人IL-6 mRNA的实时定量检测法

目的：建立TaqMan-MGB技术实时定量检测人IL-6 mRNA的方法，并用于检测人外周血单个核细胞在牛膝多糖诱导下IL-6 mRNA的表达。方法：（1）抽取人外周静脉血，EDTA抗凝，用淋巴细胞分离液分离外周血单个核细胞，用Trizol裂解液提取总RNA，将总RNA逆转录为cDNA，PCR扩增IL-6后纯化PCR产物，与pMD 18-T载体进行连接，转化宿主菌DH-5α。提取重组质粒DNA，作为实时荧光定量检测的标准品。建立TaqMan-MGB技术实时定量检测IL-6 mRNA的方法。（2）终浓度分别为0、100、200、400、800和1 600 mg/L的牛膝多糖作用于人外周血单个核细胞，定量检测IL-6 mRNA的表达。结果：（1）酶切分析、PCR扩增以及测序结果均证实IL-6 cDNA成功重组到pMD 18-T载体上。（2）TaqMan-MGB技术检测IL-6 mRNA线性范围广；灵敏度高；特异性好；重复性好。（3）牛膝多糖作用于人外周血单个核细胞后IL-6 mRNA表达增高。结论：TaqMan-MGB技术能较为精确地定量IL-6 mRNA。     

人TLR2和TLR4胞外区cDNA的克隆

目的 从脂多糖诱导的外周血单个核细胞克隆人Toll样受体2(TLR2)和Toll样受体4(TLR4)胞外区cDNA.方法 用不同浓度脂多糖在不同时间刺激单个核细胞后提取总RNA,RT-PCR方法半定量测定TLR2和TLR4的表达,应用pUCm-T载体克隆胞外区cDNA,双酶切以及DNA测序进行鉴定.结果 单个核细胞在四种浓度脂多糖刺激3 h,6 h和12 h后.TLR2和TLR4表达并不相同.15 μg/mL脂多糖作用6 h TLR2表达最高,10 μg/mL脂多糖作用3 hTLR4表达最高.经RT-PCR扩增TLR2和TLR4胞外区cDNA分别为1 700 bp和1 900 bp,T载体克隆、酶切鉴定及测序分析后证实,目的 片段与GenBank中序列一致.结论 脂多糖的诱导对外周血单个核细胞TLR2和TLR4表达有显著影响,并且成功克隆了胞外区cDNA.     

人T细胞活化衔接因子基因真核表达载体的构建

目的构建人T细胞活化衔接因子（LAT）基因的真核表达载体。方法以人外周血单个核细胞的cDNA为模板,采用聚合酶链式反应（PCR）扩增LAT基因编码区的全部序列,克隆人真核细胞表达载体pCMV—Myc中,测序鉴定目的基因并运用核转染的方法转染人外周血T淋巴细胞。结果PCR扩增的特异性片段长度为729bp,以此构建重组质粒pCMV—Myc—LAT,测序结果与Genbank中的人LAT基因cDNA序列一致。转染人外周血T淋巴细胞后可检测到LAT蛋白的表达。结论成功构建了pCMV—Myc—LAT重组真核表达载体。     

TCR Dβ-Jβ sjTRECs检测方法的建立和应用

目的：建立检测TCR Dβ-Jβ sjTRECs的方法，并了解不同T细胞受体Dβ-Jβ之间T细胞受体删除DNA环(sjTRECs)在胸腺细胞和外周血T细胞中的形成情况。方法：利用巢式PCR分别扩增10例正常人外周血单个核细胞和3例正常胸腺细胞DNA中不同的Dβ片段和Jβ重排时形成的sjTRECs的情况，PCR产物进一步进行克隆和序列分析以确定结合区的位置。结果：可检测到Dβ1与5个Jβ1基因片段、Dβ2与4个Jβ2基因片段分别形成的sjTRECs，其中以Dβ1-Jβ1S1、Dβ1-Jβ1S2和Dβ2-Jβ2S2sjTRECs最常见，PCR产物经克隆和序列分析证实其形成Dβ-jβsjTRECs的情况。结论：成功地建立了检测Dβ-Jβ-sjTRECs的方法，为分析各TCRβ亚家族的sjTRECs含量和确定TCRβ naive细胞提供了新方法。     

