人淋巴细胞趋化因子真核表达质粒的构建及体外转染的生物学活性测定

目的：构建人淋巴细胞趋化因子(HLptn)真核表达质粒，在膀胱肿瘤细胞株BIU-87中表达重组质粒，并检测趋化活性。方法：用RT-PCR法自活化的人外周血淋巴细胞扩增HLptn含编码区序列的cDNA，克隆至pGM-T Easy T载体，测序正确后，将目的片段插入pcDNA3．1( )载体，获得阳性克隆pcDNA3．1( )．HLptn；用脂质体介导其转染BIU-87细胞，用Western-blot检测转染后细胞中HLptn的表达；取转染后的上清液，采用Boyden小室法检测表达的HLptn趋化CD4^ 、CD8^ T淋巴细胞的生物学活性。结果：克隆的cDNA序列与GenBank中U23772的序列编码区内第225位碱基不同，系同义突变，构建了真核重组表达载体pcDNA3．1( )-HLptn；转染的BIU-87细胞表达HLptn，其培养上清对CD4^ 、CD8^ T淋巴细胞具有趋化活性。结论：成功构建的HLptn真核表达系统可在人膀胱肿瘤细胞株BIU-87中表达。     

Radiation-induced bystander effect in immune response

Objective Since most reports on bystander effect have been only concerned with radiation-induced damage,the present paper aimed at disclosing whether low dose radiation could induce a stimulatory or beneficial bystander effect. Methods A co-culture system containing irradiated antigen presenting cells (J774A,1) and unirradiated T lymphocytes (EL-4) was established to observe the effect of J774A, I cells exposed to both low and high doses of X-rays on the unirradiated EL-4 cells, Incorporation of ^3H-TdR was used to assess the proliferation of the EL-4 cells expression of CD80/86 and CD48 on J774A. 1 cells was measured with immunohistochemistry and flow cytometry, respectively.NO release from J774A.1 cells was estimated with nitrate reduction method.Results Low dose-irradiated J774A.I cells could stimulate the proliferation of the unirradiated EL-4 cells while the high dose-irradiated J774A.1 cells exerted an inhibitory effect on the proliferation of the unirradiated EL-4 cells. Preliminary mechanistic studies illustrated that the differential changes in CD48 expression and NO production by the irradiated J774A.1 cells after high and low dose radiation might be important factors underlying the differential bystander effect elicited by different doses of radiation. Conclusion Stimulatory bystander effect can be induced in immune cells by low dose radiation,     

Surface expression of complement regulatory proteins, decay-accelerating factor (DAF) and CD59, on cultured human endothelial cells

To clarify the response of endothelial cells to complement, we studied not only the reaction of endothelial cells against complement lysis sensitivity (CLS) test, but also the expression of complement regulatory proteins by two-color flow cytometric analysis and backscattered scanning immunoelectron microscopic analysis using monoclonal antibodies to decay-accelerating factor (DAF) and/or CD59. Complement activation didn't lead to the cell death of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs). In control, two-color flow cytometric analysis indicated that HAECs consisted of a single double-positive population for these proteins as well as HUVECs. Then, HUVECs and HAECs after the CLS test resulted in having same distribution by flow cytometry. Moreover, the microscopic analysis showed that DAF or CD59 was expressed with a diffuse distribution. However, DAF on HUVECs and CD59 on HAECs were present at the margin of cell surfaces more than at the other places. These findings suggest that endothelial cells have a defense mechanism for complement activation in vitro by the changes of expression of complement regulatory proteins on the membrane surface, and that the mechanism of HAECs to complement is the same as that of HUVECs.     

