免疫抑制药对体外骨髓前体细胞向巨噬细胞分化的影响

目的 探讨细胞培养环境中添加免疫抑制药对骨髓前体细胞向巨噬细胞分化的影响. 方法 取C57BL/6小鼠颈椎脱臼致死,无菌操作制备骨髓前体细胞,在体外细胞培养体系中加入巨噬细胞集落刺激因子和三种免疫抑制药(雷帕菌素、环孢菌素A和紫杉醇),通过显微镜和流式细胞仪观察与检测诱导分化细胞的形态和细胞表面分子表达. 结果 骨髓前体细胞诱导分化的细胞具有典型巨噬细胞形态,细胞表面分子F4/80和CD11b表达都在95%以上,而CD11c表达极低,与对照组比较,诱导分化的巨噬细胞数明显减少,环孢菌素A对分化的巨噬细胞周期无影响,但紫杉醇明显增加诱导分化的巨噬细胞凋亡. 结论 3种免疫抑制药可能影响骨髓前体细胞向巨噬细胞分化.     

白介素25及其相关因子在肝纤维化中的作用

白介素25(IL-25)是白介素17家族成员之一,在慢性炎性反应和肿瘤中具有重要作用。IL-25能够活化肝星状细胞并且可以诱导下游促纤维化细胞因子IL-4、IL-13的表达;IL-25可以促进M2型巨噬细胞极化并且可能会招募LY-6Clo单核细胞来源的巨噬细胞分化,而M2型巨噬细胞和LY-6Clo单核细胞来源的巨噬细胞都是抗肝纤维化细胞。IL-25及其相关因子与肝纤维化的关系还不甚清楚。     

结直肠癌细胞来源的外泌体miR-34a对M2型巨噬细胞极化的影响北大核心CSCD

目的:探究结直肠癌来源的外泌体对巨噬细胞极化的影响及其作用机制。方法:建立THP-1来源的M2型巨噬细胞体外诱导分化模型,利用流式细胞染色实验检测细胞分化效率;通过与结直肠癌细胞共培养、结直肠癌细胞外泌体干预M2型巨噬细胞分化,利用实时荧光定量PCR实验检测ARG1、IL-10和NLRP3 mRNA的表达水平。结果:体外诱导分化的M2型THP-1巨噬细胞CD163阳性细胞比例显著升高;与对照组相比,与结直肠癌细胞HCT8和HCT116共培养的M2型巨噬细胞ARG1、IL-10表达水平显著升高,NLRP3表达水平下降。与转染阴性对照microRNA(miR-NC)组相比,转染miR-34a inhibitor的HCT8和HCT116共培养或外泌体刺激下,M2型巨噬细胞ARG1、IL-10表达水平下降,NLRP3表达水平升高;与miR-NC组相比,转染miR-34a inhibitor的THP-1细胞ARG1、IL-10表达水平显著下降,NLRP3表达水平显著升高。结论:结直肠癌细胞来源的外泌体miR-34a可以抑制巨噬细胞NLRP3的表达,促进巨噬细胞M2型极化。     

IL-22 对骨髓来源巨噬细胞相关炎症因子表达的影响

目的:探讨IL-22对骨髓来源巨噬细胞相关炎症因子表达的影响。方法:从小鼠的股骨分离骨髓细胞,经过分化培养获得骨髓来源巨噬细胞,再用脂多糖(LPS)刺激诱导为M1型巨噬细胞。IL-22干预M1型巨噬细胞后,采用流式细胞术检测巨噬细胞标志物;用ELISA分别检测IL-1β和TNF-α的分泌水平;用RT-PCR分别检测炎症因子i NOS、IL-1β和TNF-α的mRNA表达水平。结果:经过分化培养,骨髓来源巨噬细胞的纯率为(99. 3±0. 4)%,IL-22干预M1型巨噬细胞后,与LPS组相比,LPS+IL-22组的M1型巨噬细胞比例下降(P0. 05),细胞炎症因子i NOS、IL-1β和TNF-αmRNA表达下降(P0. 05),IL-1β和TNF-α分泌水平降低(P0. 05)。结论:IL-22可抑制LPS诱导的骨髓来源巨噬细胞炎症因子的表达,减轻其炎性反应。     

