实验性自身免疫性脑脊髓炎小鼠脾脏和肝脏中NKT细胞调变的研究

目的:观察NKT细胞在实验性自身免疫性脑脊髓炎(EAE)小鼠脾脏和肝脏中所占百分比的变化特点,探讨NKT细胞在EAE模型中的免疫调节作用.方法:以MOG35-5521肽诱导C57BL/6小鼠建立EAE模型并进行临床评分.于发病高峰期处死小鼠,分离脾脏和肝脏淋巴细胞,采用免疫荧光染色和流式细胞术(FCM)分析,观察EAE小鼠与正常小鼠脾脏和肝脏中NKT细胞在全部淋巴细胞中所占百分率的变化.结果:在EAE小鼠不同器官中,NKT细胞占淋巴细胞的百分率均较正常小鼠减少.脾脏NKT细胞百分率(%)从正常组2.22±0.14下降到EAE模型组1.94±0.07(P<0.05),肝脏NKT细胞百分率(%)从正常组5.52±2.17下降到2.67±1.41(P<0.05).结论:NKT细胞在EAE模型C57BL/6小鼠脾脏和肝脏中增殖受抑,提示EAE发病可能通过对NKT细胞数量的调节进而影响其对免疫应答的调节.     

神经肽K物质,P物质对小鼠脾脏和胸腺淋巴细胞的影响

研究了速激肽SK和SP对小鼠脾脏和胸腺淋巴细胞的作用,观察到SK与SP在亚适量的ConA或LPS共同作用下,能促进脾脏细胞的体外增殖和抗体生成。此外,5×10~(-7)M的SK对ConA激活的胸腺细胞有促增殖作用,而SP没有这个效应。FACS分析的结果表明,SK能促使ConA激活的脾脏和胸腺T淋巴细胞亚群发生变化,使脾脏细胞中lyt2~ 细胞所占比例上升,而L_3T_4~ 细胞比例不变;使胸腺细胞lyt 2~ 和L_4~ 比例都下降。以上结果表明神经肽SK、SP对免疫反应有调节作用。     

微小RNA-7敲减对小鼠脾脏中T淋巴细胞体外功能的影响

观察微小RNA-7(microRNA-7,miR-7)敲减(Knock down,KD)对小鼠脾脏T淋巴细胞体外功能的影响并探讨其意义。常规分离野生型(wild type,WT)小鼠脾脏T淋巴细胞,经CD3和CD28抗体刺激后,Real-time PCR检测不同时间点(0h;24h;48h)细胞中miR-7的表达变化;进一步用Con A、CD3和CD28抗体刺激miR-7KD小鼠脾脏T淋巴细胞,CCK8检测细胞增殖率;Real-time PCR检测miR-7KD小鼠脾脏T淋巴细胞IL-12、IL-4、IL-6、TNF-α、IFN-γ和IL-10表达的变化;FACS检测CD4+T和CD8+T细胞的数量变化及CD4+T细胞膜分子CD44、CD62L和IL-4、IFN-γ的表达变化。结果显示,WT小鼠脾脏T淋巴细胞活化后,miR-7的表达水平显著上调(P0.05);与WT小鼠相比,在Con A、CD3和CD28抗体作用下miR-7KD小鼠脾脏T淋巴细胞增殖明显增加(P0.05);miR-7KD小鼠脾脏T淋巴细胞IL-12、IL-4、IL-6、TNF-α和IFN-γ水平均明显上调(P0.05),而IL-10表达显著下调(P0.05);FACS检测结果显示CD4+T细胞比例明显上调(P0.05),而CD8+T细胞的比例变化不显著(P0.05);CD4+T细胞膜分子CD62L水平显著下降,CD69及IL-4、IFN-γ的表达水平均显著上调(P0.05)。结果表明,miR-7敲减以后可显著影响小鼠脾脏T淋巴细胞的功能,本实验为后续深入探讨其在T淋巴细胞功能调控中的作用提供实验依据。     

小鼠脾脏树突状细胞免疫功能的体外研究

本实验通过对小鼠脾脏分离到的树突状细胞免疫功能的研究表明,在ConA,PHA刺激的淋巴细胞增殖反应和混合淋巴细胞反应(MLR)中树突状细胞具有明显强于巨噬细胞的抗原递呈作用(P<0.001或P<0.005)。树突状细胞的这种作用可被特异性的单克隆抗体所抑制(抑制率>95％)。少量的巨噬细胞可促进其作用。     