Suppression of allogeneic T cells proliferation by CD3/CD46-induced T-regulatory 1 cells

CD46 is not only identified as a complement regulatory protein which protects host cells from complement attack,but also a new co-stimulatory molecule for human T cells.CD3/CD46 co-stimulation can induce a T-regulatory 1 cell(Tr1)-specific cytokine phenotype in human CD4+ T cells.However,the role of CD46 as a co-stimulatory molecule in the modulation of the acquired immunity,such as transplant immunology,remains unclear.In this study,CD4+ T cells were isolated from human CD46-transgenic C57BL/6 mice by magn...     

电离辐射对EL-4细胞倍增时间的影响

研究电离辐射对EL－4淋巴瘤细胞倍增时间的影响。方法：体外传代培养EL－4细胞并计数。按下式计算细胞倍增时间：TD＝0．693（T2-T1）／ln（N2／N1）。结果：0．1～4．0GyX射线单次照射使EL－4细胞倍增时间明显延长。0．05Gy低剂量预照射可明显缩短2．0Gy大剂量照射所致细胞倍增时间的延长。结论：0．1Gy以上剂量X射线使EL－4细胞倍增时间延长。0．05Gy预照射可诱导适应性反应。     

猫创伤性ALI和ARDS中CD59和CD46的表达和意义

目的 观察补体调节蛋白ＣＤ５９和ＣＤ４６在创伤性急件肺损伤（ＡＬＩ）和急性呼吸窘迫综合征（ＡＲＤＳ）动物肺组织中的表达变化,并探讨其在ＡＬＩ和ＡＲＤＳ发病机制中的意义。方法 采用右胸撞击建立创伤性ＡＬＩ和ＡＲＤＳ模型,用免疫组化和原位杂交技术检查ＡＬＩ和ＡＲＤＳ家猫肺组织中ＣＤ５９,ＣＤ４６的表达情况。结果 伤后１２ｈ ＡＬＩ动物肺组织中ＣＤ５９,ＣＤ４６表达上调,２４ｈ最为明显,４８～７２ｈ减弱     

“补肾生髓”法通过Notch信号通路调节DCs介导的CD4+ T细胞分化及对EAE的干预作用研究

目的：研究“补肾生髓”法方药（BSD）对通过调节树突状细胞（Dendritic cells,DCs）Notch信号通路对CD4+ T细胞分化及实验性自身免疫性脑脊髓炎（Experimental autoimmune encephalomyelitis,EAE）模型小鼠发病的干预作用。 方法：主动免疫法诱导2D2小鼠EAE后,磁珠分选收集脾脏和淋巴结中 CD4+ T 细胞,体外分离培养及诱导 C57BL/6 （C57）小鼠骨髓来源树突状细胞（BMDC）成熟,分组干预后,与CD4+ T 细胞共培养后,被动注射至C57小鼠体内,观察小鼠发病情况。实验分组为：对照（Control）组、BSD组、阳性药物γ分泌酶抑制剂（gamma-secretase inhibitor,GSI）组。采用Western blot检测各组DCs Notch通路蛋白表达情况,采用流式细胞术分析各组中CD4+ T 细胞分化情况, 结果：BSD可抑制DCs Notch1,Jagged1,MAML蛋白水平,通过DCs诱导CD4+T细胞Treg分化增多,Th17分化减少,并改善被动免疫诱导的小鼠EAE症状评分。 结论：BSD可通过抑制DCs Notch信号通路活化,减少DCs介导的CD4+T细胞分化,进而缓解EAE残疾症状。     