低氧对巨噬细胞外泌体的表达及其对骨肉瘤细胞的化疗抗性影响的体外研究

目的在体外探究低氧对巨噬细胞外泌体分泌的影响及对骨肉瘤细胞顺铂耐药性的改变。方法Transwell侵袭实验检测不同氧浓度(1%O2的低氧与常氧)条件下骨肉瘤细胞MG63对巨噬细胞的趋化能力;在低氧与常氧条件下将MG63与巨噬细胞进行共培养,流式细胞术及RT-PCR检测巨噬细胞的分化;分离纯化来源于不同氧浓度条件下培养的M2型巨噬细胞外泌体,利用透射电镜、纳米粒径、Western blot进行鉴定,双免疫荧光进行示踪MG63对外泌体的摄取;克隆形成实验及流式细胞术检测来源于不同氧浓度下的巨噬细胞外泌体对MG63的增殖、凋亡影响;CCK-8检测来源于不同氧浓度下的巨噬细胞外泌体对MG63顺铂耐药性的影响。结果低氧能够促进骨肉瘤细胞对巨噬细胞的趋化能力,并进一步诱导巨噬细胞向M2型分化;成功分离不同氧浓度下培养的M2巨噬细胞外泌体,且低氧能够显著上调巨噬细胞对外泌体的分泌;低氧能够明显上调巨噬细胞外泌体对骨肉瘤细胞增殖能力的促进作用,抑制骨肉瘤细胞发生凋亡,并上调骨肉细胞对顺铂的耐药性。结论在体外低氧能够促进骨肉瘤细胞对巨噬细胞的趋化能力,并进一步通过诱导巨噬细胞向M2型分化及提高M2型巨噬细胞外泌体的分泌,进而上调骨肉瘤细胞的增殖能力,抑制其凋亡,最终导致骨肉瘤细胞产生顺铂耐药性。     

基于差异接种细胞数的高效骨髓来源巨噬细胞分离方法的建立与评价

目的 探索不同铺板细胞数接种骨髓细胞对分离小鼠骨髓来源巨噬细胞得率的影响。方法 依据文献中给定的常用细胞铺板范围，将分离的小鼠骨髓细胞按六种不同细胞数接种在细胞培养皿中，使用巨噬细胞集落刺激因子(M-CSF)诱导骨髓细胞分化为巨噬细胞后，利用细胞计数法比较各组间贴壁细胞的比例差异，采用流式细胞术、间接免疫荧光法检测巨噬细胞的标志物F4/80,根据F4/80来判断巨噬细胞分化的阳性率，并最终比较各组之间骨髓来源巨噬细胞的得率。结果 在6.7×10⁵个/cm²～14.7×10⁵个/cm²细胞数范围内，按照6.7×10⁵个/cm²细胞数进行铺板，能够获得最高的贴壁细胞比例且贴壁细胞中F4/80⁺巨噬细胞得率最高，在获得相同数量巨噬细胞的前提下，该细胞数能够节省40%～53%制备巨噬细胞所需要的模型小鼠。结论 按照6.7×10⁵个/cm²的铺板细胞数进行骨髓细胞铺板，骨髓来源巨噬细胞的得率最高。     

系统性红斑狼疮患者巨噬细胞表型和功能初步研究

为了比较健康者与SLE患者巨噬细胞表型和吞噬功能的不同,收集性别、年龄匹配的健康对照者及SLE患者外周血,密度梯度离心法分离单个核细胞,磁珠分选CD14+单核细胞,巨噬细胞集落刺激因子诱导7d,分化为成熟巨噬细胞。FACS检测巨噬细胞表面CD163表达。取健康人CD4+T细胞标记CFSE,在抗CD3、CD28抗体刺激下,以5∶1比例分别与SLE和HC巨噬细胞共培养4d,FACS检测CD4+T细胞增殖比例。紫外辐照Jurkat T细胞株诱导人来源凋亡细胞,将pHrodo Green标记的凋亡细胞(5∶1)与SLE和HC巨噬细胞共培养2h,FACS检测pHrodo+CD14+巨噬细胞的百分率。结果显示,与HC相比,SLE巨噬细胞CD163表达量显著下降;SLE巨噬细胞对CD4+T细胞增殖抑制能力显著降低;SLE巨噬细胞对凋亡细胞的吞噬功能显著下降。这些结果提示SLE患者巨噬细胞表型、免疫调节能力和吞噬功能存在异常,可能参与其发病。     

