CD11c和NLDC-145单克隆抗体标记的阳性细胞的观察

目的 探讨CD11c和NLDC145单克隆抗体标记胸腺内何种间质细胞，并是否具有特异性。方法 应用CD11c单克隆抗体及NLDC-145单克隆进行标记，采用免疫荧光双重染色法及免疫电镜法对胸腺内间质细胞进行观察。结果 CD11c阳性细胞为指状嵌入突起细胞(interdigitating cell，IDC)及少数巨噬细胞。NLDC-145阳性细胞为胸腺上皮细胞。结论 CD11c标记IDC，NLDC-145标记胸腺上皮细胞。胸腺巨噬细胞中存在不同亚群。     

BALB/c小鼠胸腺巨噬细胞不同亚群特性研究

目的研究小鼠胸腺巨噬细胞组成、分布、形态、功能。方法糖皮质激素诱导小鼠胸腺细胞凋亡,采用免疫荧光染色法,免疫组织化学和酸性磷酸酶(ACP)双重染色方法,发现小鼠胸腺巨噬细胞的不同表型。结果发现小鼠胸腺巨噬细胞存在四种表型:树状突起型细胞:可被Mac-2,F4/80单克隆抗体标记,数量最多,分布于整个胸腺实质且有吞噬作用;扁平型细胞:可被F4/80单克隆抗体标记,分布于皮质被膜下方;小卵圆型细胞:可被Mac-2单克隆抗体标记,分布于胸腺髓质和皮髓交界(CMR),此类细胞未见吞噬现象。ED1~+细胞:细胞形态不规则,主要分布于皮髓质交界区。以上不同的巨噬细胞亚群均可显示酸性磷酸酶活性。结论小鼠胸腺巨噬细胞存在不同亚群,表现在其组成、形态、分布、特点及功能差异等方面。     

HIV-1重组核酸疫苗质粒诱导小鼠免疫应答的研究

目的：研究白细胞介素6（IL－6）对人类免疫缺陷病毒（HIV）gp120基因免疫小鼠的调节作用。方法：构建表达中国流行株HIV－1B亚型gp120基因的核酸疫苗质粒pGP及共表达中国流行株HIV－1B亚型gp120基因与IL－6基因的核酸疫苗质粒pGPIL-6，检测其在哺乳动物细胞中的表达并 将上述两种质粒注射Balb/c鼠，检测小鼠产生的CD4^ 和CD8^ T细胞数情况及诱导CTL反应能力。结果：以间接免疫荧光法（IFA）检测到gp120在哺乳动物细胞中得到表达。pGPIL-6免疫鼠，小鼠产生的CD4^＋和CD8^ T细胞数及诱导产生CTL反应的能力明显高于单纯注射gp120的小鼠。结论：协同应用IL－6基因可明显加强HIV－1gp120基因免疫效果，为中国流行株HIV－1核酸疫苗的可行性提供了重要实验依据。     

混合谱系激酶结构域样假激酶在小鼠脑缺血后脑水肿组织中的表达

目的:研究混合谱系激酶结构域样假激酶(MLKL)在C57BL/6J小鼠脑缺血后脑水肿组织中的表达及作用。方法:通过向野生型(WT)和MLKL敲除(MLKL~-/~-) C57BL/6J小鼠尾静脉注射浓度为6 mg/ml玫瑰红溶液(剂量为40 mg/kg)构建缺血后脑水肿模型。一周后取组织,利用免疫荧光染色技术观察MLKL在缺血后脑水肿区的表达和分布,Western Blot检测MLKL在小鼠缺血后脑水肿区的表达水平,采用免疫电镜技术观察MLKL的亚细胞水平定位,苏木精-伊红染色(HE)观察组织缺血坏死及水肿情况。结果:免疫荧光染色结果显示正常WT小鼠大脑皮质无MLKL表达,缺血后脑损伤区的WT小鼠有大量MLKL阳性细胞,MLKL主要表达于GFAP阳性星形胶质细胞; Western Blot结果显示脑缺血后脑损伤区的MLKL表达显著增加;免疫电镜结果显示脑水肿区域血管周围的星形胶质细胞内有大量MLKL蛋白胶体金颗粒分布; HE染色结果显示MLKL敲除可显著减轻脑水肿程度。结论:脑缺血可上调WT小鼠缺血部位MLKL表达,提示MLKL可能参与缺血后的脑水肿发生。     

