转基因树突状细胞激活细胞毒性T细胞特异性杀伤淋巴瘤的体外实验

目的 探讨转基因树突状细胞激活细胞毒性T细胞产生抗淋巴瘤的特异性细胞免疫反应。方法 采用人骨髓来源的髓系前体细胞,在人细胞因子IL-4、GM-CSF和INF-α诱导下,在体外生成大量树突状细胞。将制备好的含有IgVH1核酸质粒,用脂质体法转染树突状细胞。转染成功的树突状细胞与外周血T淋巴细胞共培养,激活特异性CTL细胞,与阳性表达IgVH1的人淋巴瘤Namalwa细胞反应,用3H-TdR掺入法观察CLLs对瘤细胞的特异性杀伤效应。结果 树突状细胞能够用脂质体方法转染IgVH1核酸质粒,并且有效递呈给外周血T细胞,对表达IgVH1的人淋巴瘤Namalwa细胞产生特异性免疫杀伤活性,与对照组相比差异有显著意义（P<0.01）。结论 体外诱导扩增的 DC能够转染 IgVH1核酸质粒,体外激活T淋巴细胞,产生特异性细胞毒效应。     

Automation of cytokine flow cytometry assays

Cytokine flow cytometry (CFC) is a multiparameter assay of antigen-specific T cell function, potentially useful in the monitoring of experimental vaccines and progression of infectious diseases and cancer. Automation of CFC assays would greatly facilitate their use in clinical trials and involves several components. We describe here the migration of these assays to 96-well plates, the use of sample-handling robotics, and the use of lyophilized antigen and antibody plates to help automate CFC. Together, these elements can produce an integrated system capable of walkaway automation of an entire assay, resulting in the reproducible processing of potentially hundreds of samples per day. Implementation of such systems has begun to be undertaken by our group and others.     

The Role of the Lymphatic System in the Pathogenesis and Treatment of Inflammatory Bowel Disease

Although the number of therapeutic options for the treatment of inflammatory bowel disease (IBD) has increased in recent years, patients suffer from decreased quality of life due to non-response or loss of response to the currently available treatments. An increased understanding of the disease’s etiology could provide novel insights for treatment strategies in IBD. Lymphatic system components are generally linked to immune responses and presumably related to inflammatory diseases pathophysiology. This review aims to summarize findings on immune-mediated mechanisms in lymphoid tissues linked with IBD pathogenesis and (potential) novel treatments. Enhanced innate and adaptive immune responses were observed in mesenteric lymph nodes (MLNs) and other lymphoid structures, such as Peyer’s patches, in patients with IBD and in animal models. Furthermore, the phenomenon of lymphatic obstruction in the form of granulomas in MLNs and lymphatic vessels correlates with disease activity. There is also evidence that abnormalities in the lymphatic stromal components and lymph node microbiome are common in IBD and could be exploited therapeutically. Finally, novel agents targeting lymphocyte trafficking have been added to the treatment armamentarium in the field of IBD. Overall, gut-associated lymphoid tissue plays a key role in IBD immunopathogenesis, which could offer novel therapeutic targets.     

重组人淋巴细胞活化基因-3蛋白胞外段的真核表达及鉴定

目的真核表达重组人淋巴细胞活化基因-3(lymphocyte activation gene-3,LAG-3)蛋白胞外段,并进行鉴定。方法用植物血球凝集素(phytohaemagg lutinin,PHA)刺激Jurkat细胞,流式细胞术检测Jurkat细胞中LAG-3蛋白的表达;提取Jurkat细胞总mRNA,RT-PCR法扩增人LAG-3蛋白胞外段基因片段,同时在蛋白C-末端引入His标签,将其克隆入载体pcDNA3. 1+,构建重组质粒,转染Expi293F真核细胞,当细胞活率低于50%时收获细胞上清,经镍柱亲合层析纯化。纯化产物进行4%~20%SDS-PAGE、HPLC及Western blot分析,BCA法测定浓度。结果经菌液PCR、双酶切及测序鉴定,表明质粒构建正确。重组表达蛋白的相对分子质量约60 000,纯化后纯度达95%以上,与鼠抗LAG-3单克隆抗体可发生特异性结合,浓度为2. 4 mg/mL。结论成功构建了重组真核表达质粒LAG-3/pcDNA3. 1+,并于Expi293F细胞中表达,纯化获得了纯度较高的LAG-3蛋白,为后期LAG-3蛋白的相关研究及其单抗的制备奠定了基础。     

响应面法优化猪脾免疫活性多肽提取工艺研究

目的通过响应面法,优化猪脾中免疫活性多肽的提取工艺。方法选择适当的酶水解猪脾脏,并在单因素试验的基础上,采用Box—Behnken设计优化提取工艺,建立各因素与提取液中多肽含量的数学模型,最后采用淋巴细胞转化试验初步验证其活性。结果获得了猪脾中免疫活性多肽提取工艺：料液比1：2．6g／mL,温度40℃,pH7．0,胰蛋白酶浓度485U／g,中性蛋白酶浓度1711U／g,每100g含活性多肽2．930g,与理论计算值2．929g基本一致,活性约为对照组的1．5倍。结论确定了猪脾免疫活性多肽最佳提取工艺。     

