FasL基因重组慢病毒载体感染SD大鼠树突状细胞的研究

目的 探讨FasL基因重组慢病毒载体感染SD大鼠树突状细胞的效率和FasI 蛋白的表达情况,为进一步研究转FasL基因在同种异体器官移植中诱导免疫耐受和保护移植物打下基础.方法 将培养一周的细胞重铺于六孔板中,每孔细胞数量为5×105,24 h后观察,细胞适合感染,按照MOI=10感染细胞,使用GFP阳性对照质粒作对照实验,感染24 h后,培养皿中添加1 ml新鲜培养基,每隔1 d加细胞因子继续培养,荧光显微镜观察荧光强度和数量,添加病毒液后10 d收集细胞进行实时定量检测和WB检测.结果 FasL基困重组慢病毒载体感染DC 8 d后,细胞开始出现荧光,10 d感染效率为100%;实时定量PCR检测瞬时转染后目的 基因的表达显示以细胞的1.00%为参照,Cell＋FasL质粒为167.03%;免疫印迹检测转染后FasL蛋白的表达显示以细胞的1.00%为参照,细胞＋FasL质粒为34.15%.结论 FasL基因重组慢病毒载体成功感染DC,实时定量PCR及Western印迹证实感染的Dc表达FasL明显提高.为进一步研究转FasL摹因在同种异体器官移植中诱导免疫耐受和保护移植物打下基础.     

常用病毒载体转染对树突状细胞的影响

树突状细胞是功能最强的抗原提呈细胞,在介导免疫应答和维持免疫耐受中起着极其重要的作用。用携带目的基因的病毒载体转染树突状细胞已广泛应用于抗肿瘤和诱导耐受等方面的治疗。但在病毒载体转染树突状细胞过程中,病毒本身常能影响树突状细胞作为抗原提呈细胞的功能。因此,了解各种病毒载体的特点和对树突状细胞产生影响的作用机制,并选用合适的病毒载体,成为针对DCs进行基因治疗成功的关键。     

肝细胞生长因子基因转染对人淋巴瘤细胞凋亡的影响

探讨肝细胞生长因子（HGF）基因转染人淋巴瘤细胞系Raji细胞后,拮抗足叶乙甙（VP－16）诱导细胞凋亡的研究。将三种细胞：未转染Raji细胞、空载体pVITR02－mcs转染细胞和HGF基因转染细胞,分成正常对照组和经vP－16处理的药物组。采用Westernblot法验证HGF蛋白的表达：CCK－8法检测诱导Raji细胞凋亡的药物浓度;通过透射电镜、流式细胞术、吖啶橙（A0）染色、苏木精咿红（HE）染色等方法观察Raji细胞的凋亡情况,并进行相关分析。结果显示：Westernblot法验证了HGF蛋白质的表达;CCK．8法显示100μg／mL足叶乙甙可明显抑制Raii细胞增殖;透射电镜下可发现典型的凋亡细胞;流式检测结果表明：给药组与正常组相比,三组细胞的凋亡率明显升高（P〈0．01）,提示VP－16具有诱导细胞凋亡的作用：但给药组间：HGF基因转染组凋亡率明显低于未转染组（P＜0．05）和空载体pVITR02．mcs转染组（P〈0．05）,提示嬲F基因转染可明显抑制VP-16诱导的Raji细胞的凋亡,AO染色和HE染色结果也同样提示HGF具有拮抗VP－16诱导的细胞凋亡效应。     

胸腺树突状细胞对胸腺细胞凋亡的促进作用

小鼠胸腺细胞在体外培养一定时间后自发出现凋亡,在与小鼠胸腺树突状细胞(MTSC4)共育后,其凋亡过程可被明显加速;与此相反,小鼠胸腺上皮细胞(MTECI)有抑制凋亡的作用.与MTSC4共育后的胸腺细胞中不仅CD4⁺CD8⁺双阳性细胞明显减小,而且CD4⁺CD8⁺单阳性细胞也减少.提示胸腺基质细胞可通过其对细胞凋亡的促进作用参与胸腺内的阴性选择,并提示阴性选择可能在胸腺髓质区仍在进行.     

胎盘发生过程中的细胞凋亡

细胞凋亡是一种正常的生理现象,在胚泡着床过程中,伴随有大量细胞凋亡。研究表明,胎盘发生过程中的细胞凋亡,对调控子宫内膜基质细胞的蜕膜化和滋养层细胞的浸润以形成胎盘具有重要意义;另外,由Fas/FasL系统介导的细胞凋亡可能与母体对胎儿的免疫耐受性有关。本文主要评述细胞凋亡的一般通路以及胎盘发生过程中细胞凋亡的调控。     

耐受性树突状细胞

树突状细胞(DC)是体内功能最强的抗原提呈细胞,是一异质性的细胞群体,在免疫调节中扮演着双重角色.随着免疫学的发展,它在诱导器官移植耐受中的作用越来越受到重视,对其作用机制的研究也有了很大的进展.如何利用DC诱导抗原特异性免疫耐受并维持其耐受状态成为研究热点,并在此基础上提出了耐受性DC的概念.     