小鼠胸腺细胞核骨架肌动蛋白的研究

以细胞分级抽提结合间接免疫荧光染色法，观察小鼠胸腺细胞核骨架肌动蛋白，结果显示：间期细胞核骨架肌动蛋白遍布全核，Ｆ－肌动蛋白主要分布于核周，构成环层，细胞有丝分裂期间，核骨架肌动蛋白经抽提后依然保留，这与胞质肌动蛋白有明显不同。ＳＤＳ－ＰＡＧＥ及免疫印迹反应结果表明，肌动蛋白是小鼠胸腺细胞核骨架的主要成分之一，双向电泳进一步说明，核骨架肌动蛋白除含有β和ｒ肌动蛋白外，还含有核骨架所特有的肌动蛋白，     

IL-1β在成人牙周炎患者牙龈组织中表达

目的　探讨IL 1β在成人牙周炎性牙龈组织的分布及与炎症细胞浸润的关系。 方法　免疫组织化学SP方法。结果　 19例牙周炎组炎性牙龈标本中均有IL 1β蛋白阳性表达 ,阳性细胞数与正常对照组牙龈相比有非常显著差异 (P <0 0 1)。IL 1β阳性细胞主要为巨噬细胞。 结论　IL 1β可能参与了成人牙周炎的发生和发展。巨噬细胞是成人牙周炎炎性牙龈组织中表达细胞因子IL 1β的主要细胞类型 ,与成人牙周炎的发生和发展有关     

结核病小鼠病理学与TH1/TH2细胞动力学及iNOS表达关系的研究

目的 从分子病理学角度探讨结核病小鼠病理学与免疫学的相关性。方法 将BALB／c小鼠随机分为两组：结核病模型组（A）和正常对照组（N）。经尾静脉注射H37RV建立结核病小鼠模型。采用免疫组化染色和常规HE染色，探讨IFN－γ、IL－4、iNOS表达与肺脏组织损伤的类型和程度的关系。免疫组化结果采用图象分析计算阳性细胞面密度。结果 结核病小鼠病程中可观察到两个阶段：感染2周内为急性炎症明，以肺泡－毛细血管间质和血管周围炎性渗出为特征，可观察到明显的TH1细胞占优势，免疫组织化学染色结果显示，在炎性渗出区内IFN－γ阳性细胞较IL－4阳性细胞明显增多；感染第4周到实验结束为慢性进展期，以肺实质内广泛肺炎伴灶性干酷坏死为特征，免疫反应表现为THO平衡，在肺损伤组织中IFN－γ和IL－4阳性细胞基本相当。iNOS表达在急性期增多慢性期减少。结核病感染过程中TH1／TH2细胞动力学和iNOS3表达与组织损伤的类型和程度关系密切。     

胎儿胸腺发育过程中T细胞抗原受体的表达

目的:检测不同胎龄胎儿胸腺组织内T细胞抗原受体(TCR)的表达及发育规律.方法:18～40周胎儿胸腺组织,采用免疫组织化学SP法检测TCRαβ和TCRγδ的表达. 结果:胚胎21周,胸腺组织开始出现TCRαβ和TCRγδ阳性细胞.TCRαβ阳性细胞主要分布被膜下及小叶间隔中,围绕血管成群分布.TCRγδ阳性细胞的分布与TCRαβ阳性细胞相似, 但细胞数量较TCRαβ阳性细胞少.38周以后胎儿胸腺内TCR阳性细胞主要分布在皮髓质交界处,被膜下及小叶间隔中的TCR阳性细胞较早期少.结论:TCR阳性细胞在胚胎早期开始出现,其分布部位提示局部微环境有利于这些细胞的分化发育.     

