淋巴细胞

Methods: Density gradient centrifugalization and absorption technique wererespectively applied to detach peripheral blood mononuclear cells andperipheral blood lymphocytes,T lymphocytes were got by Nylon postabsorption method, and CD4+T and CD8+T lymphocytes divided with anti- CD4 andanti-CD8 antibody by alexin cytotoxic method.

密度梯度离心法分离外周血单个核细胞，玻璃器皿吸附法分离外周血淋巴细胞，尼龙棉柱法分离 T 淋巴细胞，补体介导的细胞毒法分别制备 CD4+T和 CD8+T 淋巴细胞，采用 T 淋巴细胞与角质形成细胞共培养技术研究银屑病患者外周血 T 细胞对角质形成细胞的影响，免疫组化法检测共培养角质形成细胞 Ki67、c-Myc 及 Bcl-xL 蛋白的表达。

Routine detecting techniquefor cell immunity such as white blood count and differential, lymphocyte blastogenesis test has beenused, and combine with flow cell technology to detect the level of cell immunization after advancedimmunization at several important aspect, and tracking experiments to observe changes in the numberof the WBC and lymphocytes, lymphocyte proliferation, the dynamic changes of CD4~+ and CD8~+lymphocyte subsets andγ-IFN; and detected dynamic changes rules of IgG, IgM in humor andmucosa with indirect ELISA, then compare with ordinary immunization methods.

主要采用白细胞计数和分类计数、淋巴细胞转化试验等常规细胞免疫检测技术结合先进的流式细胞技术从细胞免疫的几个重要方面入手检测猪瘟超前免疫后细胞免疫的水平，并进行跟踪试验，观察其白细胞、淋巴细胞数目变化情况，淋巴细胞增殖情况，CD~(4+)和CD~(8+)淋巴细胞亚群及γ-IFN的动态变化规律；同时通过间接ELISA方法检测体液和黏膜中各IgG、IgM的动态变化规律，同时与普通免疫方法进行比较。

The following methods were used in the study, such as viral inoculation of animal, pathological method, flow cytometry, RT-PCR, in situ hybridization, immunohistochemistry and terminal deoxy nucleotidyl transferase mediated dUTP nick end labling . Morphological changes of the infected chickling, dynamic changes of T, B cells and T subsets, changes of adhesion molecules on the lymphocyte surface, necrosis and apoptosis of lymphocyte and neurons, changes of MIP-1β and IL-8 producing cells in the brain, cell types of perivascular tuffing in cerebral tissue were systematically studied.

在研究过程中，采用了病毒接种技术、普通病理学研究方法、流式细胞仪技术、RT-PCR技术、原位杂交技术、免疫组化技术、凋亡细胞末端标记技术，系统研究了不同日龄的SPF雏鸡人工感染AEV-NH937株后的病理变化，T和B淋巴细胞、T淋巴细胞亚群的动态变化规律，淋巴细胞表面某些粘附分子的变化，淋巴细胞和神经元的坏死与凋亡，雏鸡脑组织中产生趋化因子MIP-1β和IL-8细胞的变化规律，脑组织中围官性细胞浸润的细胞类型。

The following methods were used in the study, such as viral inoculation of animal, pathological method, flow cytometry, RT-PCR, in situ hybridization, immunohistochemistry and terminal deoxy nucleotidyl transferase mediated dUTP nick end labling. Morphological changes of the infected chickling, dynamic changes of T, B cells and T subsets, changes of adhesion molecules on the lymphocyte surface, necrosis and apoptosis of lymphocyte and neurons, changes of MIP-1β and IL-8 producing cells in the brain, cell types of perivascular tuffing in cerebral tissue were systematically studied. The key research results were.(1)The average percentage of CD19+cell in blood, bursa and spleen, after 3, 5 days of inoculation, was significantly Phybridization revealed that the number of IL-8 and MIP-1βproducing cells was increased in the infected brain.

