人外周血树突状细胞诱导及免疫学特性研究

[目的]从人外周血分离、纯化、扩增树突状细胞(DC),并对其形态学和免疫学特性进行初步探讨.[方法]从人外周血分离DC前体细胞(主要为CD14 细胞)用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素-4(rhIL-4)联合培养,诱导扩增成熟DC.观察DC形态、分析DC表型、核型及检测DC激发同种异体淋巴细胞增殖能力.[结果]分离的DC前体经rhGM-CSF和rhIL-4共同培养1周后,可获得大量成熟DC,扩增了24.5倍,纯度达90%以上.DC高表达分化抗原CD86、CD40、HLA-DR、CD83、CDIa,能强烈激活同种异体T淋巴细胞增殖.[结论]人外周血CD14 细胞经体外诱导培养,可以生成大量功能成熟的DC,从而为进一步开展DC的基础研究和临床应用打下基础.     

转染癌胚抗原基因的人树突状细胞疫苗表达的研究

目的　探索使用质粒转导制备转基因DCs疫苗的方法。方法　采用细胞因子rhIL 4和rhGM CSF诱导培养法制备人外周血树突状细胞 ,用Lipofectine向人DCs转染含人CEA真核表达质粒。结果　转染组DCs培养上清CEA含量明显高于未转染组培养上清CEA含量。结论　人CEA真核表达质粒转染DCs可内源性表达CEA     

转移性癌细胞体外水解酶表达及影响因素的作用

目的　为阐明树突状细胞肉瘤 (DCS)细胞在体外培养条件下其水解酶表达的类型及一些影响因素的作用。方法　采用水解空斑法检测 10种特异性蛋白酶抑制剂、不同抗体及基质对 DCS细胞的体外蛋白水解作用的影响。结果　抑肽酶 (aprotinin)、乙二胺四乙酸钠 (EDTA- Na2 )、抑胃酶 ((pepstatin)可显著抑制 DCS细胞的蛋白水解作用。抗泛素抗体、抗 DCS抗体对 DCS的蛋白水解作用无明显影响。在纤粘连蛋白 (FN)、层粘连蛋白 (L N)基质上 DCS细胞铺展良好 ,蛋白水解作用不明显。结论　本实验条件下 ,DCS细胞可表达丝氨酸蛋白酶、金属蛋白酶(非基质水解酶 )、天门冬氨酸蛋白酶的活性。抗泛素抗体、抗 DCS抗体对 DCS细胞的蛋白水解作用无明显影响。不同基质上 DCS细胞水解酶表达状态不同     

CD73 and Adhesion of B-Cells to Follicular Dendritic Cells

CD73, otherwise known as ecto-5'-nucleotidase, is a lymphocyte maturation marker which is involved in intracellular signalling, lymphocyte proliferation and activation. In addition, we have demonstrated that CD73 is involved in mediating lymphocyte binding to in vitro cultured endothelial cells and in controlling adhesion between freshly isolated germinal center B-cells and follicular dendritic cells (FDC). In secondary lymphoid tissues, CD73 is expressed on FDC in the light zone of the germinal center as well as on small, resting mantle zone B-cells but not on B-cells within germinal centers. In this review article, the potential role of CD73 in controlling B-cell-FDC interactions and B-cell maturation will be discussed.     

人龋病牙髓中神经肽与抗原呈递细胞的动态研究

目的 :研究人龋病牙髓中降钙素基因相关肽 (Calicitoningene -relatedpeptide,CGRP)和树突状细胞(Dendriticcells,DC)、P物质 (SubstanceP,SP)和血管内皮细胞(Endothelialcells,EC)之间的空间变化特点 ,以探明牙髓病理状态下神经免疫间的相互作用。方法:采用免疫组化法观察龋病不同阶段牙髓中上述物质的变化 ,并对前、后两种物质分别进行双标免疫组化染色。结果:(1)4个指标的数量在不同组间比较差别有统计学意义 (P<0.05)。 (2)双标染色显示 :龋病牙髓中神经肽与抗原呈递细胞共聚集在龋坏相应冠髓的造牙本质细胞层下 ,CGRP接触或包绕树突状细胞 ;而SP随龋坏深入逐渐接近血管并与管壁接触。结论:龋源性刺激引起牙髓中CGRP、SP、DC、EC的局部相互作用加强共同调节牙髓免疫反应     

