融合IL2的慢病毒实现对T细胞的高效基因转导

目的 制备融合IL2的慢病毒以提高对人原代T淋巴细胞的转导效率.方法 将白细胞介素-2(IL2)基因定向克隆至水疱性口炎病毒G蛋白(VSV-G)编码基因5′端,构建新型慢病毒包装辅助质粒pMD2.IL2-G.使用不同剂量pMD2.IL2-G进行慢病毒包装,应用免疫印迹和ELISA比较病毒滴度,流式细胞术检测病毒对T细胞...     

rhTPO对巨核系的定向分化效应

目的 :探讨重组人促血小板生成素 (rhTPO)对外周造血干细胞中巨核系祖细胞的定向诱导分化能力。方法 :经化疗及G CSF动员后分离的外周造血干细胞 ,进行液体和集落培养 ,研究rhTPO单独及与IL 3、IL 6、SCF的协同作用。结果 :液体培养后 ,TPO组单个核细胞数 (MNC)扩增了 ( 1.73± 0 .49)倍 ,IL 3 IL 6 SCF组MNC比种植时增加 ( 4.2 0± 1.14)倍 ,IL 3 IL 6 SCF TPO组MNC增加了 ( 4.5 3± 1.2 7)倍。单独应用TPO及TPO与IL 3、IL 6、SCF合用产生高比例的CD41a 细胞 ,TPO组培养前CD41a 细胞为 11.70 %± 5 .2 3% ,培养后CD41a 细胞为 19.17%± 6 .2 6 % ( P <0 .0 5 )。CD41a 细胞数在TPO组扩增了 ( 3.5 2± 1.18)倍 ,IL 3 IL 6 SCF组扩增了 ( 5 .32± 1.79)倍 ,IL 3 IL 6 SCF TPO组扩增了 ( 6 .94± 2 .19)倍。结论 :TPO可以定向诱导巨核系细胞的分化 ,IL 3、IL 6、SCF可以协同TPO的作用 ,这在造血调控研究 ,体外定向扩增外周血干细胞 ,促进外周造血干细胞移植患者血小板的恢复具有应用前景     

儿童肾脏病患者血清白介素-2、白介素-6和胰岛素样生长因子-Ⅱ检测及其临床意义

为观察儿童肾脏病患者血清白介素 - 2 (IL - 2 )、白介素 - 6 (IL - 6 )、胰岛素样生长因子 -Ⅱ (IGF -Ⅱ )的含量 ,探讨细胞因子与肾脏病的关系 ,采用RIA法检测了不同肾脏病儿童患者血清IL - 2、IL - 6、IGF -Ⅱ的水平。结果显示 ,正常儿童血清IL - 2含量为 1 .6 0± 0 .32 μg/L、IL - 6为 37.0± 6 .0ng/L、IGF -Ⅱ为 0 .30±0 .1 5μg/L。正常男女性儿童之间血清三个细胞因子含量无显著性差异 (P >0 .0 5 )。正常儿童与成年人之间三个细胞因子有显著性差异 (P <0 .0 1 )。慢性肾小球肾炎血清IL - 2含量为 1 .96± 0 .4 4 μg/L、IL - 6为 5 4 .9± 2 0 .9ng/L、IGF -Ⅱ为 0 .5 2± 0 .1 5 μg/L ;急性肾炎血清IL - 2含量为 1 .92± 0 .4 7μg/L、IL - 6为 76 .3± 36 .2ng/L、IGF -Ⅱ为 0 .6 2± 0 .2 2 μg/L ;紫癜性肾炎血清IL - 2含量为 1 .91± 0 .33μg/L、IL - 6为 5 5 .5± 1 5 .1ng/L、IGF -Ⅱ为 0 .5 0± 0 .1 9μg/L ;肾病血清IL - 2含量为 1 .96± 0 .6 5 μg/L、IL - 6为 5 6 .2± 2 8.4ng/L、IGF -Ⅱ为 0 .5 9±0 .2 8μg/L ;不同肾脏病儿童三个细胞因子与正常人相比有显著差异 (P <0 .0 1 )。提示 :不同肾脏病儿童血清IL- 2、IL - 6、IGF -Ⅱ含量升高表明 ,这三个细胞因子均积极参与     