在研究过程中，采用了病毒接种技术、普通病理学研究方法、流式细胞仪技术、RT-PCR技术、原位杂交技术、免疫组化技术、凋亡细胞末端标记技术，系统研究了不同日龄的SPF雏鸡人工感染AEV-NH937株后的病理变化，T和B淋巴细胞、T淋巴细胞亚群的动态变化规律，淋巴细胞表面某些粘附分子的变化，淋巴细胞和神经元的坏死与凋亡，雏鸡脑组织中产生趋化因子MIP-1β和IL-8细胞的变化规律，脑组织中围官性细胞浸润的细胞类型。

The studies on the capacities of immune responses of lymphocytes in peripheral blood and immune organs of Grass Carp (Ctenpharyngoden idellus Cuvier et Valencinnes) to PHA and SPA with incorporation of 〓H-TdR and active reaction of Acid α-Naphthyl Acetate Esterase indicated that there were T and B lymphocytes in peripheral blood of grass carp, but only T lymphocyte and no B lymphocyte in the thymus of grass carp. It was justified that the T lymphocytes in peripheral blood was originated from the thymus, and B lymphocyte could be from the spleen and anterior kidney of grass carp.

运用〓H-TdR掺入法和酸性醋酸萘酯酶活性检测法分别研究了草鱼外周血以及免疫器官中的淋巴细胞对PHA和SPA的免疫应答以及化学标志特征，发现草鱼外周血中存在着类似于哺乳动物的T、B两类不同的淋巴细胞亚群；同时也发现胸腺中只有T淋巴细胞，而无B淋巴细胞，提示了胸腺中淋巴细胞可能是外周血中T细胞的来源，而B淋巴细胞可能来自脾脏或前肾。

Get high purity DCs by Cultured plastic-adherent monocytes isolated from healthy human peripheral blood with GM-CSF and IL-4 for 7 days. To observe the morphology of DCs by inverted phase contrast microscope ,electron microscope and laser confocal microscope. Analyse phenotype of DCs with flow cytometry. Investigate the endocytosis ability of DCs as a group by Horseradish peroxidase endocytosis assay. To appraise allogeneic mixed lymphocytes reaction of DCs by MTT reduction assay. Analyse the levels of IL-12 and TNF in liquids of cultured medium by ELISA and MTT reduction assay respectively. Soluble antigens of HCCs was obtained by 3 freeze-and-thaw cycles. Biological characteristics of HC soluble antigens pulsed DCs were monitored by flow cytometry. According to MTT reduction assay estimated the cell proliferation of self lymphocytes activated by HC antigens pulsed DCs. Get high purity BCG HSP 70 protein by SDS-PAGE electrophoresis and determined its biological activity with ELISA. Analyse phenotype of antigen pulsed DCs primed by BCG HSP70 with flow cytometry. By MTT reduction assay estimated the cell proliferation of self lymphocytes and the MLR of DC based vaccine. Analyse expression of HLA-DR molecule on surface of HCC lines. The IFN-γ mRNA in lymphocytes after actived by DC vaccine and the Fas-L expression on DC and DC vaccine primed lymphocytes were detected by in situ hybridization and flow cytometry respectively. Specific cytotoxity lysis of T lymphocytes and nonspecific inhibition of liquids in culture medium against HCC lines were also tested. Detect expression of hAFP on four HCC lines with Cell-ELISA. Induce apoptosis of HCCs with actinomycin-D. Interaction of DCs and apoptotic cells was observed under transmission electron microscope. Growth inhibition test of DC against HCC lines was also performed. Establish the nude mouse model bearing human HC xenografts and indentify the characteristic of tumour by histochemistry and immunohistochemistry techniques. Prevent and treat transplanted human HC on nude mouse with Freezing and anabiotic HC specific lymphocytes.