人乳头瘤病毒16 E6转染冻融人外周血树突状细胞疫苗体外抗宫颈癌作用

目的：制备来源于人外周血并转染人乳头瘤病毒（HPV）16 E6基因的冻融树突状细胞（DC）疫苗，检测其细胞形态。转染情况及体外诱导的免疫效应。方法：细胞因子扩增人外周血DC，低温-70℃冻存；Lipofectamine转染HPV16 E6制备DC疫苗。动态形态学观察，免疫细胞化学检测E6分子表达，体外诱导并测定细胞毒性T淋巴细胞活性。结果：转染E6基因的人外周血冻融DC呈形态迥异的多突起状，免疫细胞化学染色显示E6蛋白表达阳性；MTT法检测人宫颈癌细胞株（CaSKIi细胞）的杀伤活性明显高于对照组（P〈0．01），与新鲜DC疫苗比无统计学意义（P〉0．05）。结论：转染历基因的人外周血冻融DC疫苗保持了功能成熟DC的形态特征，且内源性表达E6蛋白，并在体外诱导出高效特异性的抗宫颈癌免疫反应，为宫颈癌的主动特异性免疫治疗提供了理想的疫苗和实验依据。     

Immunotoxic effects of short-term atrazine exposure in young male C57BL/6 mice.

The herbicide atrazine (ATR) is a very widely used pesticide; yet the immunotoxicological potential of ATR has not been studied extensively. Our objective was to examine the effect of ATR on selected immune parameters in juvenile mice. ATR (up to 250 mg/kg) was administered by oral gavage for 14 days to one-month-old male C57BL/6 mice. One day, one week, and seven weeks after the last ATR dose, mice were sacrificed, and blood, spleens, and thymuses were collected and processed for cell counting and flow cytometry. Thymus and spleen weights were decreased by ATR, with the thymus being more sensitive than the spleen; this effect was still present at seven days, but not at seven weeks after the last ATR dose. Similarly, organ cellularity was persistently decreased in the thymus and in the spleen, with the splenic, but not thymic cellularity still being depressed at seven weeks post ATR. Peripheral blood leukocyte counts were not affected by ATR. There were also alterations in the cell phenotypes in that ATR exposure decreased all phenotypes in the thymus, with the number of CD4(+)/CD8(+) being affected the least. At the higher doses, the decreases in the thymic T-cell populations were still present one week after the last ATR dose. In the spleen, the CD8(+) were increased and MHC-II(+) and CD19(+) cells were decreased one day after the last ATR dose. Also, ATR treatment decreased the number of splenic na?ve T helper and T cytotoxic cells, whereas it increased the percentage of highly activated cytotoxic/memory T cells. Interestingly, the proportion of mature splenic dendritic cells (DC; CD11c(high)), was also decreased and it persisted for at least one week, suggesting that ATR inhibited DC maturation. In the circulation, ATR exposure decreased CD4(+) lymphocytes at one day, whereas at seven days after the last ATR dose, in addition to the decrease in CD4(+) lymphocytes, the MHC-II(+) cells were also decreased at the 250 mg/kg dose. Thus, ATR exposure appears to be detrimental to the immune system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with certain effects persisting long after exposure has been terminated.     

DC联合小剂量IL-2大量扩增白血病特异性CTL的实验研究

目的： 建立和优化体外大量扩增白血病特异性CTL的方法。 方法： 用1×107FBL3细胞冻融产物（FBL3LY）冲击的DC每10天1次对C57BL/6小鼠反复接种，在第3次接种5 d后处死小鼠制备脾T细胞，每周用FBL3LY冲击的DC刺激，体外培养10 d后加入小剂量的IL2。 结果： 共培养至21 d， T细胞可扩增100倍以上，其中CD8+细胞比率明显增高。乳酸脱氢酶释放试验证实扩增所得CTL对FBL3细胞具有高效的杀伤作用（81.84±8.68%），而对K562细胞无特异性杀伤作用。 结论： FBL3LY冲击的DC联合小剂量IL2可大量扩增FBL3白血病特异性CTL，该方法的建立对提高白血病特异性CTL免疫治疗的临床疗效具有重要意义。