肿瘤源性因子对肿瘤相关免疫抑制细胞的影响

肿瘤相关树突状细胞(TADC)、髓系来源抑制细胞(MDSC)和肿瘤相关巨噬细胞(TAM)等免疫抑制细胞在肿瘤免疫逃逸过程中能够诱导免疫抑制反应.肿瘤源性因子不仅影响这些细胞的分化和功能,而且影响这些细胞的迁移.这些在肿瘤免疫中的研究进展能够为发展新的抗肿瘤免疫治疗策略提供依据.     

CsA通过抑制免疫突触的形成促进类风湿关节炎巨噬细胞的凋亡

目的:探讨免疫抑制剂环抱素A (CsA)对类风湿关节炎(RA)患者体内参与免疫突触形成的巨噬细胞凋亡的影响.方法:将人单核细胞株THP-1(human acute monocytic leukemia cell line)诱导分化的巨噬细胞经葡萄球菌肠毒素(staphylococcal enterotoxin B,SEB) ( 100μg/L)包被后与活化的Jurkat T细胞(human acute T-cell leukemia cell line)共培养促免疫突触形成,加或不加CsA(1 mg/L),置于无血清RP-M11640培养液诱导凋亡16 h后,以Annexin V-PI染色,用流式细胞术检测巨噬细胞的凋亡情况.临床收集10例健康对照及10例确诊活动期RA患者外周血,采用免疫磁珠法阴性分选CD4+T细胞并将其与诱导分化的巨噬细胞共培养,亦设加或不加人CsA(1 mg/L)组,研究CsA对免疫突触调控巨噬细胞凋亡作用的影响.结果:在细胞株、健康人及RA患者外周血由单核细胞诱导分化的巨噬细胞和CD4+T细胞相互作用形成免疫突触后巨噬细胞的凋亡率分别为(32.9±2.8)%,(24.7±1.6)%,(14.5%±1.2)%,较相应单独培养的巨噬细胞的凋亡率(61.4±2.4)%、(45.5土2.6)%、(22.9±1.5)%显著降低(P<0.05).而在加CsA组,细胞株来源的巨噬细胞及RA患者来源的巨噬细胞的凋亡率分别为(48.8 +2.0)%、(16.9土1.1)%,较相应参与免疫突触形成的巨噬细胞的凋亡率有所升高(P<0.05),而加人CsA对健康对照巨噬细胞凋亡率的影响差异无统计学意义.结论:RA患者外周血诱导分化的巨噬细胞参与免疫突触的形成后可显著抑制其凋亡率,而CsA能减弱突触对巨噬细胞的这一保护功能,起到促进巨噬细胞凋亡的作用,该结论为减少RA炎性因子的分泌、减弱关节破坏提供了有利的理论依据.     

小鼠骨髓来源巨噬细胞体外培养及极化相关实验方法的建立

目的 建立小鼠骨髓来源巨噬细胞体外培养,并将其极化为M1和M2型巨噬细胞的方法.方法 获取小鼠骨髓细胞,以GM-CSF诱导其中单核细胞向巨噬细胞分化发育,并辅以LPS和IFN-γ以及IL-4刺激巨噬细胞分别向M1型和M2型分化.采用流式细胞术、实时定量PCR和ELISA的实验检测极化后细胞内以及细胞培养上清中的标志性细胞因子如IL-12、IL-10、Argl、Yml、MMR等的表达情况.结果 形态学观察发现骨髓获取的细胞经诱导分化后呈现巨噬细胞特征性的贴壁形态；流式细胞术行胞内染色表明,极化后的M1型与M2型巨噬细胞各自表达其特征性的细胞因子IL-12和Argl；实时定量PCR和ELISA分别检测所培养细胞胞内以及培养上清中的细胞因子,结果表明所刺激的M1型巨噬细胞高表达IL-12、iNOS、TNF-α等炎性因子,而M2型巨噬细胞表达MMR、Argl、Yml等标志因子.结论 建立了稳定的M1和M2型巨噬细胞体外培养体系.     