脾脏巨噬细胞对凋亡细胞诱导免疫反应的影响

为研究小鼠脾脏巨噬细胞(Mφ)对凋亡细胞诱导免疫应答的影响,我们用氯磷酸二钠脂质体(clodronate-liposomes,CL)清除小鼠脾脏Mφ,于不同时间点对三组(nave、PBSL、CL)小鼠选择性回输e450标记的DO11.10CD4~+T细胞、凋亡细胞及其相关抗原,然后用流式细胞仪检测小鼠血液与脾脏中免疫细胞的清除率、DO11.10CD4~+T细胞(即KJ126+T细胞)的增殖及Foxp3+Treg细胞的增殖。结果显示:(1)注射CL 1d后,外周血单核细胞的清除率约93%,脾脏中F4/80+Mφ、单核细胞与树突细胞的清除率分别为99%、90%、54%;(2)PBSL组与CL组中,KJ126+T细胞占CD4+T细胞的比率分别为1.32%、0.56%,差异有统计学意义(P0.0001);Foxp3+T细胞占CD4~+T细胞的比率分别为0.69%、0.89%,差异有统计学意义(P=0.0004)。表明CL可以有效清除血液与脾脏中的免疫细胞;清除脾脏Mφ可以抑制KJ126+T细胞的增殖和促进Foxp3+Treg细胞的增殖。提示脾脏Mφ参与了提呈凋亡细胞相关抗原的过程,参与调节凋亡细胞诱导的抗原特异性T细胞的免疫应答能力及凋亡细胞诱导的免疫耐受。说明脾脏Mφ在凋亡细胞诱导的免疫反应中起着重要的作用。     

JM细胞培养上清对人B淋巴细胞和小鼠脾细胞增殖的免疫调节作用

JM 是来自人胸腺不成熟 T 细胞的急性淋巴细胞白血病细胞系.本文以 SAC 刺激的人外周血纯化 B 淋巴细胞和小鼠脾细胞增殖为模型,观察了 JM 细胞培养上清(SPN_(JM))对人 B 淋巴细胞和小鼠脾细胞增殖的免疫调节作用.发现具有 T 细胞抑制活性的 SPN_(JM)对人和小鼠 B 淋巴细胞的增殖具有促进作用。在无 SAC 诱导时,SPN_(JM)与 HrIL—2协同对 B 细胞仍有促增殖作用.本实验还发现,高浓度时对 T 细胞具有抑制作用的 SPN_(JM),在低浓度时(1:640)对 T 细胞增殖亦具有促进作用.     

超重症联合免疫缺陷小鼠细胞免疫功能特征研究

超重症联合免疫缺陷小鼠(B.C.B-17scid-beige)是将自然杀伤细胞(NK)缺陷基因(bg 基因)导入重症联合免疫缺陷病小鼠(C.B-17scid)体内得到的一种新品系小鼠.为确定该鼠基本细胞免疫特征,我们测定了该鼠 T、NK 和淋巴因子激活的杀伤细胞(LAK)等的功能,以及聚肌胞(poly I-C)对该鼠 NK 细胞活性的影响。该小鼠脾淋巴细胞不能针对 T 细胞有丝分裂原刀豆蛋白(ConA)的刺激而产生增殖反应。同正常对照小鼠相差非常显著(P<0.01)。该鼠 NK 细胞活性(对 YAC-1细胞杀伤活性)明显低于亲代 scid 小鼠和正常小鼠(P<0.01),经聚肌胞(100μg/只)预先刺激后该鼠 NK 细胞活性未见增加.而亲代 scid 小鼠则有明显增加。该鼠脾脏细胞经白细胞介素—2刺激后,LAK 细胞活性(对 P815细胞杀伤活性)明显低于亲代 scid 小鼠和正常小鼠(P<0.01)。上述结果表明 B、C、B-17scid-beige 小鼠是一种 T、NK和 LAK 细胞联合免疫缺陷小鼠,该鼠 B 细胞功能也是缺陷的(详见它文)。上述缺陷是 scid 基因和 bg 基因共同作用的结果,该动物为建立人—免疫缺陷动物模型提供了一种更为理想的受体小鼠,并为基础免疫学研究提供了一种新的研究工具。     