共抑制分子PD-1在类风湿关节炎模型免疫系统及滑膜组织的表达

目的:探讨共抑制分子PD-1在牛二型胶原(CII)诱导的类风湿关节炎模型(CIA)小鼠免疫器官、关节及RA患者滑膜组织内的表达.方法:使用牛二型胶原免疫DBA-1/j小鼠,建立小鼠类风湿关节炎模型;免疫组化检测类风湿关节炎标本PD-1的表达变化;流式细胞分析术(FACs)检测对PD-1阳性细胞进行定位.结果:免疫组化的结果证实,小鼠的骨髓、胸腺、脾脏及淋巴结有中有大量的PD-1阳性细胞,FACs分析结果表明这些PD-1+ 细胞主要为CD3+T细胞,B220+B细胞,CD68+巨噬细胞及CD11c+树突状细胞,统计分析表明CIA小鼠体内PD-1表达及分布相对于对照组小鼠没有显著提高.此外,RA患者及CIA小鼠的滑膜组织内仅发现少量PD-1阳性的细胞. 结论:PD-1阳性细胞可能不参与类风湿性关节炎的发生发展过程.     

抗鸭胸腺细胞单克隆抗体的制备及其免疫组织化学研究

目的：研究鸭胸腺细胞表面标志。方法：利用杂交瘤细胞融合技术，制备抗鸭胸腺细胞单克隆抗体，并利用免疫组织化学方法研究其反应阳性细胞的组织分布和发生。结果：单克隆抗体C12反应阳性细胞在胸腺，法氏囊和脾脏组织中均有分布，阳性细胞类型不同。结论：C12株杂交瘤细胞分泌的抗体所反应的阳性细胞是成熟的T淋巴细胞和树突状细胞，配体是两者的共同表面标志。     

Essential microenvironment for thymopoiesis is preserved in human adult and aged thymus

Normal human thymuses at various ages were immunohistologically examined in order to determine whether adult or aged thymus maintained the microenvironment for the T cell development and thymopoiesis was really ongoing. To analyze the thymic microenvironment, two monoclonal antibodies (MoAb) were employed. One is MoAb to IL-1 receptor (IL-1R) recognizing medullary and subcapsular cortical epithelial cells of normal infant human thymus. The other is UH-1 MoAb recognizing thymic epithelial cells within the cortex, which are negative with IL-1R-MoAb. Thymus of subjects over 20 years of age was split into many fragments and dispersed in the fatty tissue. However, the microenvironment of each fragment was composed of both IL-1R positive and UH-1 positive epithelial cells, and the UH-1 positive portion was populated with lymphocytes showing a follicle-like appearance. Lymphocytes in these follicle-like portions were mostly CD4+CD8+ double positive cells and contained many proliferating cells as well as apoptotic cells. Thus these follicle-like portions in adult and aged thymus were considered to be functioning as cortex as in infant thymus. Proliferative activity of thymocytes in the thymic cortex and the follicle-like portions definitely declined with advance of age, while incidence of apoptotic thymocytes increased with aging.     