中性粒细胞诱导适应性免疫应答过程中树突状细胞成熟活化

目的 探讨T辅助（Th）1细胞免疫应答过程中,中性粒细胞（PMN）对树突状细胞（DC）的调节作用。方法 将脂多糖（LPS）刺激的中性粒细胞（LPS-PMN）和未成熟树突状细胞（imDC）共培养,流式细胞术检测DC表面CD40和CD86,酶联免疫吸附试验（ELISA）检测白细胞介素12（IL-12）、肿瘤坏死因子α（TNF-α）。将与LPS—PMN共培养后DC（PMN-DC）提纯,与FITC标记的卵清蛋白（FITC-OVA）培养,检测其吞噬功能。将PMN-DC与D011．10T细胞和OVA17肽共培养,计活细胞数,胞内染色测干扰素γ（IFN-γ）、白细胞介素4（IL-4）。结果 LPS-PMN可刺激imDC CD40和CD86上调并分泌IL-12、肿瘤坏死因子（TNF）-α,且这种能力能被抗TNF-α单抗所抑制。与imDC对照,PMN—DC吞噬功能下降,能显著刺激D011．10T细胞增殖,分泌高水平IFN-γ、少量IL-4。结论 LPS-PMN具有促使imDC成熟活化,刺激其分泌细胞因子,发挥其在Th1免疫应答中抗原呈递,促进Th1分化的作用。     

系统性红斑狼疮患者外周血T细胞CTLA-4表达及其临床意义的研究#

目的：研究系统性红斑狼疮（Systemic Lupus Erythematosus, SLE）T细胞中CTLA-4的表达及临床意义。 方法：分离正常人和SLE患者的外周血单个核细胞（PBMCs）,以抗-CD3、抗-CD28刺激培养48h,刺激培养前后收取细胞,以流式细胞术（FCM）检测CD4+和CD8+T细胞中CTLA-4+细胞比例,并分析其与SLE疾病活动性指数（SLE Disease Activity Index,SLEDAI）和肾损害的关系。再以ELISA法检测培养上清中游离的CTLA-4水平。 结果：SLE患者刺激前的CD4+和CD8+T细胞中、主要是CD25+T细胞中CTLA-4+细胞的比例较正常人显著增高,且与SLEDAI呈正相关;而其CD8+CD28- T细胞中CTLA-4+细胞比例也显著高于正常人,但与SLEDAI之间无显著相关性;经抗-CD3、抗-CD28抗体刺激后,其CD4+CD25+T细胞、CD8+CD25+T细胞或CD8+CD28- T细胞中CTLA-4+细胞的比例却显著低于正常人,但与SLEDAI之间无显著相关性,仅在活动性SLE患者中有肾损组的CD8+CD28- T细胞中CTLA-4+细胞的比例显著低于非肾损组;而且经刺激培养后SLE患者PBMC上清中游离的CTLA-4水平也显著低于正常人。结论：SLE患者新鲜分离的T细胞（CD4+及CD8+）中CTLA-4表达异常增高,反映T细胞异常活化和疾病活动;另一方面,SLE患者T细胞又存在CTLA-4诱导性表达障碍,可能与SLE T细胞体外再活化的能力减弱有关。     

Screening of aplastic anaemia-related genes in bone marrow CD4+ T cells by suppressive subtractive hybridization

Background CD4(+) T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4(+) T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4(+) T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia. Methods The bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as “tester” and donor as “driver”, their CD4(+ )T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library.Results PCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4(+ )T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4(+) T cells in aplastic anaemia. Conclusions Screening and cloning genes, which regulate functions of CD4(+) T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4(+) T cells in aplastic anaemia.     