转染FasL基因树突状细胞诱导异基因小鼠脾脏T细胞凋亡的研究

目的 制备稳定表达FasL蛋白的小鼠骨髓源树突状细胞(dendritic cell,DC)并探讨其诱导异基因小鼠脾脏T细胞凋亡的机理.方法 采用培养基选择法体外培养小鼠骨髓源DC,脂质体法转染FasL基因至小鼠成熟DC,实时定量PCR检测转染前后FasL mRNA的表达,流式细胞仪和免疫蛋白印迹检测转染前后FasL蛋白的表达.异系小鼠静脉分别输注未转染DC、转染空质粒DC和转染FasL的DC,7 d后TdT介导的原位末端标记法(TUNEL)和流式细胞仪检测脾脏中T淋巴细胞凋亡.结果 体外培养可获得成熟的小鼠骨髓源DC,转染FasL基因的DC较未转染DC FasL mRNA和FasL蛋白表达明显升高.对脾脏中T淋巴细胞凋亡的检测发现,转染FasL的DC组凋亡指数(11.67±1.53)明显高于未转染DC组(2.67±0.58)和转染空质粒组(3.33±0.58),P＜0.01.结论 培养基选择法可收获大量骨髓源DC,脂质体转染FasL基因至小鼠骨髓源DC,可以使DC高表达FasL蛋白.转染FasL的DC输注能明显诱导异系小鼠脾脏T淋巴细胞凋亡.     

NO介导的巨噬细胞对脾脏T细胞增殖的抑制作用

本文研究一氧化氮(NO)在介导小鼠巨噬细胞抑制脾脏T细胞增殖中的作用。结果显示NO 供体硝普钠(SNP) 呈剂量依赖性抑制脾脏T细胞对ConA增殖反应, 小鼠腹腔巨噬细胞与脾细胞的混合培养也可抑制T细胞增殖, 这种抑制作用与NO产量正相关, NO合酶抑制剂L 硝基 精氨酸(L NNA) 可部分逆转这种抑制作用     

驱虫斑鸠菊对正常小鼠免疫功能的影响

目的探讨驱虫斑鸠菊对小鼠免疫功能的影响,揭示其免疫作用机理.方法利用[3H]-TdR掺入法分别测定了驱虫斑鸠菊对环胞菌素A(Con A)诱导的小鼠脾脏T淋巴细胞和细菌脂多糖(LPS)诱导的B淋巴细胞增殖活性以及脾脏T淋巴细胞分泌白细胞介素-2(IL-2)活性的影响,运用酶联免疫吸附试验测定了驱虫斑鸠菊对小鼠血清中抗体活性的影响;采用流式细胞仪测定脾脏B淋巴细胞表面抗原CD19的表达水平.结果驱虫斑鸠菊的低、中、高3个剂量体内对脾脏T、B淋巴细胞的增殖活性、血清总抗体和针对人A375黑素瘤细胞抗原特异性抗体含量、B细胞表面抗原CD19的表达以及对脾脏T淋巴细胞分泌IL-2活性都具有明显抑制作用.结论驱虫斑鸠菊对机体体液免疫和细胞免疫功能都具有明显抑制作用.     

Heat-labile B-cell mitogen obtained from Listeria monocytogenes.

A water-soluble extract of Listeria monocytogenes strain 10403 acts as a mitogen on cultured mouse spleen lymphocytes. This mitogen induced a response six to nine times that of controls, as measured by [3H]thymidine incorporation. The mitogen extract was derived from washed bacterial cells which were mechanically disrupted with a French press. The extract was centrifuged at 105,000 X g and filtered through a 0.22-micrometer filter. Similar levels of lymphocyte stimulation were observed in lymphocyte cultures prepared from spleens of nude mice, indicating the effect of this mitogen on B-cells. The mitogenic property of this extract was destroyed by heating to 56 degrees C. This heat treatment does not destroy the antigens in the extract, which stimulate spleen cell cultures obtained from specifically immune mice. Similarly prepared extracts from Staphylococcus epidermidis and Salmonella typhimurium did not show similar levels of mitogenic activity. The mitogenic property of the L. monocytogenes extract was present in two strains of Listeria tested and was not associated with mouse virulence.     

Effect of the mannose-binding artocarpus integer lectin on the cellular proliferation of murine lymphocytes

The effect of the mannose-binding champedak (Artocarpus integer) lectin-M on the cellular proliferation of murine lymphocytes was investigated in this study. Our data demonstrated that the lectin was the main mitogenic component in the crude extract of the champedak seeds. It stimulated the proliferation of murine T cells at an optimal concentration of 2.5 μg/ml in a 3 day culture. Lectin-M appeared to be a T-cell mitogen as it does not induce significant DNA synthesis when cultured with spleen cells from the nude mouse. In the absence of T cells, the lectin was incapable of inducing resting B cells to differentiate into immunoglobulin secreting plasma cells.     