Estrogen Blocks Early T Cell Development in the Thymus

PROBLEM: Pregnancy and estrogen are known to suppress B lymphopoiesis as well as lead to thymic involution in the mouse. Additionally, estrogen deficiency by oophorectomy reportedly causes a selective increase in the B220+ B cells in the murine bone marrow. The purpose of this study was to determine if estrogens played a regulatory role in T cell development. METHODS: The first experimental group consisted of 5–6-week-old Balb/c mice that received subcutaneous pellets of placebo, estriol, estradiol, or progesterone. The thymus glands were examined 2–4 weeks after treatment. The second group consisted of 6-week-old Balb/c mice who underwent either bilateral oophorectomy or a sham procedure. Two weeks after the surgery, extensive phenotypic characterization of the thymus and spleen cells was performed by flow cytometry using monoclonal antibodies to surface markers of T cell subsets. RESULTS: Estrogen treatment causes a dramatic reduction of thymic size and cellularity. All defined T cell subsets of CD4 and CD8 were reduced, with a disproportionate loss of CD4+CD8+ double positive cells. Examination of the triple negative (CD3-CD4-CD8-) subset revealed a striking loss of TN developmental progression of the early precursor cells. Based on the expression of CD44 (pgp-1) and CD25 (IL-2Rα) markers, the TN thymic compartment was composed almost entirely of the earliest population (CD44+, CD25-), with the remaining maturational stages (CD44+, CD25+; CD44-, CD25+; CD44-, CD25-) depleted. In contrast, all T cell developmental stages in the thymus were found to be in normal proportions in the oophorectomized mice, with no differences in the splenic T and B cell subsets. CONCLUSIONS: The study demonstrates that estrogen but not progesterone blocks T cell development in the thymus. However, contrary to our expectation, estrogen deprivation by oophorectomy does not enhance T cell development.     

Comparative analysis and anatomic distribution of ras p21, IL-2R, and MEL-14 in malignant and hyperplastic murine thymus.

The distribution and localization of thymocytes positive for p21 ras, the lymphocyte homing receptor antigen MEL-14, and IL-2 receptors were studied by immunohistology and flow cytometry. Comparisons were made between age-matched normal mice, carcinogen-treated mice at early (stage II) and late (stage III) stages of disease, and cortisone-treated mice. In normal thymus, the majority of cortical and medullary thymocytes are p21 ras positive. MEL-14hi- and IL-2R-positive cells are located in the cortex and comprise less than 5% of the thymus population. Stage II carcinogen-treated animals consistently show increased numbers of MEL-14hi cells in the thymus, with fewer animals having increased numbers of IL-2R positive cells. These populations appear to be different from one another. All stage III animals have MEL-14hi-positive tumor cells, which in 70% of the cases also express IL-2R. Cortisone treatment was used to study non-malignant proliferation. After cortisone treatment there is a marked increase of p21 ras staining in both the cortex and medulla during the first 72-hour interval. Within 24 hours, 50% of the thymocytes are IL-2R positive, but MEL-14hi cells are not detected. By 48 hours, 90% of the thymus population expresses IL-2R and 50% of the cells are MEL-14hi positive, and this results in a substantial population of cells positive for both IL-2R positive:MEL-14hi markers. This population rapidly disappears by 72 hours, leaving 90% of the cells MEL-14hi positive and less than 10% IL-2R positive. The staining of p21 ras at 72 hours is unusual, showing a speckled, cytoplasmic pattern. In light of our findings, we propose that the first step in thymic lymphomagenesis in carcinogen-treated C57BL/6 mice involves the rare cortical MEL-14hi subpopulation and is thymic dependent. A late stage involves expression of IL-2 receptors by a subset of MEL-14hi cells, thus conferring the potential for autonomous growth and malignancy.     