系统性红斑狼疮患者外周血单个核细胞TRAF1 mRNA的表达及其意义

目的：研究肿瘤坏死因子受体相关因子1(TRAF1)mRNA表达在SLE患者的外周血单个核细胞中的表达是否异常，以及该基因mRNA表达与SLE活动指数是否相关。方法：TRAF1 mRNA表达采用RT-PCR半定量法；SLE活动指数以SLEDAI为判断标准。结果：TRAF1 mRNA在SLE患者的外周血单个核细胞中的表达较正常人为高，但与SLEDAI指数无相关。结论：TRAF1基因可能在SLE的发病机制中起重要作用。     

小鼠GM-CSF基因真核表达载体的构建及其表达活性的鉴定

目的：构建小鼠GM－GSF基因的高效真核表达载体，筛选导入该载体后高水平表达GM－CSF的小鼠淋巴瘤细胞系RMA，并探讨转GM－CSF基因瘤苗治疗小鼠T淋巴细胞瘤的方法。方法：PCR扩增小鼠GM－CSF cDNA3‘端770bp的片段，将其插入真核表达载体pcDNA3；用电穿孔法将构建的载体导入小鼠淋巴瘤细胞系RNA，有限稀释法制备单个细胞克隆，经RT－PCR、骨髓祖细胞增殖实验和集落形成实验筛选相对高表达GM－CSF的RNA克隆，该克隆细胞经丝裂霉素灭活后免疫小鼠以诱导其产生抵抗RMA肿瘤细胞再攻击的能力。结果：构建的重组质粒含有预期片段，插入方向正确，核酸序列无误，且获得了高表达GM－CSF的RMA克隆，将其用丝裂霉素灭活免疫小鼠后使它们产生了抗肿瘤免疫保护力。结论：转GM－CSF基因瘤苗可能作为有效的抗T淋巴细胞瘤瘤苗。     

TCR Vβ2、Vβ5和Vβ17-Dβ1 sjTRECs在正常人胸腺、脐血和外周血中的检测情况

目的建立检测TCR Vβ2、Vβ5和Vβ17-Dβ1 sjTRECs的方法，分析其在胸腺细胞、脐血和正常人外周血T细胞中的存在情况，从而了解近期胸腺输出相应的TCR Vβ亚家族naive T细胞的情况。方法利用半巢式PCR分别扩增3例正常胸腺细胞、10例脐血和10例正常人外周血单个核细胞DNA中的Vβ2、Vβ5和Vβ17与Dβ1基因片段重排时产生的sjTRECs。结果在正常胸腺细胞、脐血和正常人外周血单个核细胞中均可检测到Vβ2、Vβ5和Vβ17与Dβ1片段形成的sjTRECs，其中胸腺细胞和脐血单个核细胞DNA中3种删除环的检出率均为100％，正常人外周血3种删除环的检出率分别为50％、70％和40％。结论成功地建立检测3种Vβ-Dβ1 sjTRECs的方法，并提供了Vβ2、Vβ5和Vβ17亚家族sjTRECs在胸腺、脐血和外周血中的检测情况。     

Tactivin疗法对外伤后骨髓病人IL-1β,TNFα的调节

采用Tactivin疗法对5例下肢外伤后骨髓炎病人治疗前后IL-1β，TNFα的变化进行了观察，对照组为健康人。于Tactivin疗法治疗前后采病人血液，分别检测：①血清IL-1β,TNFα水平；②体外培养外周血单个核细胞自发性和经LPS刺激后分泌的IL-IB，TNFα水平（用免疫酶方法检测）；③外周血单个核细胞的1IL-1β，TNFαmRNA基因表型的检测，用N．Gough方法抽提外周血单个核细胞的mRNA，与用’‘P标记的IL－lpDNA探针、TNFaDNA探针分别进行杂交，用放射自显影方法检测。实验结果表明：①来治疗的骨髓病人血清IL－16水平升高的同…     