Mitogenic activity of staphylococcal peptidoglycan.

Staphylococcus aureus peptidoglycan displayed a marked dose-dependent mitogenic activity for mouse splenocytes and human peripheral blood lymphocytes in vitro, as measured by increased [3H]thymidine incorporation. Similarly it was mitogenic for athymic nude mouse spleen cells, whereas no blastogenic effect was observed in T cell-enriched and B cell-depleted mouse lymphocyte cultures. These data demonstrate that peptidoglycan-responding cells in mouse spleen cell cultures are B lymphocytes.     

连翘提取物对小鼠T淋巴细胞体外活化与增殖的影响

目的:分析连翘(FS)提取物对小鼠淋巴结T细胞的体外活化与增殖的影响,初步探讨其免疫抑制作用机制.方法:无菌分离小鼠淋巴结细胞,加入多克隆刺激剂刀豆蛋白A(ConA)进行刺激,利用荧光标记的单克隆抗体(mAb)染色结合流式细胞术(FCM),检测小鼠T淋巴细胞的表达的活化抗原CD69、CD25、CD71的表达情况;以羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFDA-SE)染色,以FCM分析FS对淋巴细胞体外增殖的影响.结果:终浓度为40、80、160 mg/L的FS均对ConA刺激诱导的T细胞CD69、CD25和CD71的表达有降低作用(P＜0.05).CFDA-SE染色分析显示,上述浓度的FS对ConA诱导的小鼠T淋巴细胞增殖具有抑制作用(P＜0.05).结论:FS对ConA诱导的T细胞早、中、后期活化和体外增殖有抑制作用.     

胚芽乳杆菌胞壁肽聚糖对细胞免疫的作用

用体外刺激、~8H-TdR掺入测定淋巴细胞增殖的方法观察到胚芽乳杆菌的胞壁肽聚糖(PG)可以直接诱导小鼠脾淋巴细胞的增殖反应,在所采用剂量范围内细胞增殖的倍数与PG剂量呈线性关系;但对胸腺细胞和分纯的脾T淋巴细胞则无直接作用。还观察到PG有协同ConA诱导小鼠脾细胞和胸腺细胞增殖作用。可见PG具有B细胞的丝裂原性,并有增强T细胞丝裂原的作用。     

Activation of B lymphocytes by mycoplasma mitogen(s).

Various strains of the murine mycoplasma M. neurolyticum have been shown to induce extensive blast transformation of mouse lymphocytes, comparable in strength to mitogenicity exerted by these mycoplasma species on rat lymphocytes. The data summarized in this report demonstrate that this mitogenic effect is non-specific. Lymphoid cells from mycoplasma free, germ-free mice were activated to the same extent as those lymphocytes obtained from conventionally bred animals. Lymph node cell suspensions obtained from athymic nude mice were strongly activated by M. neurolyticum mitogen. Furthermore, mouse thymocytes and mouse T-cell enriched populations, were not stimulated by these mitogens. It was thus suggested that M. neurolyticum activates mouse B lymphocytes.     

Effect of liposomes on lymphocytes: induction of proliferation of B lymphocytes and potentiation of the cytotoxic response of T lymphocytes to alloantigens

Liposomes of certain lipid composition prepared by the detergent removal method (Brunner, J. et al., Biochim. Biophys. Acta 1976. 455: 322) induced the proliferation of spleen cells from different mouse strains. Spleen cell populations enriched in B lymphocytes and those obtained from nude mice were induced to proliferate, whereas spleen cell fractions enriched in T lymphocytes and thymocytes were not. The mitogenic effect of liposomes resembled that of lipopolysaccharide (LPS) and it depended upon their lipid composition. Liposomes prepared from dimyristoyl lecithin (DML), 2:1 dimyristoyl lecithin:cholesterol (DML:C), 2:1 dioleoyl lecithin: cholesterol (DOL:C), and 2:1 egg yolk lecithin:cholesterol (EYL:C) were mitogenic, whereas liposomes prepared from egg yolk lecithin (EYL) alone were not mitogenic for spleen cells. The mitogenic effects of these liposome preparations were in the decreasing order DML greater than DOL:C greater than or equal to EYL:C greater than DML:C greater than EYL. The results suggest a correlation between the membrane fluidity of liposomes and their mitogenic effect. Although no proliferative response was induced on T lymphocytes, two of these liposomes, DML and EYL:C, had the ability to potentiate the cytotoxic response of T lymphocytes to alloantigens in mixed leukocyte culture. In responder-stimulator combinations which differed for the H-2K, H-2D or the entire H-2 region, these liposomes potentiated the cytotoxic response significantly. The results suggest that liposomes have an ability to modulate T lymphocyte response.     