人白细胞介素(IL)-28和IL-29基因的克隆与序列分析

人IL 2 8A、IL 2 8B和IL 2 9是由外周血单个核细胞(PMBC)和树突状细胞 (DC)等产生的细胞因子 ,组成一个新的细胞因子家族[1,2 ] 。其基因结构与IL 10相近似 ,但氨基酸水平与干扰素 (IFN)更接近 ,故Kotenko等[2 ] 也将它们相应地称为IFN λ1(IL 2 9)、IFN λ2 (IL 2 8A)和IFN λ     

SARS患者血清细胞因子临床意义探讨

目的　探讨严重急性呼吸综合征 (SARS)患者病情进展的宿主免疫机制。方法　使用流式细胞仪检测急性SARS感染患者 5 0例和正常对照 2 0例的细胞因子 (IL 2、IL 4、IL 6、IL 10、IFN γ、TNF α)。按照疾病预后分成死亡组和治愈组 ,前瞻性设计分析细胞因子在 2组患者中的异同及对病情发展的影响。结果　治愈组与死亡组患者的细胞因子 (IL 2、IL 4、IL 6、IL 10、IFN γ、TNF α)均明显高于正常对照组 ,差异有显著性 (P <0 .0 5 )。治愈组和死亡组比较 :治愈组IFN γ高于死亡组 ,差异有显著性 (P <0 .0 5 ) ,死亡组IL 6、IL 10明显高于治愈组 ,差异有显著性 (P <0 .0 5 )。结论　SARS病毒感染确实引起机体的免疫系统紊乱。功能受损的程度与疾病的预后有一定相关性 ;TH2类细胞因子免疫反应过强 ,TH1类细胞因子免疫反应相对减弱 ,造成的机体免疫自稳平衡失调可能是SARS患者死亡的因素之一     

轮状病毒肠炎患儿血清和大便IL-2,IL-6,IFN-γ动态变化及与临床指标关系的研究

目的　研究轮状病毒肠炎患儿血清和大便IL 2、IL 6、IFN γ的动态变化 ,及临床指标的关系。方法　采用双抗体夹心ELISA法。结果　 119例腹泻患儿RV阳性率 6 3.0 % ,当年 11月份为高峰月。这些细胞因子在病程中呈不同的高峰时相 ,大便IFN γ达峰值最早 ,血清及大便IL 2达峰值最晚 ,大便IL 2与IL 6差异有非常显著性 (P <0 .0 1) ,大便IL 2与IFN γ、IL 6与IFN γ、血清IL 2与IL 6、腹泻恢复时间与大便IL 2、血、便IL 2及IFN γ与淋巴细胞 +单核细胞均差异有非常显著性或差异有显著性 (P <0 .0 1或P <0 .0 5 )。结论　IL 2、IL 6、IFN γ在婴幼儿轮状病毒肠炎中发挥了重要的免疫调控作用 ,且与临床指标密切相关。     

螺旋藻对实验性变应性鼻炎大鼠血清TH1/TH2细胞因子表达的影响

目的　观察螺旋藻对实验性变应性鼻炎大鼠血清TH1 TH2细胞因子表达的影响 ,以探讨其治疗机制。方法　用卵清蛋白致敏大鼠制成变应性鼻炎动物模型 ,口服螺旋藻治疗 ,观察其行为学差异 ;组织病理学方法观察鼻黏膜改变 ;用酶联免疫吸附法 (ELISA)对血清中TH1细胞因子IFN γ、IL 2和TH2细胞因子IL 4、IL 5进行检测。结果　行为学得分 ,阳性对照组 (7.8± 1.0 3)明显高于实验组 (1.6± 1.35 )和正常对照组 (0 ) ,差异有显著性 (P <0 .0 1) ;实验组鼻黏膜炎性反应明显轻于阳性对照组 ;实验组IFN γ、IL 2水平明显高于阳性对照组 ;差异有显著性 (P <0 .0 1) ;实验组IL 4、IL 5表达水平明显低于阳性对照组 ;差异有极显著性意义 (P <0 .0 1)。结论　螺旋藻通过调节TH1和TH2细胞因子的表达 ,纠正失衡的TH1 TH2的细胞因子网络而对变应性鼻炎产生治疗作用。     

