单核细胞源性树突状细胞表达淋巴细胞趋化因子mRNA的初步研究

目的:体外诱导、培养单核细胞源性树突状细胞(DC),研究其淋巴细胞趋化因子(lymphotactin,Lptn)mRNA表达的动态变化。方法:采用密度梯度离心的方法分离人外周血中的单个核细胞(PBMC),用重组人粒细胞-巨噬细胞集落群体刺激因子(rhGM-CSF)、重组人白细胞介素-4(rhIL-4)刺激贴壁的单核细胞,诱导培养DC,第6天用重组人肿瘤坏死因子-α(rhTNF-α)诱导DC成熟。用流式细胞术检测成熟和未成熟的DC表面分子CD1a和CD83;在电镜下观察成熟DC的形态;以RT-PCR法扩增其LptncDNA并克隆至pGM-TEasyT载体中,测序;以RT-PCR结合凝胶成像分析系统,半定量分析培养3、5及7dDC的LptnmRNA表达的强度。结果:电镜观察培养7d的细胞具有典型的DC形态,流式细胞术检测DC表面分子CD83呈高水平表达。用RT-PCR法克隆的cDNA序列与GenBank中U23772(登陆号)提供的序列一致。培养3d的DC不表达LptnmRNA,培养7d的DC较培养5d的DCLptnmRNA表达增强。结论:单核细胞源性DC能表达LptnmRNA,随着DC的成熟,LptnmRNA的表达增强。     

多器官功能障碍综合征小鼠肺间质树突状细胞的病理学观察

目的探讨肺间质树突状细胞在多器官功能障碍综合征（MODS）免疫紊乱及脏器损伤机制中的变化与作用。方法C57BL／6小鼠腹腔注射酵母多糖复制MODS模型,分为正常、3—6h（致伤早期）、12～48h[失控性全身炎性反应（SIRS）期]、5～7d（恢复期）和10～12d（MODS期）组。光镜与电镜观察各组小鼠肺及间质树突状细胞的病变;运用免疫组织化学方法检测间质树突状细胞表面标记物CD11c和CD205,共刺激分子CD80和CD86在肺中的表达水平;逆转录-聚合酶链反应法检测趋化因子SLC及其受体CCR7在肺中的表达情况;流式细胞术检测MODS各期小鼠外周血CD4^＋与CD8^＋的T淋巴细胞数量与比值。结果致伤早期,肺间质树突状细胞显著增生,共刺激分子CD80和CD86低水平表达,趋化因子SLC及其受体CCR7在肺组织中表达水平开始上升,外周血T淋巴细胞CD4^＋／CD8^＋比值下降;SIRS期,间质树突状细胞继续增生,CD80和CD86标记阳性细胞数显著上升（与正常组比较均P〈0．01）,SLC与CCR7在肺组织中表达明显高于正常组（均P〈0．01）,外周血T淋巴细胞CD4^＋／CD8^＋比值明显下降（与正常组比较P〈0．01）;MODS期,肺间质树突状细胞高度增生,但CD80和CD86表达显著减少（与SIRS期比较P〈0．01）,肺组织中SLC表达水平继续上升,而CCR7表达水平明显下降（与SIRS期比较P〈0．01）,外周血T淋巴细胞CD4^＋／CD8^＋比值显著下降。结论肺间质树突状细胞在MODS中的变化可能参与并影响了MODS病程中的免疫失衡与免疫抑制过程。CCR7的表达水平可以作为估价间质树突状细胞迁移活性和机体免疫应答水平的一个指标。     

人CD23基因的扩增及其在HEK 293T细胞的高效表达

目的建立一个有效表达CD23的系统。方法采用RT-PCR方法,从外周血淋巴细胞中扩增人的CD23cDNA基因,并将其克隆到真核表达载体pcDNA3·1B上,构建成pcDNA3·1/CD23质粒表达载体;将pcDNA3·1/CD23质粒转染HEK293T细胞,通过流式细胞仪和Westernblot检测CD23在细胞中的表达情况。结果扩增的CD23cDNA序列与文献报道的一致;通过构建表达载体和转染细胞实现了CD23cDNA在293T细胞膜上的表达。转染效率达80%以上,并获得了较高水平的表达。结论扩增CD23cDNA基因,经过转染HEK293T细胞,实现了CD23在HEK293T细胞的高效表达。     