共刺激分子B7.1和葡萄球菌肠毒素A膜表面共修饰瘤苗抗肿瘤作用的研究

目的 观察跨膜型葡萄球菌肠毒素A(SEA-TM)和糖基化磷脂酰肌醇锚定型mB7．1(mB7．1-GPI)二种免疫分子膜表面修饰瘤苗的抗肿瘤作用是否优于单种免疫分子膜表面修饰的瘤苗。方法构建pcDNA3．1( )／mB7．1-GPI真核表达载体,转染中国仓鼠卵巢上皮细胞(CHO)中,表达和纯化mB7．1-GPI。通过蛋白转染法将SEA-TM、mB7．1-GPI单独或共同锚定到EL-4肿瘤细胞膜上,制成瘤苗,观察这些瘤苗刺激小鼠脾细胞的增殖和分泌白细胞介素(IL)-2和γ干扰素(IFN)-γ的量及抗肿瘤作用。结果mB7．1-GPI。和SEA-TM能单独或共同锚定在肿瘤细胞膜上,具有相当的稳定性;在体外有刺激小鼠脾细胞增殖和分泌IL-2、IFN-γ的功能。mB7．1-GPI、SEA-TM、mB7,1-GPI SEA-TM锚定肿瘤细胞,制成的瘤苗,能抑制荷瘤小鼠的肿瘤生长和延长荷瘤小鼠的存活时间。mB7．1-GPI和SEA-TM双锚定瘤苗,显示出比单一蛋白锚定瘤苗更强的抗肿瘤作用。结论用蛋白转染法将SEA和mB7.1二种免疫分子同时锚定到肿瘤细胞膜上所制成的瘤苗,其抗肿瘤作用优于单种免疫分子锚定的瘤苗。     

补体调节因子在异种器官移植中的作用

器官移植作为脏器终末期疾病的临床治疗手段,在过去10年中取得了突破性的进展.无论是临床器官移植实施例数,还是移植成功率、术后存活率均有显著的提高.这些成就一方面得益于器官保存方法、临床手术技巧的不断改进和完善,另一方面也得益于对影响移植效果的基本免疫过程的深入了解,以及更有效的免疫抑制药物的发现及合理使用.但是伴随着器官移植的成功,器官供体短缺的问题日渐严峻.     

CD 80(B7-1) expression on human tumor cell lines and its costimulatory signals for T cell proliferation and cytokine production.

ＣＤ８０（Ｂ７１）ｅｘｐｒｅｓｉｏｎｏｎｈｕｍａｎｔｕｍｏｒｃｅｌｌｉｎｅｓａｎｄｉｔｓｃｏｓｔｉｍｕｌａｔｏｒｙｓｉｇｎａｌｓｆｏｒＴｃｅｌｐｒｏｌｉｆｅｒａｔｉｏｎａｎｄｃｙｔｏｋｉｎｅｐｒｏｄｕｃｔｉｏｎＹａｎＪｕｎ严俊，ＭａＢａｏｌｉ马宝骊...     

CD40 ligand expression in mycobacterium bovis BCG infection and its regulation by cytokines: a direct role of interleukin 12

BACKGROUND: Activation and clonal expansion of T cells require not only the recognition of processed antigen on the surface of the antigen-presenting cell (APC) by T-cell receptor (TCR), but also involve co-stimulatory signals that are provided by the simultaneous engagement of cell surface molecules expressed by both the APC and the T cell. Interaction between CD40 and its ligand (CD40L) is known to mediate host immune response and T-cell-mediated effector functions in mycobacterial infections in mice. In this work, we investigated the capacity of Mycobacterium bovis (M. bovis) BCG to induce the expression of CD40L on human T cells. METHODS: Human cells were obtained from healthy adults by centrifugation using Ficoll/Hypaque. Cells (1 x 10(6)) were incubated in RPMI medium with BCG. After incubation at 37 degrees C in 5% CO(2) atmosphere for 40 h, cells were collected and double-stained with anti-CD40L-PE and anti-CD4-FITC or anti-CD8-FITC mAb. The quantification of positively stained population was based on samples stained with isotype control antibodies analyzed on a FACScan. RESULTS: M. bovis BCG stimulation induced a significant amount of CD40L expression on CD4+ T cells, while CD40L was not significantly detected on human CD8+ T cells. The highest CD40L expression on BCG-activated T cells in synergism with interleukin-12 endogenous occurred after a 40-h cell culture with a dose of 10 microg/mL of BCG (84.86 +/- 11.77; mean +/- standard deviation [SD]). This CD40L expression on BCG-activated human T cells was significantly inhibited by anti-IL-12 mAb (5.08 +/- 1.7; mean +/- SD). In contrast, anti-IFN-gamma and anti-IL-2 mAb did not have an important role in this expression. CONCLUSIONS: These results indicate that the capacity of BCG to induce CD40L expression on human cells represents a novel mechanism underlying the regulation of cellular responses against tuberculosis. Furthermore, the results showed a direct role of IL-12 to enhance the expression of CD40L on BCG-activated human cells.     