自身免疫调节因子在小鼠胚胎干细胞向胸腺上皮祖细胞分化中的表达

文题释义：自身免疫调节因子：基因分析显示由于单基因突变引起一种自身免疫病,此基因则被命名为自身免疫调节因子即AIRE基因。AIRE基因的突变或者缺失会造成胸腺内自身组织特异性抗原转录缺失,影响阴性选择,从而致使自身反应性T细胞逃逸外周,引起自身免疫反应。AIRE基因一直是免疫学相关研究中的热点。胸腺上皮细胞：胸腺上皮细胞分为髓质胸腺上皮细胞和皮质胸腺上皮细胞,二者均来源于胸腺上皮祖细胞,据研究报道髓质胸腺上皮细胞表达的AIRE基因调控着胸腺内的阴性选择,但是由于胸腺上皮祖细胞和胸腺上皮细胞不易分离且数量少,其应用和研究一直受限。该实验在体外将胚胎干细胞分化为胸腺上皮祖细胞,可以为相关研究提供细胞来源。  摘要背景：自身免疫性疾病主要是由于胸腺内自身组织特异性抗原持续表达缺失而引起强烈免疫应答的一类疾病,而胸腺功能减退和胸腺内组织特异性抗原的不稳定表达会限制治疗效果。胸腺组织主要由胸腺上皮细胞组成,但胸腺内成熟胸腺上皮细胞和胸腺上皮祖细胞有限的数量来源极大限制了相关研究。目的：研究小鼠胚胎干细胞向胸腺上皮祖细胞分化过程中自身免疫调节因子的表达变化。方法：采用两步分化方法定向诱导小鼠胚胎干细胞分化为内胚层再分化为胸腺上皮祖细胞,分别收集定向诱导分化第0,3,13天细胞,采用细胞免疫荧光、流式细胞术、Western blot、Real-Time PCR 检测相关基因及蛋白的表达变化。结果与结论：①诱导分化第0天,免疫荧光检测OCT4、SSEA1表达阳性;诱导分化第3天,免疫荧光检测SOX17、FoxA2呈双阳性表达;诱导分化第13天,流式细胞术检测EpCAM1、K5、K8表达阳性;②Real-time PCR检测小鼠胚胎干细胞定向分化过程中PAX1、PAX9、FOXN1、PLET1基因表达逐渐增高;③Real-time PCR检测分化第0,3,13天AIRE基因表达升高,INS2、GAD67基因表达也升高;④Western blot检测分化第0,3,13天AIRE蛋白表达降低,胰岛素蛋白、GAD67蛋白均无表达;⑤结果表明,小鼠胚胎干细胞成功分化为胸腺上皮祖细胞,且分化而来的胸腺上皮祖细胞中AIRE基因表达很高,促进了INS2、GAD67基因的表达,为细胞移植治疗自身免疫性疾病提供评价依据。ORCID: 0000-0001-5253-3085(胡蓉) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Thymic Stromal-Cell Abnormalities and Dysregulated T-Cell Development in IL-2-Deficient Mice

The role that interleukin-2 (IL-2) plays in T-cell development is not known. To address this issue, we have investigated the nature of the abnormal thymic development and autoimmune disorders that occurs in IL-2-deficient (IL-2^–/–) mice. After 4 to 5 weeks of birth, IL-2^–/– mice progressively develop a thymic disorder resulting in the disruption of thymocyte maturation. This disorder is characterized by a dramatic reduction in cellularity, the selective loss of immature CD4^-8^- (double negative; DN) and CD4⁺8⁺ (double positive; DP) thymocytes and defects in the thymic stromal-cell compartment. Immunohistochemical staining of sections of thymuses from specific pathogen-free and germ-free IL-2^–/– mice of various ages showed a progressive ,loss of cortical epithelial cells, MHC class II-expressing cells, monocytes, and macrophages. Reduced numbers of macrophages were apparent as early as week after birth. Since IL-2^–/– thymocyte progenitor populations could mature normally on transfer into a normal thymus, the thymic defect in IL-2^–/– mice appears to be due to abnormalities among thymic stromal cells. These results underscore the role of IL-2 in maintaining functional microenvironments that are necessary to support thymocyte growth, development, and selection.     

小鼠胸腺雌激素受体α与β亚型的免疫组织化学研究

目的:探讨雌激素受体α与β亚型在不同年龄段小鼠胸腺内的表达。方法:免疫组化分析雌激素受体α与β亚型在3、8及15月龄段小鼠胸腺内的表达水平;双色免疫荧光分析雌激素受体α与Treg细胞标志Foxp3表达的关系。结果:3月龄小鼠的胸腺皮质区及髓质区内有大量的雌激素受体β及α亚型的阳性细胞。然而,8月龄及15月龄的小鼠胸腺则缺乏雌激素受体β亚型表达而雌激素受体α亚型阳性细胞仍然可发现于这些胸腺的皮质区内。使用雌激素受体α/β及Foxp3(调节性T细胞的标志)抗体对标本进行双色标记结果证实雌激素受体α/β阳性的细胞为Foxp3阴性。结论:成年小鼠胸腺缺乏雌激素受体β,但雌激素受体α为阳性,提示雌激素受体α是介导雌激素引发的胸腺萎缩的关键激素受体。同时也证实雌激素受体α/β不参与Foxp3调节性T细胞的发育。     