端粒酶在小儿急性白血病中表达的意义

目的 探讨端粒酶在小儿急性白血病中的表达及作为预测病情，判断预后标志物的可能性。方法 采用端粒重复序列扩增-微孔杂交法（TRAP-Kyb)，检测16例急性白血病患儿骨髓和外周血单个核细胞端粒酶活性，对照组为10例非白血病血液病患儿的骨髓及5例正常健康小儿的外周血单个核细胞，结果 16例急性白血病患儿表达阳性率83.1%)；10例非白血病血液病患儿有2例呈低度阳性表达，5例正常健康小儿外周血单个核细胞中未见阳性表达。结论 端粒酶活性在急性白血病患儿骨髓和外周血单个核细胞中明显升高，表明端粒酶的活性与急性白血病的发生可能有着密切关系。检测其活性可能成为一种评估急性白血病病情及预后的有用指标。     

伏马菌素B1对人外周血单个核细胞表面HLA-Ⅰ分子表达的影响

目的：探讨伏马菌素B1（FB1）对体外培养的人外周血单个核细胞表面（HPBMc）HLA-I分子表达的影响。方法：采用流式细胞术（FCM）、Western blot及半定量RT-PCR方法，研究不同浓度FB1（10和50μmoL／L）处理后人外周血单个核细胞表面HLA-I分子表达的变化。结果：FCM定量分析结果表明，经FB1处理24h后，两组FB1处理细胞HLA-I的平均荧光强度均较对照组降低（P〈0．05），但是在两个处理组之间无统计学意义。Westernblot也证实了上述结果。在mRNA水平上，分别检测了HLA-I分子3个等位基因HLA-A，HLA-B，HLA—C的表达情况。结果显示，FB1处理后，HLA—A、HLA-B mRNA的表达均没有明显影响，仅HLA-C mRNA的表达较对照组降低。结论：10和50μmol／L FB1处理24h可抑制人外周血单个核细胞表面HLA-I分子的表达。     

编码与大鼠ERα-甘露糖苷酶同源的人6A8 cDNA在真核细胞的表达

目的观察６Ａ８ｃＤＮＡ（１３５８ｂｐ）在真核细胞的表达。方法Ｎｏｒｔｈｅｒｎｂｌｏｔ，构建６Ａ８ｃＤＮＡ表达载体ｐＲｃ／ＣＭＶ－６Ａ８ｃＤＮＡ，转染ＣＯＳ－７细胞，及基因免疫小鼠。结果６Ａ８ｃＤＮＡ有４．３ｋｂ和３．５ｋｂ两种ｍＲＮＡ转录形式。４．３ｋｂｍＲＮＡ以外周血白细胞最丰富，其次为淋巴结、脾脏和骨髓，胸腺组织表达较少，胎肝则缺如。３．５ｋｂ者也以外周血白细胞最丰富，其次为肝脏、淋巴结和骨髓，胸腺和脾脏表达量最少。ｐＲｃ／ＣＭＶ－６Ａ８ｃＤＮＡ在ＣＯＳ－７细胞表达了分子量４５０００左右的蛋白质，与单抗６Ａ８有反应。ｐＲｃ／ＣＭＶ－６Ａ８ｃＤＮＡ基因免疫小鼠，６／１０只小鼠血清与人活化Ｂ细胞株３Ｄ５细胞膜有反应，５／５只对照小鼠血清呈阴性反应。结论６Ａ８ｃＤＮＡ（１３５８ｂｐ）在真核细胞获得表达，但此ｃＤＮＡ非为全长。     

Monoclonal antibody to a human leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat

The monoclonal antibody (F10–89–4) described in this study recognizes an antigen which by quantitative absorption analysis is absent from human brain, kidney, liver, heart, erythrocytes, platelets and normal serum, but is present on spleen, lymph node, chronic lymphatic leukemia cells, bone marrow, thymus and granulocytes at a ratio of 1 : 1 : 0.8 : 0.3 : 0.3 : 0.1, respectively. Analysis with the fluorescence-activated cell sorter showed that 100% of thymocytes, lymph node lymphocytes, blood mononuclear cells and granulocytes carry the antigen, while 83% of bone marrow cells are positive. There was marked heterogeneity in the amount of labeling of thymocytes, with 3 major peaks. There was also heterogeneity of labeling of blood mononuclear cells and lymph node lymphocytes, with a weakly staining hump containing approximately 20% of the cells in the case of lymph node lymphocytes. Double labeling experiments demonstrated that the weakly staining cells of blood and lymph node were B lymphocytes, while frozen sections of thymus showed that the antigen was expressed most weakly in subcapsular cortical thymocytes, and most strongly on medullary thymocytes. Biochemical studies established that the antigen bound to lentil lectin columns, and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies using NaB³ H₄-labeled blood mononuclear cells established that the antigen was a major glycoprotein of lymphocytes, and that its molecular weight was in the region of 190 000 to 215 000.     

Antigen-binding small lymphocytes in the guinea-pig: I. The immunological response to human thyroglobulin

The time course of the relative distribution of small lymphocytes binding ¹²⁵I-labelled human thyroglobulin (HTg) in cell suspensions from the peripheral blood and various lymphoid organs was studied in guinea-pigs at progressive intervals up to 28 days after immunization with an emulsion of HTg and BCG in Freund's incomplete adjuvant (FIA). Small lymphocytes binding ¹²⁵I-labelled HTg were first detected in peripheral blood, popliteal (draining) lymph node, spleen and bone marrow preparations on the 10th day, and in mesenteric (distant) lymph node and thymus preparations on the 14th day after primary immunization. In general, the percentage of these cells increased progressively thereafter until the end of the period of study. Blocking experiments with unlabelled antigens indicated that the binding of ¹²⁵I-labelled HTg by small lymphocytes was specific. An anti-HTg antibody cytophilic for guinea-pig small lymphocytes was demonstrated by the passive transfer of antigen-binding capacity to lymphocytes of unimmunized animals with hyperimmune guinea-pig serum. It is proposed that, in these experiments, anti-HTg cytophilic antibody was bound first to small lymphocytes in the tissues participating actively in the immune response (popliteal node, spleen and bone marrow) before spilling over into the general circulation to coat lymphocytes at other sites (mesenteric node and thymus).     

Porcine DC-SIGN: molecular cloning, gene structure, tissue distribution and binding characteristics

DC-SIGN, a human C-type lectin, is involved in the transmission of many enveloped viruses. Here we report the cloning and characterization of the cDNA and gene encoding porcine DC-SIGN (pDC-SIGN). The full-length pDC-SIGN cDNA encodes a type II transmembrane protein of 240 amino acids. Phylogenetic analysis revealed that pDC-SIGN, together with bovine, canis and equine DC-SIGN, are more closely related to mouse SIGNR7 and SIGNR8 than to human DC-SIGN. pDC-SIGN has the same gene structure as bovine, canis DC-SIGN and mouse SIGNR8 with eight exons. pDC-SIGN mRNA expression was detected in pig spleen, thymus, lymph node, lung, bone marrow and muscles. pDC-SIGN protein was found to express on the surface of monocyte-derived macrophages and dendritic cells, alveolar macrophages, lymph node sinusoidal macrophage-like, dendritic-like and endothelial cells but not of monocytes, peripheral blood lymphocytes or lymph node lymphocytes. A BHK cell line stably expressing pDC-SIGN binds to human ICAM-3 and ICAM-2 immunoadhesins in a calcium-dependent manner, and enhances the transmission of porcine reproductive and respiratory syndrome virus (PRRSV) to target cells in trans. The results will help better understand the biological role(s) of DC-SIGN family in innate immunity during the evolutionary process.     