趋化因子受体4基因重组腺病毒载体的构建及鉴定

背景：目前研究发现,基质细胞衍生因子1/趋化因子受体4轴具有介导骨髓间充质干细胞定向迁移的作用。 目的：构建小鼠趋化因子受体4基因重组腺病毒载体。 方法：从C57BL/6小鼠中提取总RNA,以RT-PCR方法获得小鼠趋化因子受体4基因全长1 080 bp的完整编码序列,通过穿梭质粒pAdTrack-CMV,将目的片段克隆入AdEasy-1腺病毒DNA中,获得重组腺病毒DNA,通过脂质体转染293细胞,经包装扩增后,获得重组腺病毒pAdTrack-CMV-CXCR4,并感染293细胞,PCR鉴定、Western blot检测蛋白表达。 结果与结论：含趋化因子受体4基因的重组腺病毒pAdTrack-CMV-CXCR4构建成功,经PCR鉴定鉴定重组病毒中含有趋化因子受体4cDNA全长,经序列测定表明趋化因子受体4cDNA序列正确无丢失和错配现象,病毒滴度高,Western blot检测感染后293细胞趋化因子受体4的蛋白表达较未感染293细胞明显增加。     

人CD154基因真核细胞表达载体的构建、鉴定于序列分析

目的 构建人CD154基因真核细胞表达载体。方法 采用RT-PCR方法,从激活的人外周血淋巴细胞的总cDNA中扩增得到820bp的人CD154cDNA片段,再用BamHI和EcoRI双酶切后定向克隆到真核细胞表达载体PcDNA3.1中,限制性内切酶酶切分析重组质粒和DNA序列分析。结果 人CD154cDNA已经正确克隆到真核细胞表达载体pcDNA3.1中。结论本研究为探讨CD154分子与淋巴细胞活化的关系及其信号传导机制。为进一步用于抗移植排斥反应的研究,或对由于CD154遗传缺陷所致的免疫缺陷病的基因治疗奠定了基础。     

人IgG Fc基因的扩增及其在真核细胞中的分泌性表达

目的:通过扩增人IgG Fc基因并与人CD5基因信号肽序列融合,构建真核分泌型表达载体,实现在真核细胞中高效表达,为利用Fc基因制备融合抗原,研究抗原基因和抗原蛋白的免疫治疗奠定了基础.方法:从外科手术切除的扁桃体中分离人淋巴细胞,用Trizol试剂提取淋巴细胞的总RNA,通过RT-PCR方法从淋巴细胞中扩增人IgG Fc 片段的基因序列,并将其克隆到pMD-T18 质粒载体上.然后以上述含有Fc片段序列的质粒载体和含有人CD5基因的质粒为模板,通过重叠延伸PCR方法将Fc片段与CD5信号肽基因序列进行融合,并将其插入到pcDNA3.1A 真核表达载体上,构建成与CD5信号肽融合的Fc片段基因的分泌型表达载体.经磷酸钙介导转染293T细胞使其表达.结果:测序表明扩增的Fc基因序列与GeneBank发表的序列完全一致,Western blot检测结果表明,本实验构建的Fc基因分泌型表达载体能够在真核细胞进行高效分泌性表达,表达水平在转染后48小时可达50 μg/1×106细胞.结论:采用RT-PCR扩增了人IgG Fc cDNA 基因,经过与人CD5信号肽序列融合构建了分泌型表达载体,实现了在293T细胞中的高效表达,这为今后构建特定抗原基因与IgG Fc基因融合表达载体,研究DNA疫苗及Fc的生物佐剂提供了实验依据.     