Alpha 1-microglobulin is mitogenic to human peripheral blood lymphocytes. Regulation by both enhancing and suppressive serum factors.

Human alpha 1-microglobulin (alpha 1-m), a 26 kilodalton serum glycoprotein, was found to exert mitogenic effects on human peripheral blood lymphocytes (PBL) in serum-free medium. Purified T cells, but not B cells, responded with proliferation to alpha 1-m, but only in the presence of monocytes. The mitogenic activity could be partially neutralized by a mouse monoclonal antibody against alpha 1-m. The mitogenicity was species-specific, since alpha 1-m homologues from rats, guinea pigs and rabbits had no effect on human PBL. In a previous study, no effect of alpha 1-m was seen on PBL in the presence of 20% serum, and, therefore, we studied the influence of different concentrations of serum on the alpha 1-m-induced mitogenicity. Thus, human serum enhanced the mitogenic effects of alpha 1-m on human PBL at 1% concentration (v/v) and suppressed the effects at 10%. The suppressing effect of serum at 10%, but not the enhancing effect at 1%, seemed to be conserved among several species. To test the effect of serum proteins of different molecular sizes, human autologous serum was separated by gel chromatography on Sephadex G-200 into four fractions. Fractions 1 and 2 (roughly containing proteins larger than 100 kilodaltons) suppressed the mitogenic effects of alpha 1-m, while fractions 3 and 4 enhanced the stimulation by alpha 1-m, at 0.5% and concentrations above. It is concluded that the mitogenic effect of alpha 1-m on lymphocytes is regulated by several serum factors, both enhancing and suppressive, that does not have any proliferative effect of their own. It can be speculated that the balance between enhancing and suppressing co-factors in the blood determines the degree of the stimulation of lymphocytes by alpha 1-m. This is compatible with an immunomodulatory role for alpha 1-m, in spite of its relatively constant plasma levels in health and disease.     

Analysis of Mature Guinea Pig T Cells with a MOnoclonal Antibody directed against a Framework Determinant of the T-Cell Receptor for Antigen

Rats were immunized with guinea pig T lymphocytes and the spleen cells were fused with cells of a mouse myeloma line. The resulting hybrids were screened for the production of antibodies selectively reacting with guinea pig T cells. The monoclonal antibody (MoAb) H159 was analysed in detail because it bound to T lymphocytes, but not to B lymphocytes or macrophages. Cellular ELISA, cytofluorometry and immunohistology revealed that the antigen detected by H159 is selectively expressed on the majority of peripheral mature T lymphocytes (about 95%). In contrast, it stained only a minor population of thymocytes in FACS analysis. H159 precipitated from NP40 lysate of T cells a protein with a molecular weight of about 90 kDa when separated under non-reducing conditions in SDS-PAGE. Under reducing conditions bands with molecular weights of about 50 kDa were found. After binding to anti-rat Ig coated beads, the MoAb H159 had a mitogenic effect for guinea pig T lymphocytes whereas soluble MoAb H159 in the presence or absence of macrophages was not mitogenic. The cellular expression and molecular characteristics of the H159 antigen together with the mitogenic activity of the antibody for T cells indicate that the MoAb H159 recognizes the guinea pig T-cell receptor for antigen via a constant region determinant.     

Mitogenic activity in human embryonic fibroblasts early after infection by human cytomegalovirus.

We have characterized the nonspecific lymphocyte stimulation by extracts of human cytomegalovirus-infected human embryonic fibroblasts. Cell extracts prepared at 5 h postinfection (early extract) and 72 h postinfection (late extract) were both highly mitogenic in lymphocyte preparations from adult blood, cord blood, and rabbit blood. Maximum stimulation of the lymphocytes was observed on day 3 after the addition of early or late extract under optimal conditions. Early extract stimulated both the E-rosetting and the EAC-rosetting subpopulations of human lymphocytes. The mitogenic activity appeared before 5 h postinfection and was fairly stable at 30 degrees C for 5 h.