Numbers and phenotype of lymphocytes emigrating from sheep bone marrow after in situ labelling with fluorescein isothiocyanate

In normal young lambs the bone marrow was selectively labelled with fluorescein isothiocyanate by a temporary perfusion of one hind-leg. One day later, the incidence of bone marrow emigrants in different lymph nodes, spleen, Peyer's patches, thymus, non-perfused bone marrow and blood was determined. The emigrants were also phenotyped by the use of monoclonal antibodies and classified into monocytes or lymphocyte subsets. Large numbers of lymphocytes left the bone marrow of the perfused leg during 1 day. Considerable numbers of cells migrated to other bone marrow compartments. Varying numbers of mononuclear emigrants were found in peripheral lymphoid organs, with labelling indices ranging from 1.06% in the blood to 0.004% in the thymus. In the spleen, comparable numbers of B- and T-lymphocyte emigrants from the bone marrow were found, whereas in the blood, lymph nodes and jejunal Peyer's patches many more emigrants were T lymphocytes than B lymphocytes. In the prescapular lymph nodes, for instance, 90.4% of emigrants were T cells but only 9.6% were B cells. Based on the large numbers of lymphocytes emigrating from the bone marrow, their phenotypes and their entry into other bone marrow compartments, it it can be concluded that the bone marrow of young lambs is an integral part of the migratory route of lymphocytes.     

Monoclonal antibody to a human brain-granulocyte-T lymphocyte antigen probably homologous to the W 3/13 antigen of the rat

The monoclonal antibody (F10–44–2) described in this report recognizes an antigen which by quantitative absorption analysis is found predominantly on spleen, lymph node, bone marrow, thymus, granulocytes and brain, the amount of antigen on these tissues being approximately the same within a factor of 2 or 3. Analysis with the fluorescence-activated cell sorter showed that 29% of thymus cells, 61% of bone marrow cells, 95% of blood mononuclear cells, 98% of lymph node lymphocytes and 100% of granulocytes carried the antigen. With blood mononuclear cells and lymph node lymphocytes, there were two distinct peaks, with one peak labeling very weakly. Double labeling experiments established that the weakly labeled peak contained the B lymphocytes. Studies on frozen sections of thymus established that positive thymocytes were found only in the medulla indicating that the antigen appears late in T lymphocyte maturation. The lymphatic nodules (B lymphocyte areas) of spleen and lymph node appeared virtually negative on frozen sections showing that there was too little antigen on the B lymphocyte surface for confident detection by fluorescence microscopy. Sodium dodecyl sulfate polyacrylamide gel eletrophoresis of NaB³ H₄-labeled blood mononuclear cells established that the antigen was a major glycoprotein of the leukocyte membrane and that its mol. wt. was 105 000. This antigen shows a striking similarity in biochemistry and tissue distribution to the W3/13 antigen of the rat and is likely to be the human homologue of this antigen.     

Proliferation of lymphocyte subsets in the adult rat: a comparison of different lymphoid organs

Adult, male Lewis rats received a single injection of 5-bromo-2'-deoxyuridine (BrdUrd) i.v. to label proliferating cells in the S phase of the cell cycle. After 1 and 24 h the thymus, bone marrow, blood, spleen, peripheral, cervical and mesenteric lymph nodes as well as Peyer's patches were removed. In cell suspensions surface staining was performed for B, T, T helper (Th) and cytotoxic/suppressor (Tc/s) T lymphocytes by identifying kappa light chain, CD5+, CD4+ and CD8+ cells, respectively. On the same slide the DNA label BrdUrd was demonstrated by a monoclonal antibody. B, T, Th and Tc/s lymphocytes proliferate locally both in central lymphoid organs such as the thymus and the bone marrow, and in peripheral lymphoid organs such as the spleen, lymph nodes and Peyer's patches. Within an organ the amount of proliferation among the lymphocyte subsets is similar, differing not more than threefold. Although concerning only a small fraction of cells within the organ, an unexpected finding is the high percentage of BrdUrd-labeled cells among B lymphocytes in the thymus (3%) and among T lymphocytes in the bone marrow (3%). One day after injection of BrdUrd the thymus contains 25% BrdUrd+ T lymphocytes, while the other organs investigated do not show more than about 2% BrdUrd+ B and T lymphocytes. Many of the newly formed lymphocyte subsets leave their organ of birth within 24 h. Thus the amount of proliferation in the lymphocyte subsets investigated is very similar and the differences between central (thymus and bone marrow) and peripheral lymphoid organs are much smaller than expected.     