天然调节T细胞与结核病免疫病理关联

目的 检测调节性T细胞(Tr)在结核病病人是否增高,确证Tr与结核免疫病理的关联性,为Tr功能研究提供疾病模型.方法 流式细胞术(FACS)分选CD3+CD4+T及CD3+CD8+T等主要淋巴细胞亚群,通过RT-PCR测定Foxp3基因在正常和结核病病人T细胞亚群中的表达,FACS多色分析Foxp3 T细胞在CD4+CD25+细胞群体中的分布,累积病例分析Tr细胞在外周血扩增情况,免疫组化方法分析Tr在结核病理的浸润,结合临床资料分析Tr数量的改变与结核病的关联性.结果 Foxp3基因在正常和结核病病人CD3+ CD4+T细胞中特异性扩增,FACS分析表明foxp3主要表达于CD4+ CD25high亚群中,占CD4+ CD25highT细胞的80%以上,以CD4+CD2high Foxp3+三联标志检测40例正常与51例活动性结核病病人外周血Tr占CD4+T细胞比例分别为(0.23±0.20)%和(1.01±1.00)%,结核病病人较正常人高4.4倍,Tr在结核病明显扩增(P<0.01),结核病理组织中有高频率Foxp3+细胞浸润.Tr扩增与结核病有效治疗和预后的关联性有深入探讨价值.结论 Tr细胞在结核病病人增高,并与结核免疫病理损伤相关,结核病可作为Tr细胞功能研究的疾病模型.     

急性心肌梗死大鼠心肌组织趋化因子及其受体mRNA表达的观察

目的:探讨急性心肌梗死大鼠心肌局部趋化因子和T细胞趋化因子受体的表达,揭示AMI后T细胞浸润心肌组织的机制。方法:结扎冠脉左前降支建立AMI大鼠模型,用半定量RT-PCR方法分析心肌梗死区和非梗死区趋化因子的表达,包括γ干扰素诱导的单核因子(MIG),正常T细胞表达和分泌、活化时表达下降的因子(RANTES),巨噬细胞炎性蛋白-1α(MIP-1α),以及T细胞趋化因子受体的表达(包括CXCR3、CCR3、CCR5)。HE染色切片进行心肌梗死区和非梗死区淋巴细胞计数分析。结果:心肌梗死区和非梗死区趋化因子RANTES、MIP-1α、MIG的mRNA表达于术后3天开始升高,1周达峰值,然后开始下降,8周降至正常,而趋化因子受体的表达没有明显改变。AMI大鼠心脏梗死区和非梗死区均可见淋巴细胞浸润,梗死区1周达高峰(81.0±10.3vs2.6±1.1,P<0.05),非梗死区2周达高峰(19.0±8.0vs3.2±0.8,P<0.05)。RANTES和MIP-1α的表达与淋巴细胞浸润显著相关。结论:AMI后心肌局部趋化因子表达增高,可能是诱导T细胞浸润心肌组织的始动因子。     

小鼠CD226(PTA1)的基因克隆及其异型

目的 :克隆小鼠CD2 2 6 (PTA1)分子。方法 :从GenBank中检索出与人CD2 2 6分子在氨基酸水平上有 5 1%同源性的EST序列 ,设计并合成特异性引物 ,采用快速扩增cDNA末端的RACE方法 ,从 4周龄BALB c小鼠的胸腺中扩增小鼠CD2 2 6cDNA序列。结果 :克隆出完整的小鼠CD2 2 6cDNA ,长 2 2 2 3bp ,其中开放读框为 10 0 2bp ,编码含信号肽在内的 333个氨基酸 ,属免疫球蛋白超家族分子 ,较人CD2 2 6分子少了 3个氨基酸 ,在氨基酸水平上有 5 3%的同源性。此外 ,还克隆了小鼠CD2 2 6分子的 3种异型。结论 :成功克隆出小鼠CD2 2 6 (PTA1)cDNA ,并发现 3种异型 ,全面探讨该分子生物学特性 ,进行体内功能实验 ,基因敲除小鼠和转基因小鼠的研究提供了坚实的基础。     

