HIV infection.     

HMBA对人成骨肉瘤MG-63细胞形态结构和终末分化指标表达的影响

应用环六亚甲基双乙酰胺处理人成骨肉瘤MG-63细胞,观察MG-63细胞处理前后形态与超微结构及其相关终末分化指标的表达变化.实验结果显示,经5 mmol/L HMBA处理后,MG-63细胞体积增大,趋于扁平铺展状态,细胞大小较为一致,排列较为规则,细胞核形态规则,核质比例减小,核仁减少,核内异染色质减少,常染色质增多,细胞核内的线粒体和高尔基体较为发达,内质网数量增多,细胞表面的微绒毛减少,在成熟细胞中可见钙化糖原颗粒,细胞表面出现钙化小泡沉积,并且形成典型的骨结节.常规细胞化学和免疫细胞化学检测显示,HMBA处理后的细胞中Ⅰ型胶原纤维、骨钙素、骨粘素的表达显著增加,实验结果表明HMBA能够有效诱导人成骨肉瘤细胞的分化,并促进其终末分化指标的表达.     

菠萝蛋白酶诱导K562细胞分化及其对P53基因表达的影响

为进一步证实菠萝蛋白酶对肿瘤细胞的诱导分化作用,采用形态观察计数、血红蛋白定量测定、发光法测定细胞吞噬能力和Ｎｏｒｔｈｅｒｎ原位杂交法观察和检测了菠萝蛋白酶诱导Ｋ５６２细胞分化及其ｐ５３基因表达变化．结果显示,菠萝蛋白酶可诱导Ｋ５６２细胞向红系和粒／巨噬系两个方向分化;在菠萝蛋白酶作用下,ｐ５３基因于给药后８ｈ转录表达增强,２４ｈ达高峰,此后又下降,其时相变化先于分化发生．这些结果提示ｐ５３基因在Ｂｒｏｍｅｌａｉｎ诱导Ｋ５６２细胞分化过程中可能起启动分化的重要作用,有关机制尚待深入研究．     

Engagement of the CD4 molecule influences cell surface expression of the T-cell receptor on thymocytes

The intrathymic differentiation process by which precursor cells derived from the bone marrow develop into immuno-competent T lymphocytes is poorly understood. Most thymocytes express both CD4 and CD8 accessory molecules, yet little is known about either the function of these molecules or the responsiveness of the CD4+8+ double positive thymocytes that bear them. Here, we address the possibility that CD4 engagement influences T-cell receptor (TCR) expression on developing thymocytes. We engaged CD4 molecules on murine thymocytes by in vivo injection of an anti-CD4 monoclonal antibody, which reduced the surface expression of CD4 on CD4+ thymocytes. More importantly, CD4 engagement also affected TCR expression on CD4+ thymocytes, but the effect on CD4+8+ double positive and CD4+8- single positive thymocytes was very different. CD4+8+ thymocytes responded to CD4 engagement by dramatically increasing surface expression of TCR, whereas CD4+8- thymocytes decreased surface expression of TCR. These results demonstrate that the effect of CD4 engagement on TCR expression is dependent upon the developmental state of the responding thymocyte, and, most interestingly, results in increased TCR expression by double positive thymocytes.     

nm23-H1基因与人早幼粒白血病细胞HL-60增殖的相关性分析

目的探讨nm23-H1基因的表达与白血病细胞HL-60增殖之间的关系。方法：以25ng／mL阿糖胞苷处理HL-60细胞，MTT法测定细胞生长抑制率，NBT还原比色法判断细胞分化状况，RT-PCR检测nm23-H1基因表达的变化；构建nm23-H1基因的真核表达质粒pEGFP-N1-nm23-H1，转染HL-60细胞，通过细胞生长曲线和血清依赖性实验检测nm23-H1基因的过表达对HL-60细胞生长的影响。结果：小剂量Ara-C对HL-60细胞的生长呈时间依赖性抑制，作用4d后细胞NBT还原能力增强且nm23-H1基因的表达下调；转染nm23-H1基因的HL-60细胞生长加快、血清依赖性下降。结论：Ara-C对HL-60细胞增殖的抑制作用与下调nm23-H1基因的表达有-定关系；nm23-H1基因在HL-60细胞中的过表达有促细胞增殖的作用，即增高了HL-60细胞的恶性程度。