Human prolactin improves engraftment and reconstitution of human peripheral blood lymphocytes in SCID mice

Recombinant human prolactin (rhPRL) was administered to huPBL-SCID mice to determine its effects on human immunologic reconstitution and function. The huPBL-SCID mice were given 10 microg i.p. injection of rhPRL every other day for a total of 10 injections after huPBL were transferred. The results demonstrated that rhPRL improved the engraftment of lymphocytes into thymus, lymph nodes and spleens, showing that the cellularities of these organs increased although the cellularities tended to vary depending on the donor. The amounts of human T cells (HLA-ABC+/CD3+) increased greatly in thymus (14.2 folds), spleen (4.16 folds) and lymph nodes (40.18 folds) after rhPRL injections. The amounts of human B cells (HLA-ABC+/CD19+) also increased greatly in lymph nodes (42.5 folds) and spleen (5.78 folds). The lymph node cells from the rhPRL-treated huPBL-SCID mice were more sensitive to PHA stimulation ([3H] thymidine incorporation). The supernatant of PHA-stimulated PBL from rhPRL-treated huPBL/SCID chimerism contained more cytokines (IFN-gamma and IL-2). The natural cytotoxicity against human sensitive target cells, K562 cells, from spleen and bone marrow of hPBL/SCID chimerism was significantly enhanced by rhPRL administration. The lymph node cells were stimulated with LPS in vitro for 3 days and the lymphocytes from the rhPRL-treated huPBL-SCID mice were more sensitive to mitogen stimulation. Both serum total IgG level and IgM level of rhPRL-treated huPBL/SCID chimerism were increased, and even without DT-rechallenge the base line of DT-specific IgG was elevated after rhPRL treatment in huPBL-SCID mice. Thus, rhPRL stimulation promotes reconstitution of human immune system in huPBL-SCID mice.     

Immunodepression in trypanosome-infected mice. VI. Comparison of immune responses of different lymphoid organs

Mitogen stimulation of cells from various lymphoid organs of C3H/He mice chronically infected with an isolate of Trypanosoma congolense was studied at different time intervals after infection, using concanavalin A (Con A) and lipopolysaccharide (LPS). At the same time, changes in the percentages of T, B and null lymphocytes in these organs were determined by immunofluorescence staining. The responses of T and B lymphocytes in the spleen were totally depressed, and the cellular composition was drastically altered by day 14 after infection. Unlike the spleen, the lymph nodes showed minor changes in their T and B lymphocyte responses and cell composition during the course of the infection, except the B cell response and composition which were altered late in the infection. The thymus and bone marrow did not show any appreciable changes in their mitogen responses and cell composition throughout the infection. The peripheral blood lymphocytes showed reduced B cell responses. Spleen cells from chronically infected mice suppressed lymphocyte stimulation induced in normal spleen and lymph node cell populations by Con A, LPS and allogeneic stimulator cells. Lymph node cells from the same group of mice did not exhibit any such suppressor activity. In the experimental system used here, the spleen is the primary site of immune depression, and other lymphoid organs such as the lymph nodes and thymus are very little affected.     

Proliferation and emigration of newly formed lymphocytes from pig spleens during an immune response

Normal young pigs were immunized intravenously with sheep red blood cells (SRBC). At various times after a second SRBC injection the spleens were connected to an extracorporeal perfusion system, and proliferating lymphoid cells in the spleens were selectively labelled with tritiated thymidine. One day later the relative and absolute numbers of spleen-derived lymphocytes were determined by autoradiography in the following organs: various parts of the spleen, mesenteric and cervical lymph nodes, thymus, bone marrow, Peyer's patches, tonsils, intestine, lung, liver and blood. From 1 to 7 days after the second SRBC injection, the spleens produced increasing numbers of lymphocytes, and labelled cells were found especially in the blood and bone marrow. The newly formed splenic lymphocytes migrated preferentially to T- but also to B-cell areas in lymph nodes, Peyer's patches and tonsils. In all organs outside the spleen nearly all labelled spleen-derived lymphocytes were small lymphocytes. However, the bone marrow contained a high proportion of labelled immature and mature plasma cells. The spleen produced large numbers of lymphocytes during the secondary immune response, many of which migrated to different organs probably as memory cells, while others were found in the bone marrow as effector cells from the immune response.