淋巴细胞趋化因子增强肝癌树突状细胞融合瘤苗体外免疫作用

目的：观察淋巴细胞趋化因子(lymphotactin, Lptn)基因修饰的肝癌树突状细胞(dendritic cells, DC)融合瘤苗的体外生物学特征和免疫作用。方法： 以重组Lptn基因修饰小鼠骨髓来源的DC，在聚乙二醇(polyethylene glycol，PEG)作用下与H22小鼠肝癌细胞融合，分别以RT-PCR及ELISA方法检测Lptn mRNA及蛋白水平表达，流式细胞仪分析细胞表面免疫分子表达。MTT法检测Lptn基因修饰的肝癌树突状细胞融合瘤苗(DCLptn/H22)对同种异体T淋巴细胞的体外刺激作用。LDH法检测DCLptn/H22融合瘤苗诱导产生的杀伤性T淋巴细胞活性。结果： Lptn基因修饰的DC能分泌较高浓度的淋巴细胞趋化因子，并且具有明显的趋化淋巴细胞功能。DCLptn/H22不但增强融合瘤苗刺激同种异体T淋巴细胞增殖, 而且能增强杀伤性T淋巴细胞活性。结论： 淋巴细胞趋化因子基因修饰能增强融合瘤苗体外免疫刺激作用。     

Identification of multinucleated cells in human kidney cortex: A way for tissue repairing?

The presence of multinucleated cells has never been demonstrated in renal tissue, although, polyploid cells were recently observed in the tubules of normal and pathological human kidney. Therefore, the aim of the present study is to identify and quantify, by electron microscopy, multinucleated cells in the cortical tissue of normal human kidney i.e., in the three compartments of renal tubule: the proximal tubule (PT), the distal tubule (DT), and the collecting duct (CD), as well as, in the glomerulus (podocytes). The percentage of the multinucleated cells observed was 5% (95%CI: 3.6%–6.7%) in renal cortical tubules with distribution in each tubular compartment of 6% in PT, 4% in DT and 3% in CD with no statistically significant difference in the distribution of multinucleated cells according to tubular compartments. Four percent of analysed podocytes (in total 149 podocytes) were multinucleated (95%CI: 1.5%−8.6%). In conclusion, multinucleated cells were identified and quantified in functionally normal kidneys, as previously demonstrated in other organs such as the liver.     

Characterization of the protective T-cell response generated in CD4-deficient mice by a live attenuated Mycobacterium tuberculosis vaccine

The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.     

Normal mouse kidneys contain activated and CD3+CD4- CD8- double-negative T lymphocytes with a distinct TCR repertoire

Healthy liver, intestine, lung, and skin harbor resident lymphocytes with conventional and unconventional phenotypes. Lymphocytes also have been detected in healthy mice kidneys; however, these cells have not been well studied and have been largely overlooked. To better characterize the intra-renal lymphocytes, we extensively perfused C57BL/6J mice with PBS and then isolated mononuclear cells for flow cytometry analysis. We observed T cells, B cells, and NK cells in normal mice kidneys after extensive perfusion. Approximately 50% of kidney T lymphocytes expressed intermediate levels of CD3 (CD3int T cells). Similar to liver and lung, a high percentage of unconventional CD3+CD4(-)CD8(-) double-negative T cells was observed in normal mice kidneys, from which 11% expressed B220 antigen. Unlike the spleen and blood, the classic CD4+ and CD8+ T lymphocytes in the kidney had a high proportion of activated CD69+ and effector/memory CD44- CD62L ligand phenotypes. Also, a small percentage of CD4+CD25+forkhead box p3+ and NKT cells was observed in perfused and exanguinated kidneys. In addition, a distinct TCR repertoire was found on intra-renal conventional and unconventional T cells compared with those from the spleen. Finally, after 24 h of renal ischemia reperfusion injury (IRI), increased production of cytokines IFN-gamma and TNF-alpha by CD4+ and CD8+ T cells, isolated from perfused kidneys, was observed. These data suggest that some of these cells harbored in the kidney could be implicated in the immune response of the IRI pathogenic process.     