用GM-CSF和IL-4从健康人外周血诱导DC；分别用倒置相差显微镜、电子显微镜及激光共聚焦显微镜观察DC形态；流式细胞术检测DC表型；HRP吞噬实验测定DC的群体内吞能力；MTT法检测同种异体混合淋巴细胞反应；ELISA法和MTT法分别测定DC培养上清液中IL-12和TNF水平；冻融法制备肝癌细胞可溶性抗原；流式细胞术检测负载肝癌可溶性抗原后DC的生物学特性；MTT法检测DC负载肝癌抗原后对自身淋巴细胞增殖的影响；SDS-PAGE制备电泳纯化BCG HSP70并鉴定纯度，ELISA测定活性；流式细胞术检测负载抗原DC经BCGHSP 70活化后的表型；MTT法检测肝癌DC疫苗对自身淋巴细胞增殖的影响和混合淋巴细胞反应；流式细胞术检测肝癌细胞表面HLA-DR表达；MTT法检测肝癌DC疫苗对自身淋巴细胞的活化；原位杂交法检测肝癌DC疫苗活化后的淋巴细胞IFN-γmRNA表达；流式细胞术检测DC和肝癌DC疫苗活化后淋巴细胞表面Fas-L；MTT法分别检测肝癌DC疫苗活化的淋巴细胞和其培养上清对肝癌细胞的特异性杀伤和非特异性抑制作用；Cell-ELISA检测人肝癌细胞hAFP表达；MTT法检测负载AFP表位肽和凋亡肝癌细胞DC对自身淋巴细胞增殖的影响；ELISA法和MTT法分别测定活化后淋巴细胞培养上清中TNF和IL-12水平；肝癌细胞凋亡的诱导和检测；DC吞噬凋亡肝癌细胞后的电子显微镜观察；DC对肝癌细胞的生长抑制试验；人肝癌裸鼠皮下移植瘤动物模型的建立及其组织学和免疫组织化学鉴定；DC及肝癌特异性淋巴细胞预防和治疗人肝癌裸鼠皮下移植瘤；冻存和复苏后的肝癌特异性淋巴细胞预防和治疗人肝癌裸鼠皮下移植瘤。

The results of study show:1. White pulp and red pulp in parenchyma of spleen of embryo can be obviously discerned after 18 days. Periarterial lymphoid sheath and ellipsoid periarterial lymphoid sheath also can be obviously discerned in spleen of 4 days chicken. T, B lymphocytes in appendix basement of embryo emerge after 20 days. It is the initial shape of cecal tonsil. Crypt structure of conjunction of esophago and stomachus glandularis form obviously at 4 days. It is the initial shape of esophago tonsil. The germinal center firstly emerges in these three organs at 14 days. With the increase of day age, the characteristic structure peripheral immune organs gradually develop mature. Spleen achieve mature at 21 days and cecal tonsil at 35 days.2. IgM~+ and IgA~+ cells in spleen of embryo emerge at 15 days. IgG~+ cell, CD3~+ and CD8~+T lymphocytes of embryo emerge at 20 days. CD3~+, CD8~+ and IgM~+ cells in cecal tonsil of embryo emerge at 20 days. However CD4~+, IgG~+ and IgA~+ cells all emerge in 1 day age chicken out of crust. CD3~+, CD4~+, CD8~+, IgM~+ and IgG~+ cells in esophago tonsil of embryo all emerge at 20 days. However IgA~+ cells emerge in 1 day age chicken out of crust.3. The amount of T, B lymphocytes in peripheral immune organs increase follow with the increase of day age, and hold an upgrade tendency. The amount of T, B lymphocytes in spleen achieved stabilization at 21 days, and in tonsil of esophago and appendix at 35 days.