胸腺及其投射神经元一氧化氮的分布

本实验用 NADPH-d组织化学方法及 n NOS免疫细胞化学方法 ,对大鼠胸腺内一氧化氮 (NO)阳性细胞的分布进行了研究。采用 CB-HRP逆行追踪结合 n NOS免疫细胞化学双重反应技术 ,观察了大鼠胸腺投射神经元 n NOS的分布。结果显示 :(1)在脑干的疑核、面后核内有 CB-HRP与 n NOS双重阳性细胞 ;(2 )在胸腺内有多种 NADPH-d和 n NOS阳性细胞 ,按其形态可分为 :髓质上皮样细胞、胸腺树突样细胞、神经元样细胞、胸腺细胞样细胞及胸腺小体 ;(3 )在胸腺的被膜下、小梁内、皮髓质交界处、小血管的周围有丰富的 n NOS阳性纤维。提示 ,胸腺内 NO的来源不同 ,其在调节胸腺的各种活动中可能发挥的作用也不同     

Increases in CD4+ T lymphocytes occur without increases in thymic size in HIV-infected subjects receiving interleukin-2 therapy

OBJECTIVE: To examine the potential contribution of the thymus to CD4+ T-lymphocyte increases in HIV-infected patients receiving intermittent interleukin-2 (IL-2) therapy. DESIGN: Fifteen HIV-infected patients treated with antiretroviral regimens who were enrolled in a study of intermittent IL-2 therapy and were willing to undergo serial thymic computed tomography (CT) were prospectively studied. METHODS: Thymic CT was performed before and approximately 6 and 12-17 months after intermittent IL-2 therapy was started. Scans were graded in a blinded manner. Changes in lymphocyte subpopulations were determined by flow cytometry. RESULTS: Statistically significant increases in CD4+ T lymphocytes occurred with IL-2 administration, with a preferential increase in naive relative to memory CD4+ T cells. Despite this increase in naive CD4+ T cells, overall there was a modest decrease in thymic volume observed during the study period. No correlation was found between changes in thymic volume indices and total, naive, or memory CD4+ T-lymphocyte counts. CONCLUSIONS: These findings demonstrate that the profound CD4+ T-lymphocyte increases seen with intermittent IL-2 administration are not associated with increases in thymic volume and more likely are due to peripheral expansion rather than increased thymic output.     

Effect of Pregnancy on Thymic T Cell Development

PROBLEM: The thymus gland decreases in size during pregnancy. The significance of this alteration is not known. METHOD: In this report, we examined thymic function by evaluating the development of T lymphocytes in the thymus of pregnant Balb/c mice at 15 and 20 days gestation using multi-color flow cytometry. Comparative analysis was made with non-pregnant mice, postpartum lactating mice, and postpartum non-lactating mice. RESULTS: Progressive reduction of thymic size and cellularity during pregnancy was observed. All of the CD4 and CD8 defined subsets were reduced, with a disproportionate loss of CD4+, CD8+ double positive cells. Examination of the CD4-, CD8- double negative compartment revealed a predominance of TCR α,β+ double negative cells, and a striking loss of precursor cells. The CD3-, CD4-, CD8- triple negative thymic subset was composed almost entirely of the earliest population (CD44+, CD25-), with the remaining maturational stages (CD44+, CD25+; CD44-, CD25+; and CD44-, CD25-) depleted. At 2 weeks postpartum, the subset ratios normalized, and the total cell count showed recovery. CONCLUSION: T cell development is blocked at the precursor level during the mouse pregnancy. These effects are transient, and gradual recovery is observed in the postpartum period.