Mycobacterium tuberculosis culture filtrate protein 10-specific effector/memory CD4⁺ and CD8⁺ T cells in tubercular pleural fluid, with biased usage of T cell receptor Vβ chains

T cell-mediated immunity is critical for the control of Mycobacterium tuberculosis infection. Identifying the precise immune mechanisms that lead to control of initial M. tuberculosis infection and preventing reactivation of latent infection are crucial for combating tuberculosis. However, a detailed understanding of the role of T cells in the immune response to infection has been hindered. In addition, there are few flow cytometry studies characterizing the Vβ repertoires of T cell receptors (TCRs) at local sites of M. tuberculosis infection in adult tuberculosis. In this study, we used culture filtrate protein 10 (CFP-10) from M. tuberculosis to characterize T cells at local sites of infection. We simultaneously analyzed the correlation of the production of cytokines with TCR Vβ repertoires in CFP-10-specific CD4(+) and CD8(+) T cell subsets. For the first time, we demonstrate that CFP-10-specific CD4(+) or CD8(+) T cells from tubercular pleural fluid can produce high levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and upregulate the expression of CD107a/b on the cell surface. The CFP-10-specific cells were effector/memory cells with a CD45RO(+) CD62L(-) CCR7(-) CD27(-) expression profile. In addition, we found CFP-10-specific CD4(+) and CD8(+) T cells in tubercular pleural fluid, with biased usage of TCR Vβ9, Vβ12, or Vβ7.2. Our findings of CFP-10-specific CD4(+) and CD8(+) T cells in tubercular pleural fluid are critical for understanding the mechanisms of the local cellular immune response and developing more effective therapeutic interventions in cases of M. tuberculosis infection.     

Contribution of CD8(+) T cells to gamma interferon production in human tuberculosis

The proportions of peripheral blood mononuclear cells (PBMC), CD4(+) T cells, and CD8(+) T cells that produce gamma interferon (IFN-gamma) in response to Mycobacterium tuberculosis were markedly reduced in tuberculosis patients, particularly in those with severe disease. Depletion of CD4(+) but not CD8(+) cells prior to stimulation of PBMC with M. tuberculosis abolished IFN-gamma production. These results show that (i) IFN-gamma production by CD8(+) and CD4(+) cells correlates with the clinical manifestations of M. tuberculosis infection and (ii) IFN-gamma production by CD8(+) cells depends on CD4(+) cells.     

Human tuberculous granulomas induce peripheral lymphoid follicle-like structures to orchestrate local host defence in the lung

The human tuberculous granuloma provides the morphological basis for local immune processes central to the outcome of tuberculosis. Because of the scarcity of information in human patients, the aim of the present study was to gain insights into the functional and structural properties of infiltrated tissue. To this end, the mycobacterial load in lesions and dissemination to different tissue locations were investigated, as well as distribution, biological functions, and interactions of host immune cells. Analysis of early granuloma formation in formerly healthy lung tissue revealed a spatio-temporal sequence of cellular infiltration to sites of mycobacterial infection. A general structure of the developing granuloma was identified, comprising an inner cell layer with few CD8(+) cells surrounding the necrotic centre and an outer area of lymphocyte infiltration harbouring mycobacteria-containing antigen-presenting cells as well as CD4(+), CD8(+), and B cells in active follicle-like centres resembling secondary lymphoid organs. It is concluded that the follicular structures in the peripheral rim of granulomas serve as a morphological substrate for the orchestration of the enduring host response in pulmonary tuberculosis.     