体外转运富含GPI-蛋白的红细胞囊泡改善PNH溶血的探索

目的　在体外观察是否能将富含ＣＤ５９ 的红细胞囊泡转运到缺失ＣＤ５９的阵发性睡眠性血红蛋白尿症（ＰＮＨ）红细胞膜上,以改善ＰＮＨ 溶血,为ＰＮＨ的治疗提供新的线索。方法　采用免疫亲和层析分离技术分离ＰＮＨ ＣＤ５９－ 红细胞,采用Ｗｅｓｔｅｒｎ 印迹分析、流式细胞仪及蛇毒因子溶血实验等检测和分析红细胞与囊泡的结合。结果　Ｗｅｓｔｅｒｎ 印迹分析显示,红细胞囊泡含有ＣＤ５９;流式细胞仪测定表明,ＰＮＨ ＣＤ５９－ 红细胞与囊泡结合后,ＣＤ５９ 的荧光标记率明显增高（Ｐ＜ ０００１）;蛇毒因子溶血实验显示,加入囊泡后ＰＮＨ ＣＤ５９－ 红细胞溶血度也显著下降（Ｐ＜ ００５）。结论　红细胞囊泡上的ＣＤ５９ 可以在体外转运到ＰＮＨ ＣＤ５９－ 红细胞上,并产生抑制补体溶血的作用,使ＰＮＨ ＣＤ５９－ 红细胞在体外受补体攻击时不易溶血     

稳定表达结核分枝杆菌38抗原的EL-4细胞株的建立及鉴定

将结核分枝杆菌的38抗原基因片段经PCR方法扩增并插入到pcDNA3真核表达载体中，通过脂质体转染EL-4细胞，经G418筛选，用RT-PCR方法和ELISA方法检测整合和表达情况。结果成功地构建了pcDNA3—38抗原重组质粒，转染细胞中可检测到38抗原的存在，PCR扩增也证实38抗原基因已稳定整合于EL-4细胞的染色体中。本实验为今后在小鼠体内检测结核DNA疫苗激发的细胞毒性T淋巴细胞(CTL)反应奠定了基础。     

HIV-1感染者治疗后的T淋巴细胞异常激活研究

  目的   探讨外周全血中异常激活的T淋巴细胞亚群与人类免疫缺陷病毒1型（HIV-1）感染者免疫功能恢复的关系,探讨HIV-1 DNA病毒库的大小与T淋巴细胞亚群的关系。  方法   以2019年7月–2020年5月进行常规检测的HIV-1感染者为目标人群,入组时根据抗逆转录病毒疗法（antiretroviral therapy, ART）治疗后CD4+ T细胞是否达到500 cells/ μL,将HIV-1感染者分为两组,其中缺陷组76例,无缺陷组61例。同时选择22例未暴露且HIV-1抗体检测阴性者作为对照组。对以上3组人群的T细胞亚群进行检测,并对缺陷组44例和无缺陷组33例共计77例进行HIV-1 DNA病毒库检测。6个月后随访缺陷组和无缺陷组,收集缺陷组（74例）和无缺陷组（59例）共133例血液样本中的CD4+ T细胞检测结果。  结果   入组时,缺陷组的活化CD4+、CD8+ T细胞含量均高于无缺陷组和对照组。缺陷组衰老CD4+ T、CD8+ T细胞含量与无缺陷组相当,均高于对照组,但仅在衰老CD8+ T细胞上差异有统计学意义。缺陷组比对照组检出更高的效应记忆CD4+ T和CD8+ T细胞,无缺陷组只检出更高的效应记忆CD8+ T细胞。缺陷组和无缺陷组均表现出比对照组更低水平的中心记忆CD4+ T和CD8+ T细胞,无缺陷组中心记忆CD4+ T细胞比缺陷组还低。对于幼稚细胞而言,无缺陷组表现出更高水平的幼稚CD4+ T细胞,缺陷组和无缺陷组的幼稚CD8+ T细胞较对照组下降。HIV-1 DNA病毒库大小与CD4+ T细胞数量和各T细胞亚群均无相关。活化CD4+ T细胞、活化CD8+ T细胞、中心记忆CD4+ T细胞与6个月以后的后续CD4+ T细胞数量呈负相关,r分别为－0.378、－0.334、－0.322（P<0.05）;幼稚CD4+ T细胞、幼稚CD8+ T细胞与后续CD4+ T细胞数量呈正相关,r分别为0.350、0.267（P<0.05）。  结论   HIV-1感染者存在不同程度的T细胞亚群异常激活,部分T细胞亚群的异常激活与后续免疫功能的恢复有关,病毒库的大小与T细胞亚群无相关关系。     