卡介菌多糖核酸提高复发性生殖器疱疹患者CD8+ T细胞的细胞因子表达

目的　研究卡介菌多糖核酸 (BCG PSN)对复发性生殖器疱疹 (RGH)患者外周血CD8 T细胞IL 12、IFN γ、IL 4表达的影响。方法　应用流式细胞仪对 4 5例RGH患者和 15名健康志愿者外周血CD8 T细胞IL 12、IFN γ和IL 4的表达进行检测。结果　治疗前RGH患者外周血IFN γ和IL 12阳性CD8 T细胞百分率均显著下降 (P <0 .0 5 ) ,Tc1 Tc2平衡失调 (P <0 .0 5 )。治疗后BCG PSN组外周血IL 12和IFN γ阳性CD8 T细胞百分率显著升高 (P <0 .0 5 ) ,Tc1 Tc2恢复平衡。病例对照组IL 12、IFN γ及IL 4阳性CD8 T细胞百分率于治疗前后变化差异均无显著性 (P >0 .0 5 )。BCG PSN组与病例对照组治疗前后IL 12、IFN γ阳性CD8 T细胞百分率间差异均有显著性 (P <0 .0 5 )。BCG PSN组复发率较低 ,复发病情较轻。结论　RGH患者存在以Tc1水平低下为主的Tc1 Tc2比例失衡和IL 12表达水平低下 ;BCG PSN通过上调外周血CD8 T细胞IFN γ、IL 12的表达 ,纠正机体细胞因子失衡状态而减少疾病的复发     

白细胞介素-2的基因、分子和作用

白细胞介素-2(IL2)主要由辅助T细胞分泌产生,除具有促进T细胞增殖分化、维持T细胞体外长期培养的功能外,尚能增强Tc、Ts、NK和K细胞的活性,诱导γ-干扰素和B细胞生长因子(BCGF)的生成,刺激B细胞产生抗体.因此IL2被认为是一种重要的免疫增强因子,开始用于免疫缺损和肿瘤的实验治疗.为了更深入地了解IL2,本文拟对国外近年开展的有关IL2的基因结构、IL2的分子结构以及IL2对B细胞的作用等工作,作一简要综述.     

白细胞介素-1受体拮抗剂基因多态性与变态性鼻炎相关性探讨

目的　探讨白细胞介素 - 1受体拮抗剂 (IL -lra)基因多态性与变态性鼻炎相关性 .方法　利用聚合酶链式反应技术对 6 9例变态性鼻炎患者及 15 6例正常对照者的IL -lra基因进行扩增 .结果　变态性鼻炎患者IL - 1ra基因频率 :IL1RN1/ 10 .8116 ,IL1RN1/ 2 0 .1739,IL1RN1/ 30 .0 14 5 ,IL - 1ra等位基因频率 :,IL1RN 10 .90 5 8,IL1RN 20 .0 86 9,IL1RN 30 .0 0 72 ,与对照组无差异 (p >0 .0 5 ) .结论　IL - 1ra基因多态性的分布与变态性鼻炎无明显相关性     

重组人IL-15促人脐血CD34+造血干细胞定向分化为NK细胞

目的 探讨IL－15促CD34^ 造血干细胞定向分化为NK细胞的作用。方法 采用MACS法分离脐血中CD34^ 细胞，分别用IL－15、SCF（造血干细胞因子）和IL－15 SCF体外培养，流式细胞仪测定CD3、CD16、CD56分子，MTT法检测NK细胞杀伤活性。结果 IL－15可以促进CD34^ 细胞分化为以CD56^ CD16^-为主的NK细胞，SCF具有较强的协同作用。结论 IL－15具有促使NK细胞定向分化的作用。     

NK cells differentiated from bone marrow,cord blood and peripheral blood stem cells exhibit similar phenotype and functions

In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34⁺ stem cells using a potent culture system. Elutriated CD34⁺ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56⁺ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56⁺ CD7⁻ and a small subset expressed CD16. These in vitro differentiated CD56⁺ NK cells displayed cytolytic activity against the HLA class I ⁻ target K562. The CD56⁺ CD16⁺ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56⁺ cells expressed high amounts of transforming growth factor-β and granulocyte-macrophage colony-stimulating factor, but no IFN-γ. Investigation of NK receptor expression showed that most CD56⁺ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.     

IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.     