研究结果表明：1、在组织结构方面，脾脏实质内的白髓与红髓在胚胎18日龄后明显可辨，4日龄雏鸡脾脏中形成明显可辨的动脉周围淋巴鞘和椭球周围淋巴鞘；盲肠基部T、B淋巴细胞在胚胎20日龄时开始出现，即初步形成盲肠扁桃体；食管与腺胃结合处在4日龄时形成明显的隐窝结构，即食管扁桃体初步形成；14日龄时，三种器官中首次出现生发中心；随着日龄的增长，外周免疫器官特征结构不断发育成熟，脾脏在21日龄时达到成熟水平，盲肠扁桃体和食管扁桃体在35日龄时达到成熟水平。2、在T、B淋巴细胞出现时间方面，脾脏中IgM~+和IgA~+细胞在胚胎15日龄时开始出现，IgG~+细胞、CD3~+和CD8~+T淋巴细胞在胚胎18日龄时出现，CD4~+细胞在胚胎20日龄时出现；盲肠扁桃体中CD3~+、CD8~+和IgM~+细胞在胚胎20日龄时开始出现，而CD4~+、IgG~+和IgA~+细胞均在雏鸡出壳后1日龄时出现；食管扁桃体中CD3~+、CD4~+、CD8~+、IgM~+和IgG~+细胞均在胚胎20日龄时开始出现，而IgA~+细胞则在雏鸡出壳后1日龄时出现。3、在T、B淋巴细胞数量变化方面，外周免疫器官中T、B淋巴细胞的数量随日龄增长，整体均呈上升趋势。

CD8~+ cell is the main T lymphocyte subset in spleen, and B lymphocyte mainly is IgG~+ cell, moreover the amount of these B lymphocytes could exceed CD3~+ T lymphocyte subset after 7 days. CD8~+ cell is the main T lymphocyte subset in tonsil of appendix, and B lymphocyte is IgM~+ cell, and the amount could exceed CD3~+ T lymphocytes after 35 days. After 21 days, B lymphocytes in esophago tonsil are the main IgA~+ cells and the amount exceeds CD3~+ lymphocytes. The amount of CD4~+ lymphocytes is more than CD8+ lymphocytes.4. CD3~+、CD4~+ and CD8~+ T lymphocytes in spleen mainly distribute in periarterial lymphoid sheath. However IgM~+、IgG~+ and IgA~+ cells mainly distribute in ellipsoid periarterial lymphoid sheath and germinal center. T lymphocytes in appendix tonsil mainly distribute in middle and inferior part of mucous and the B lymphocytes mainly in middle and mucous between 4~7 days. Whereafter T, B lymphocytes equably distribute in mucous. CD4~+ cells arrange tightly and mainly occupy the central part in aggregates of T lymphocytes in esophago tonsil and CD8~+ lymphocytes mainly distribute in periphery. Meanwhile B lymphocytes encircle the periphery of aggregates of T lymphocytes. The aggregates of B lymphocytes is mainly the germinal center with lots of IgM~+、IgG~+ and IgA~+ cells. Meanwihle T lymphocytes encircle the periphery of aggregates of B lymphocytes.5. There is an intimate relationship between the development of tissue structure of peripheral immune organs and lymphcytopoiesis. The maturation of tissue structure is stimulated by the immigration of lymphocytes and the mature tissue structure provides place where lymphocytes grow mature and functionate.