脑死亡供体肾脏病理改变及移植应用标准评判的探讨

目的探讨脑死亡供体肾脏病理改变及对临床使用的指导意义。方法对13例脑死亡供体26个肾脏进行了穿刺活检,做HE染色、网状纤维染色及PAS染色。结果发现大多数患者均有不同程度的近曲小管坏死,而远曲小管、肾小球、基底膜多无改变。选择肾近曲小管上皮细胞坏死〈50%、肾小球无明显改变、肌酐〈250μmol/L、年龄〈55岁的供体26个肾脏施行了移植,均取得成功。结论脑死亡供体肾脏根据穿刺病理活检改变并结合临床表现可以作为临床移植的评判依据。     

CD8(+) T cells participate in the memory immune response to Mycobacterium tuberculosis

The contribution of CD8(+) T cells to the control of tuberculosis has been studied primarily during acute infection in mouse models. Memory or recall responses in tuberculosis are less well characterized, particularly with respect to the CD8 T-cell subset. In fact, there are published reports that CD8(+) T cells do not participate in the memory immune response to Mycobacterium tuberculosis. We examined the CD8(+) T-cell memory and local recall response to M. tuberculosis. To establish a memory immunity model, C57BL/6 mice were infected with M. tuberculosis, followed by treatment with anti-mycobacterial drugs and prolonged rest. The lungs of memory immune mice contained CD4(+) and CD8(+) T cells with the cell surface phenotype characteristic of memory cells (CD69(low) CD25(low) CD44(high)). At 1 week postchallenge with M. tuberculosis via aerosol, > or =30% of both CD4(+) and CD8(+) T cells in the lungs of immune mice expressed the activation marker CD69 and could be restimulated to produce gamma interferon (IFN-gamma). In contrast, <6% of T cells in the lungs of naive challenged mice were CD69(+) at 1 week postchallenge, and IFN-gamma production was not observed at this time point. CD8(+) T cells from the lungs of both naive and memory mice after challenge were cytotoxic toward M. tuberculosis-infected macrophages. Our data indicate that memory and recall immunity to M. tuberculosis is comprised of both CD4(+) and CD8(+) T lymphocytes and that there is a rapid response of both subsets in the lungs following challenge.     

Evaluation of T cell immune responses in multi-drug-resistant tuberculosis (MDR-TB) patients to Mycobacterium tuberculosis total lipid antigens

Mycobacterium tuberculosis lipid antigens produce significant T cell responses in healthy tuberculin reactor [purified protein derivative (PPD-positive] individuals. In the present study, proliferation and interferon (IFN)-gamma/interleukin (IL)-4 responses were analysed to M. tuberculosis total lipid antigens in T lymphocytes from 25 patients with multi-drug-resistant tuberculosis (MDR-TB). The obtained results were compared with those of 30 asymptomatic healthy PPD-positive and 30 healthy tuberculin skin test negative (PPD-negative) subjects. Peripheral blood mononuclear cells (PBMCs) and T cells (CD4(+) and CD8(+)) were stimulated using autologous immature dendritic cells. Proliferation responses were assessed using 3-{4,5-dimethylthiazol-2-yl}-2,5 diphenyl tetrazolium bromide (MTT). IFN-gamma/IL-4 concentrations in the supernatant of the CD4(+) and CD8(+)T cells were measured by enzyme-linked immunosorbent assay. Proliferation assay showed that the peripheral blood mononuclear cells and CD4(+) T cells from the MDR-TB patients responded significantly less to the M. tuberculosis total lipid antigens than to the CD4(+) T cells in the PPD-positive subjects. Total lipid antigen-specific proliferative responses in the CD8(+) T cells from the MDR-TB patients were minimally detected and the responses were similar to those of the PPD-positive subjects. IFN-gamma production by the CD4(+) T cells stimulated by total lipid antigens from the MDR-TB patients was decreased significantly compared with the PPD-positive individuals, whereas IL-4 production in the patients was elevated. IFN-gamma and IL-4 production in the CD8(+) T cells of the MDR-TB patients was similar to those of the PPD-positive subjects. In conclusion, it is suggested that stimulated CD4(+) T cells by M. tuberculosis total lipid antigens may be shifted to T helper 2 responses in MDR-TB patients.