外周血PBMC来源的树突状细胞的快速无血清培养方法及细胞信号转导机制

目的 探讨体外无血清条件下诱导人外周血单核细胞(PBMC)迅速生成树突状细胞(DC)的方法．并初步探讨钙离子载体(CI)诱导分化的信号转导途径与肿瘤坏死因子(TNF)-α所诱导的是否相同。方法 分离健康献血者的PBMC．给予无血清培养基及50ng／ml的rhGM-CSF过夜培养后，再分别给予100ng／ml的A23187或50ng／ml的TNF-α，或预先加入0．5μg／ml的环胞菌素A(CsA)30min后，再加入A23187、TNF-α，共培养40h。于相差显微镜下观察细胞形态的变化，流式细胞仪检测细胞的表面标志，MTT比色法检测不同方法处理的PBMC刺激同种异体T细胞的增殖作用。结果 健康献血者的PBMC在无血清条件下，给予rhGM-CSF及CI或TNF-α培养40h，均可获得DC的典型形态．包括CD14表达下调、CD83及共刺激分子(CD80、CD86)表达上调，以及较强刺激同种异体T细胞增殖的作用；上述由CI诱导的细胞形态的改变、表面分子的表达以及刺激T细胞增殖的作用均可被CsA所抑制。而TNF-α所诱导的细胞形态的改变、表面分子的表达以及刺激T细胞增殖的作用均不受CsA影响。结论 健康献血者的PBMCs在体外无血清条件下．司以被rhGM-CSF及CI或TNF-α迅速诱导成DC，但CI与TNF-α诱导PBMC分化为DC的细胞信号转导途径不同。     

巨细胞病毒感染对重型β-地中海贫血儿童异基因造血干细胞移植后早期T细胞亚群的影响

目的探讨巨细胞病毒(CMV)对儿童重型β-地中海贫血(地贫)儿童异基因造血干细胞移植(Allo-HSCT)后180 d外周血T细胞亚群的变化。方法对我院儿科造血干细胞移植中心2010年1月～2011年1月儿童重型地贫Allo-HSCT 35例病例半年后的T细胞亚群进行分析,比较有CMV(13例)和无CMV感染组(22例)在造血干细胞移植后早期T细胞的变化。结果半年内有CMV组CD8+比率高于无CMV感染组,但CD4+比率低。在CMV感染组伴随CD8+CD28+细胞比率降低,CD8+CD28-细胞比率和CD8+比率升高,同时发现CD8+与CD8+CD28-细胞比率呈线性相关性。结论重型β-地中海贫血儿童在异基因造血干细胞移植后,CMV感染后会导致CD8+CD28-细胞堆积,是构成CD8+细胞升高的主因,且两者比率呈线性相关。