The Role of Receptors for T Cell Products in Antibody Formation

Immunocompetent cell interactions are achieved via direct contact between functionally different cell types or via interactions between soluble factors elaborated by regulatory T cells and specific receptors on responding cells for the T cell factors. In either case, there exist certain restrictions with respect to the effective interactions, which depend on the state of differentiation and genetic background of the responding cell type. Such restrictions are considered to be mainly determined by the development and nature of the receptor site on responding cell types for different T cell factors, which is now refered to the “acceptor” for the T cell factors. The presence of such acceptor sites on different populations of both T and B cells has been demonstrated in various experimental systems, and they are now considered to be the site by which responding cells receive appropriate signal for destination of their further differentiation.

We have tried to review the nature and possible role of acceptor sites on both B and T cells for different T cell factors with respect to the induction and regulation of immune responses. A special emphasis was put on the genetic nature of the acceptor site. The observed genetic restrictions in the acceptance of T cell factors by responding cells suggest that such restrictions are needed for meaningful and unmistakable communications between funcionally different immunocompetent cells. Furthermore, the presence or absence of acceptor sites for certain T cell factors is supposed to be a very important factor for determination of the immune responsiveness of animals against certain antigens, and thus in some cases the Ir gene effect may predominantly affect the expression of acceptor site. Possible implications of acceptor site in the regulation of antibody response and in the network of immunocompetent cell interactions are discussed.     

The Role of Receptors for T Cell Products in Antibody Formation

Immunocompetent cell interactions are achieved via direct contact between functionally different cell types or via interactions between soluble factors elaborated by regulatory T cells and specific receptors on responding cells for the T cell factors. In either case, there exist certain restrictions with respect to the effective interactions, which depend on the state of differentiation and genetic background of the responding cell type. Such restrictions are considered to be mainly determined by the development and nature of the receptor site on responding cell types for different T cell factors, which is now refered to the “acceptor” for the T cell factors. The presence of such acceptor sites on different populations of both T and B cells has been demonstrated in various experimental systems, and they are now considered to be the site by which responding cells receive appropriate signal for destination of their further differentiation.

We have tried to review the nature and possible role of acceptor sites on both B and T cells for different T cell factors with respect to the induction and regulation of immune responses. A special emphasis was put on the genetic nature of the acceptor site. The observed genetic restrictions in the acceptance of T cell factors by responding cells suggest that such restrictions are needed for meaningful and unmistakable communications between funcionally different immunocompetent cells. Furthermore, the presence or absence of acceptor sites for certain T cell factors is supposed to be a very important factor for determination of the immune responsiveness of animals against certain antigens, and thus in some cases the Ir gene effect may predominantly affect the expression of acceptor site. Possible implications of acceptor site in the regulation of antibody response and in the network of immunocompetent cell interactions are discussed.     

T cell derived IgE binding factors inhibit IgE-specific rosettes on basophilic leukaemic cells and lymphocytes

T cell derived IgE-binding factors inhibited the formation of IgE-specific rosettes on both rat basophilic leukaemic cells and lymphocytes. This was demonstrated by IgE-binding factors released from either IgE-pulsed T cells or adherent cells as well as from IgE-binding factor producing hybridomas. The different binding factors inhibited the IgE-rosettes to a different degree when tested on basophils and lymphocytes, most likely due to the higher affinity for IgE of the former cell type. The results indicate that rat basophils and mouse T cells may recognize common epitopes on the Fc portion of IgE.     

Stem cell plasticity: learning from hepatogenic differentiation strategies.