Importance of stromal determinants in the generation of dendritic and natural killer cells in the human spleen

Summary The interaction between stroma and blood cells in the human spleen has received little attention, despite their well-defined roles during blood cell development in bone marrow. We have reported previously that human spleen-derived fibroblasts display a differentiated myofibroblast phenotype and constitutively express a biologically active form of membrane interleukin (IL)-15 that can drive co-cultured CD34(+) blood cells to differentiate into activated natural killer (NK) cells. Here, we show that, in addition to NK cells, CD34/fibroblast co-cultures also yield myeloid CD1a(+)CD38(+)CD68(+)CD86(+) HLA-DR(+)CD14(-)CD80(-) dendritic cells (DCs) after 3-4 weeks in culture. We found that DC development depended on endogenously secreted stromal macrophage colony-stimulating factor (M-CSF) and CD40/CD40L interaction rather than on fibroblast- and CD34-derived membrane IL-15. CD1a(+) cells were necessary for co-produced NK cells to acquire lytic functions by a mechanism involving cell-to-cell contact and DC-derived IL-12. This study highlights the importance of spleen myofibroblasts in the in vitro generation of two distinct cell types (DC and NK cells) from the innate immune system and suggests that the human spleen is involved in the generation of NK cells from circulating progenitors.     

Umbilical Cord Mesenchymal Stem Cells Increase Expansion of Cord Blood Natural Killer Cells

Natural killer (NK) cell-mediated cytotoxicity can control leukemia relapse while protecting patients from graft-versus-host disease (GVHD) after allogeneic stem cell transplant. Cord blood (CB) is rich in NK cell progenitors with similar properties of proliferation and cytotoxicity as adult blood NK cells. Hence, it is attractive to expand and potentially utilize these cells for adoptive immunotherapy. In this study, CB mononuclear cells were CD3-depleted by immunomagnetic microbead selection to remove T cells. This CD3^dep CB-MNC fraction was then plated for ex vivo expansion, with or without a feeder layer of irradiated umbilical cord mesenchymal stem cells (UC-MSC), with or without cytokines that have been shown to be critical for NK expansion: IL-2, IL-15, IL-3, and FLT-3L. At an average of 2 weeks of culture, there was significantly higher expansion (64.7 ± 8.4-fold) of CD56⁺/CD3⁻ NK cells in the presence of the UC-MSC feeder layer and cytokines compared to controls (no increase with feeder layer only and 6.4 ± 1.5-fold increase with cytokines only, P < .05). Contact between CD3^dep CB-MNC cells and UC-MSC augmented NK expansion. The combination of all 4 cytokines was superior to IL-2 alone or 2 cytokines combinations: mean 64.7 ± 8.4-fold expansion with 4 cytokines combination versus IL-2 alone, IL-2 + FLT-3L, IL-2 + IL-15 or IL-2 + IL-3 (12.2 ± 2.0, 14.4 ± 2.4, 10.4 ± 4.1, 25.2 ± 8.1 respectively). We also observed that only fresh CD3^dep CB-MNC preparations could be expanded reliably, whereas frozen and thawed CD3^dep CB-MNC cells did not expand consistently (mean fold increase 6.5 ± 3.2). Cytotoxicity of expanded NK cells was compared with NK cells from fresh and overnight IL-2 activated CD3^dep CB-MNC. Whereas fresh cells displayed no discernible killing, strong cytotoxicity against K562, Raji, REH, and SUP-B15 cells lines was noted after overnight activation in IL-2. Cytotoxicity of expanded NK cells against Raji, REH, and SUP-B15 was lower, which, however, correlated with a predominant expansion of CD56⁺/CD16⁻ cells known to have less cytolytic activity than CD56⁺/CD16⁺. To test the transfection efficiency in NK cells, fresh or expanded CD3^dep CB-MNC cells were electroporated with either DNA or mRNA constructs for GFP. DNA had a low transfection efficiency (<10%), whereas the one for mRNA reached 52%, but at the cost of significant cell death. Our results suggest that CB NK cell progenitors can be expanded to obtain large numbers by using an irradiated feeder of UC-MSC. They maintain an elevated cytotoxic profile, and may be genetically manipulated—all characteristics that make them suitable for cellular therapies.     