白介素10对人成熟树突状细胞蛋白表达的影响

利用人外周血淋巴细胞分离液密度梯度离心分离CD14~+的单核细胞,并通过IL-4、GM-CSF、LPS、IFN-γ等重组细胞因子在体外将其诱导为成熟树突状细胞(mature dendritic cells,mDCs),利用10 ng/mL白介素10 (interleukin-10,IL-10)处理mDCs 4 h,双向电泳联合MALDI-TOF/TOF MS技术分析差异表达蛋白。结果显示共筛选出35个差异蛋白点,其中21个上调,14个下调,它们的功能主要涉及糖代谢、HIF信号转导通路、细胞骨架和免疫功能等。因此,IL-10可能通过调控mDCs的代谢过程以及细胞骨架或运动能力等相关蛋白来影响其生物学功能,为进一步深入研究IL-10对mDCs的影响具有潜在作用。     

树突状细胞来源的Exosomes的免疫调节作用

Exosomes是多种细胞经晚期内体形成的一种膜性小囊泡。最初认为其功能仅为降解内吞物质,但研究发现exosomes的特异功能与其来源细胞相关,尤其是抗原提呈细胞(APCs)——树突状细胞来源的exosomes(dendritic cell-derived exosomes,DEXs)集MHC-I/MHC-II、共刺激分子、黏附分子、热休克蛋白于一身,在体内外免疫调节中起非常重要的作用。现对DEXs诱导抗肿瘤免疫应答和诱导免疫耐受两方面的功能及可能的免疫调节机制进行综述。     

人咽鳞癌Fadu细胞RNA负载人树突状细胞后体外诱导特异性CTL的研究

目的:探讨咽鳞癌细胞总RNA转染的树突状细胞(DC)疫苗体外诱导特异性抗肿瘤免疫的能力.方法:分离人脐血单个核细胞,经重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)、重组人IL-4(rhIL-4)诱导不成熟DC(iDC),提取人咽鳞癌细胞FaDu总RNA后,转染入人iDC,成为FaDu RNA/DC疫苗,用流式细胞仪检测DC表面分子CD40、CDS0、CD83、CD86、HLA-DR的表达,混合淋巴细胞反应测定DCs刺激同种异基因T细胞增殖能力.用LDH法评估转染肿瘤总RNA的DC瘤苗的细胞毒性T淋巴细胞反应.结果:与转染前比较,人咽鳞癌细胞FaDu总RNA转染后脐血单核细胞来源DCs表面CD40、CD80、CD83、CD86、HLA-DR分子水平明显升高(P均<0.05);可显著促进T细胞增殖,在体外能诱导高效而特异的抗下咽癌免疫效应(P<0.05).结论:人咽鳞癌细胞的总RNA转染的DC肿瘤疫苗能诱导CD8+,CD4+T细胞免疫,是较有临床应用前景的下咽癌免疫治疗方法.     

绿原酸对Fas/FasL途径介导的非酒精性脂肪肝细胞凋亡的影响

目的：研究绿原酸对Fas/FasL途径介导的非酒精性脂肪肝细胞凋亡的影响。方法：将40只SD雄性大鼠平均分成空白组（NC组）、高脂组（HFD组）、高脂饮食+低剂量绿原酸组（HFD-LC组）和高脂饮食+高剂量绿原酸组（HFD-HC组）。喂养12w之后处死,取血清检测总胆固醇（TC）、甘油三酯（TG）和高、低密度脂蛋白胆固醇（HDL-C、LDL-C）。取肝脏称其重量,计算肝指数,将肝脏组织分成两份,一份用于制备HE染色切片由于观察病理变化,另一份用于Fas、FasL和Caspase-8基因表达量的检测,检测方法采用半定量RT-PCR法。结果：与NC组相比,HFD组大鼠在体重、肝脏重量、肝指数以及Fas、FasL和Caspase-8基因表达量均有所上升;与HFD组相比,绿原酸干预后能够降低体重、肝脏重量、肝指数以及Fas、FasL和Caspase-8基因表达量,并且高剂量绿原酸作用效果更明显;切片结果显示,对比NC组,HFD组大鼠肝细胞内出现大量的脂肪空泡,经绿原酸干预后这一现象得以改善。结论：绿原酸能够降低Fas、FasL和Caspase-8基因表达量,对肝细胞具有保护作用。     

Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.     