脾脏在21日龄时达到稳定，食管扁桃体和盲肠扁桃体均在35日龄时达到稳定；脾脏中T淋巴细胞亚群以CD8~+细胞为主，B淋巴细胞则以IgG~+细胞为主，并在7日龄后数量超过CD3~+T淋巴细胞；盲肠扁桃体中T淋巴细胞亚群以CD8~+细胞为主，B淋巴细胞以IgM~+细胞为主，并在35日龄后数量超过CD3~+T淋巴细胞；21日龄后，食管扁桃体中B淋巴细胞以IgA~+细胞为主，数量超过CD3~+细胞，CD4~+细胞的数量多于CD8~+细胞。4、在T、B淋巴细胞组织定位方面，脾脏中CD3~+、CD4~+和CD8~+T淋巴细胞主要分布在动脉周围淋巴鞘中，而IgM~+、IgG~+和IgA~+细胞主要分布在椭球周围淋巴鞘和生发中心中；4～7日龄时，盲肠扁桃体中T淋巴细胞主要分布在粘膜固有层的中下部区域，而B淋巴细胞则主要分布在中上部区域，随后各日龄T、B淋巴细胞均匀地分布在粘膜固有层中；在食管扁桃体的T淋巴细胞聚集物中，CD4~+细胞紧密排列，主要占据中央部位，CD8~+细胞主要散布在外周，同时B淋巴细胞又环绕在整个T淋巴细胞聚集物的外周；B淋巴细胞聚集物主要为生发中心，其中存在大量IgM~+、IgG~+和IgA~+细胞，同时T淋巴细胞又环绕在整个B淋巴细胞聚集物的外周。5、外周免疫器官的组织结构发育和淋巴细胞发生之间存在密切的关系，淋巴细胞迁入淋巴器官刺激组织结构的发育成熟，同时成熟的组织结构又为淋巴细胞发育成熟并行使功能活动提供场所。

CD1a and CD83 molecules could also be expressed on the surface of CD3+ T lymphocytes and CD19+ B lymphocytes. The expression levels of CD1a and CD83 in activated lymphocytes were higher than those in the unactivated lymphocytes, and higher in B lymphocytes than in T lymphocytes.

结果该细胞表达特征性分子CD1a、CD40、CD80、CD83、CD86， CD1a 、CD83在CD3+T、CD19+B淋巴细胞上也表达，在激活的淋巴细胞上表达水平高于未激活的淋巴细胞，而且B淋巴细胞上的表达水平高于T淋巴细胞。

Results The activation of NF-κB, proliferation response, and expression of IL-4 and I L-5 mRNA and protein in T lymphocytes stimulated by PMA were significantly hig her than those of their blank control (P.01), while those indexes of T l ymphocytes stimulated by PMA and PDTC simultaneously were significantly lower th an those stimulated by PMA alone (P.01). The apoptotic index of T lympho cytes stimulated with PMA were significantly lower than that of their blank cont rol (P.01), and the apoptotic index of asthmatic guinea pig T lymphocytes stimulated with PMA and PDTC simultaneously were significantly higher than that stimulated by PMA alone (P.01). The significant positive correlations w ere found between the activation of NF-κB and the proliferation (r=0.64, P.001), and the expression of IL-4 and IL-5 mRNA and protein of T lymph ocytes, respectively (r=0.55-0.68, P.001). There was also signific ant negative correlation between the activation of NF-κB and apoptosis of T l ymphocytes (r=0.62, P.001). Conclusions NF-κB may participate in the signal conduction of PKC regulated proliferation, apoptosis and expression of IL-4 and IL-5 of T lymphocytes in asthma.

结果加入PMA培养的哮喘组T淋巴细胞NF-κB的活化、细胞增殖反应、IL-4和IL-5的mRNA和蛋白质的表达均显著高于其空白对照(P.01)，而同时加入PMA和PDTC培养的哮喘组T 淋巴细胞以上指标均显著低于只加入PMA培养的哮喘组T淋巴细胞(P.01)；加入PMA 培养的哮喘组T淋巴细胞的凋亡指数显著低于其空白对照(P.01)，而同时加入PMA 和PDTC培养的哮喘组T淋巴细胞凋亡指数显著高于只加入PMA培养的哮喘组T淋巴细胞(P 。01)。T淋巴细胞NF-κB的活化与增殖反应呈显著正相关(r=0.64，P.00 1)，与IL-4和IL-5的mRNA和蛋白质的表达也均呈显著正相关(r=0.55-0.68，P 。001)，而与凋亡指数呈显著负相关(r=-0.62，P.001)。

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