转染IL-21的CD34~+脐血造血干细胞抗裸鼠卵巢癌作用研究

目的 探讨IL-21转染的脐血造血干细胞(CD34~+UBSC·IL-21)对荷卵巢癌裸鼠的治疗作用.方法 从脐血分离CD34~+造血干细胞,体外培养扩增后用于重组体pIRES2-IL-21-EGFP转染.以肿瘤大小、荷瘤鼠生存期判断CD34~+UBSC-IL-21对荷瘤裸鼠的治疗效应.以RT-PCR、免疫荧光、ELISA、Western blot、脾细胞增殖试验及免疫组化法分别鉴定CD34~+UBSC和肿瘤组织中IL-21的表达及活性.裸鼠脾细胞中NK细胞含量及脾细胞的杀伤效应、血清中IFN-γ和TNF-α水平分别用FCM与ELISA检测.结果 pIRES2-IL-21-EGFP成功转染CD34~+UBSC.CD34~+UBSC-IL-21能抑制肿瘤生长,延长荷瘤裸鼠生存期,治疗鼠肿瘤局部能表达IL-21、血清IFN-γ和TNF-α水平升高,NK细胞含量及NK细胞杀伤活性明显增强,与其他组相比,差异有统计学意义(P<0.01).结论 转染IL-21的CD34~+UBSC有良好的抗裸鼠卵巢癌作用,该结果为临床使用UBSC为载体的基因治疗卵巢癌研究奠定了基础.     

细胞因子短期扩增对造血干/祖细胞黏附和迁移能力的影响

目的：探讨干细胞因子（SCF）+白细胞介素-6（IL-6）短期扩增对CD34+造血干/祖细胞黏附和迁移能力的影响。方法:用密度剃度离心的方法分离脐血CD34+细胞，经SCF和IL-6孵育48 h，用CCK-8方法检测CD34+细胞增殖能力；用流式细胞仪检测处理前后的CD49d（VLA-4）、CD11a（LFA-1）、CD62L（L-selectin）及CD184（CXCR4）的表达。用纤连蛋白（FN）包被96孔板，检测经或未经因子扩增的CD34+细胞的黏附能力。扩增的CD34+细胞悬浮于transwell培养板的上层，下层添加基质细胞衍生因子（SDF-1），流式细胞仪检测迁移细胞数，计算迁移率。结果:经SCF+IL-6处理48h后CD34+细胞扩增近3倍；表达CD49d、CD11a、CD62L及CD184的CD34+细胞的百分数分别由原来的26.34%±5.37%、17.63%±4.57%、46.38%±6.61%和9.58%±1.56%增加到65.67%±8.72%、56.67%±6.34%、84.76%±9.57%和19.32%±3.64%（P<0.01）。扩增后的CD34+细胞对FN的黏附能力及在SDF-1诱导下的迁移作用都显著增强（P<0.01）。结论:SCF+IL-6短期扩增CD34+ 造血干/祖细胞显著增加细胞的黏附能力，增加SDF-1诱导的迁移作用，可能是SCF+IL-6促进归巢的主要机制之一。     

Impaired NK cell differentiation of blood-derived CD34+ progenitors from patients with myeloid metaplasia with myelofibrosis

Cultured blood CD34(+) progenitors from patients with myeloid metaplasia with myelofibrosis (MMM) failed to differentiate into natural killer (NK) cells with recombinant interleukin (IL)-15. No NK cells either could be induced in coculture with IL-15-expressing fibroblasts from MMM patients' spleens. The impaired NK differentiation could be circumvented by using normal blood CD34(+) cells in the coculture. In this case, cell-to-cell contact and IL-15 interaction were crucial for NK cell differentiation. Pretreatment of normal CD34(+) progenitors with anti-IL-15 monoclonal antibody markedly reduced NK cell production while MMM fibroblast pretreatment did not. Both normal and MMM progenitors constitutively expressed IL-15. Analysis of endogenous IL-15 signaling pathway revealed a constitutive gammac/Jak3 association and STAT3 activation in the two types of progenitors. Anti-IL-15 monoclonal antibody treatment caused a downregulation of IL-15 signaling in normal but not MMM blood cells. The impaired NK differentiation in MMM may thus arise from a deregulated control of an endogenous IL-15 involved in hematopoietic progenitors' lymphoid differentiation.     

Phenotypic and Functional Heterogeneity of Natural Killer Cells from Umbilical Cord Blood Mononuclear Cells

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-γ and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the naïve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56^hiCD16^? NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-γ expression by CD56^hiCD16^? and CD56^loCD16^? subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.