再生障碍性贫血患者铜／锌比对淋巴细胞亚群的影响

目的：研究血清铜、锌及铜／锌比值在再生障碍性贫血中的水平变化,以及对外周血 T 淋巴细胞亚群的影响。方法选择50例再生障碍性贫血患者（初治再障组）,经过正规治疗,可随访患者42例[根据治疗方法不同分为标准治疗组（22例,应用雄激素＋免疫抑制剂治疗）和葡萄糖酸锌治疗组（20例,在标准治疗组基础上加用萄糖酸锌口服液治疗）]。选择同期40例健康正常人为正常对照组。采用原子吸收分光光度法测定血清铜、锌及铜／锌比值,流式细胞仪检测患者外周血 T 淋巴细胞亚群。结果初治再障组患者血清铜含量及铜／锌比值均高于正常对照组,而锌含量明显低于正常对照组（均 P ＜0．01）。再生障碍性贫血患者铜／锌比值与 CD4＋／CD8＋呈负相关（r＝"0．45,P ＝0．005）。治疗后葡萄糖酸锌治疗组患者 CD4＋／CD8＋比值恢复好于标准治疗组（P ＝0．000）。结论铜／锌比值与 T 淋巴细胞亚群失调共同参与再生障碍性贫血的发病及转归。     

纳洛酮对大鼠中重度创伤性脑损伤后细胞免疫功能影响的实验研究

目的：通过观察大鼠创伤性脑损伤（traumatic brain injury,TBI）后纳洛酮治疗前后血浆β-EP、CD4＋、CD8＋和IL-2的动态变化,探讨纳洛酮对大鼠TBI后细胞免疫功能变化的影响及作用机制。方法：采用气体冲击致大鼠中重度脑损伤模型;流式细胞术、RIA、ELISA检测血液中CD4＋、CD8＋、β-EP、IL-2的变化。结果：TBI大鼠经纳洛酮治疗后,血浆中CD4＋和IL-2含量升高,β-EP和CD8＋则降低。大、小剂量纳洛酮治疗在相应时间点比较有显著性差异（P〈0.05）。结论：纳洛酮可通过降低TBI大鼠血浆中β-EP,调节TBI后应激紊乱,竞争性抑制β-EP对免疫细胞受体的作用,从而使CD4＋、CD8＋和IL-2水平趋于正常,恢复免疫平衡,起到治疗与保护作用。     

母乳婴儿源乳杆菌的筛选及其免疫调节能力研究

以分离自母乳婴儿源的乳杆菌——鼠李糖乳杆菌BF-1、植物乳杆菌BF-15、唾液乳杆菌BF-29、干酪乳杆菌BF-55为对象,研究这些菌株对体外模拟人工胃、肠液及胆盐的耐受性,对Caco-2细胞的黏附能力、安全性和对小鼠脾淋巴细胞增殖活性的影响,探讨菌株的免疫调节活性。试验结果表明:植物乳杆菌BF-15对人工消化液和胆盐有较强的耐受性,对Caco-2细胞黏附能力[(7.10±0.30)CFU/cell]显著高于阳性对照菌株LGG[(3.90±0.30)CFU/cell](P<0.05);BF-15除对氨基糖苷类、糖肽类抗生素的固有耐药性外,对苯唑西林、头孢噻吩也有耐药性,无抗性质粒;BF-15和LGG的活性和热致死菌在一定的菌浓范围(1×10^6~10^7CFU/mL)均促进体外小鼠淋巴细胞增殖,表现出剂量依赖关系,同时活性菌株作用效果明显优于热致死菌株。在相同菌浓条件下,两株菌体外免疫调节能力没有显著性差异(P>0.05)。母乳婴儿源乳杆菌——植物乳杆菌BF-15具有较强的抗逆性,较高的黏附性和一定的免疫调节能力。     

Transplanted Neural Stem Cells Modulate Regulatory T, γδ T Cells and Corresponding Cytokines after Intracerebral Hemorrhage in Rats

The immune system, particularly T lymphocytes and cytokines, has been implicated in the progression of brain injury after intracerebral hemorrhage (ICH). Although studies have shown that transplanted neural stem cells (NSCs) protect the central nervous system (CNS) from inflammatory damage, their effects on subpopulations of T lymphocytes and their corresponding cytokines are largely unexplored. Here, rats were subjected to ICH and NSCs were intracerebrally injected at 3 h after ICH. The profiles of subpopulations of T cells in the brain and peripheral blood were analyzed by flow cytometry. We found that regulatory T (Treg) cells in the brain and peripheral blood were increased, but γδT cells (gamma delta T cells) were decreased, along with increased anti-inflammatory cytokines (IL-4, IL-10 and TGF-β) and decreased pro-inflammatory cytokines (IL-6, and IFN-γ), compared to the vehicle-treated control. Our data suggest that transplanted NSCs protect brain injury after ICH via modulation of Treg and γδT cell infiltration and anti- and pro-inflammatory cytokine release.