Many studies on stem cell plasticity are challenging the concept that stem cells contain an intrinsically predefined, unidirectional differentiation program. This means that the developmental fate of a stem cell is dependent on the general potential of the cell (pre-determined stem cell fate) as well as on microenvironmental cues, such as stimuli from growth factors (stem cell niche). Here, we reviewed reports that examined the hepatocyte differentiation ability of stem cells from two different sources: embryonic stem cells and adult stem cells. All of those stem cells revealed the ability to give rise to hepatocyte-like cells using different induction strategies. However, it is still not clear which of those stem cells would be the best source for hepatocyte replacement or which would be the best protocol. We herein present the current knowledge regarding available protocols and factors used in order to obtain functional hepatocytes from stem cells.     

成体干细胞分化的影响因素

成体干细胞(AS)分化的调控机制,多认为与微环境密切相关。其内源性调控包括干细胞内的一些结构蛋白和多肽因子调控的不对称分裂以及端粒体的长度。外源性调控主要为转化生长因子调节干细胞的增殖和分化;膜蛋白介导的细胞间相互作用,细胞膜表面分子在干细胞和周围细胞间传递信号;细胞外基质对AS细胞的调控,整合素家族介导AS细胞与细胞外基质粘附,通过激活生长因子受体,从而影响AS细胞的分布和分化方向。影响AS细胞分化的微环境,包括细胞所处的三维空间结构和相应的多维分化信号。     

Array-based functional screening of growth factors toward optimizing neural stem cell microenvironments

To gain insights into the effect of various growth factors on the behaviors of neural stem cells, cell culture assays were performed on the array that displayed five different growth factors including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, brain-derived neurotrophic factor, and ciliary neurotrophic factor. These factors were expressed in Escherichia coli as fusion proteins with a hexahistidine sequence and arrayed on a nickel ion-functionalized chip as single factors or the combination of two factors. Neural stem cells obtained from the fetal rat brain were cultured on the array to investigate their proliferation and differentiation. It was shown that the five growth factors displayed as a single component had significant impacts on cell behaviors. These effects are overall in accordance with those reported previously. On the other hand, in the case that two different growth factors were co-displayed on a single spot, the behaviors of neural stem cells could not be simply predicted from their individual effects. We performed a multivariate cluster analysis for the quantitative data on cell proliferation and differentiation. It was shown that the effect of two growth factors co-displayed was competitive, synergistic, or destructive depending on the combinations. In other peculiar cases, the effect of growth factors was totally different from those of individual factors.     

Glycosaminoglycan synthesis and shedding induced by growth factors are cell and compound specific

The interactions between growth factors and sulphated glycosaminoglycans (GAG) have been extensively studied. The aim of this study is to investigate if growth factors would show specificity of action on the synthesis and shedding of sulphated GAG, using two different cell lines: endothelial and smooth muscle cells. The cells were grown in the presence or absence of growth factors: EGF, FGF2, VEGF121, VEGF165. Transfection assays were also performed using recombinant pcDNA3.1, containing VEGF165 cDNA. In order to analyse the different types of GAG the cells were metabolically labelled with [³⁵S]-sulphate. At low doses, VEGF121 was the only growth factor able to increase both the synthesis and secretion of heparan sulphate (HS) in endothelial cells. Over expression of VEGF165 stimulated HS synthesis in both cells. The combined results showed that growth factors affect GAG synthesis in a cell specific and dose dependent manner.     

Glycosaminoglycan synthesis and shedding induced by growth factors are cell and compound specific

The interactions between growth factors and sulphated glycosaminoglycans (GAG) have been extensively studied. The aim of this study is to investigate if growth factors would show specificity of action on the synthesis and shedding of sulphated GAG, using two different cell lines: endothelial and smooth muscle cells. The cells were grown in the presence or absence of growth factors: EGF, FGF2, VEGF121, VEGF165. Transfection assays were also performed using recombinant pcDNA3.1, containing VEGF165 cDNA. In order to analyse the different types of GAG the cells were metabolically labelled with [(35)S]-sulphate. At low doses, VEGF121 was the only growth factor able to increase both the synthesis and secretion of heparan sulphate (HS) in endothelial cells. Over expression of VEGF165 stimulated HS synthesis in both cells. The combined results showed that growth factors affect GAG synthesis in a cell specific and dose dependent manner.