The Fas/Fas ligand system and cancer: immune privilege and apoptosis

The Fas/FasL system has been suggested to play an important role in the establishment of immune privilege status for tumors by inducing Fas-mediated apoptosis in tumor-specific lymphocytes. However, the role of cell-surface expressed FasL in tumor cell protection has recently become controversial. Our laboratory has focused on the study of the role of the Fas/FasL system in the normal tissue remodeling of the female reproductive tract and in immune-privileged organs. Our studies have demonstrated a connection between sex hormones and the regulation of the Fas/FasL pathway in immune and reproductive cells. More recently, we have investigated the resistance of tumor cells to Fas-mediated apoptosis. We have also characterized a new form of FasL, different from the classical membranal form, which is secreted by ovarian cancer cells. In this review we describe the main techniques used in these studies.     

Gene delivery for genetically engineered mucosal cells with enhanced function

A non-viral transfection method for oral mucosal cells was investigated using a modified transfection method and five commercial transfection reagents. The CellFECTIN^TM gave the highest expression of a transfected gene. When the mucosal cells were transfected with 0.3 ng DNA/cell, the transfection efficiency was optimal, and the production of a reporter protein increased up to ten times higher than those with the other transfection reagents.     

Dendritic cells genetically engineered to express IL-10 induce long-lasting antigen-specific tolerance in experimental asthma

Dendritic cells (DCs) are professional APCs that have a unique capacity to initiate primary immune responses, including tolerogenic responses. We have genetically engineered bone marrow-derived DCs to express the immunosuppressive cytokine IL-10 and tested the ability of these cells to control experimental asthma. A single intratracheal injection of OVA-pulsed IL-10-transduced DCs (OVA-IL-10-DCs) to naive mice before OVA sensitization and challenge prevented all of the cardinal features of airway allergy, namely, eosinophilic airway inflammation, airway hyperreactivity, and production of mucus, Ag-specific Igs, and IL-4. OVA-IL-10-DCs also reversed established experimental asthma and had long-lasting and Ag-specific effects. We furthermore showed, by using IL-10-deficient mice, that host IL-10 is required for mediating the immunomodulatory effects of OVA-IL-10-DCs and demonstrated a significant increase in the percentage of OVA-specific CD4(+)CD25(+)Foxp3(+)IL-10(+) regulatory T cells in the mediastinal lymph nodes of OVA-IL-10-DC-injected mice. Finally, adoptive transfer of CD4(+) mediastinal lymph node T cells from mice injected with OVA-IL-10-DCs protected OVA-sensitized recipients from airway eosinophilia upon OVA provocation. Our study describes a promising strategy to induce long-lasting Ag-specific tolerance in airway allergy.     

Membrane molecules in induction of apoptosis of thymocytes by mouse thymic dendritic cells which express Fas ligands

Ａｐｏｐｔｏｔｉｃｔｈｙｍｏｃｙｔｅｓｗｅｒｅｄｅｔｅｃｔｅｄｉｎｓｉｔｕｉｎｔｈｅｔｈｙｍｕｓ[1],ｗｈｉｌｅｔｈｅｅｆｆｅｃｔｓｏｆｔｈｙｍｉｃｓｔｒｏｍａｌｃｅｌｌｓｏｎｔｈｅｐｒｏｃｅｓｓｏｆｃｅｌｌｄｅａｔｈｏｆｔｈｙｍｏｃｙｔｅｓａｒｅｓｔｉｌｌｕｎｃｌｅａｒ.Ｗｅｐｒｅｖｉｏｕｓｌｙｆｏｕｎｄｔｈａｔｍｏｕｓｅｔｈｙｍｉｃｄｅｎｄｒｉｔｉｃｃｅｌｌｓ(ＭＴＳＣ4)ｅｎｈａｎｃｅｄｔｈｅａｐｏｐｔｏｓｉｓｏｆｔｈｙｍｏｃｙｔｅｓｉｎｖｉｔｒｏ[2],ａｎｄｔｈｅｓｅｅｆｆｅｃｔｓｗｅｒｅｄｅｐ…     

Serum amyloid A inhibits dendritic cell apoptosis to induce glucocorticoid resistance in CD4+ T cells

Mediators produced by the airway epithelium control the activation, recruitment, and survival of pulmonary dendritic cells (DC) that present antigen to CD4⁺ T cells during the genesis and exacerbation of allergic asthma. The epithelial-derived acute phase protein, serum amyloid A (SAA), induces DC maturation and T_H17 polarization. T_H17 responses are associated with severe forms of allergic asthma that are poorly controlled by corticosteroids. We sought to determine whether SAA would enhance the survival of DC during serum starvation and could then contribute to the development of a glucocorticoid-resistant phenotype in CD4⁺ T cells. Bone marrow-derived dendritic cells (BMDC) that were serum starved in the presence of SAA were protected from activation of caspase-3 and released less lactate dehydrogenase. In comparison with untreated serum-starved BMDC, treatment with SAA downregulated mRNA expression of the pro-apoptotic molecule Bim, increased production of the pro-survival heat shock protein 70 (HSP70), and induced secretion of pro-inflammatory cytokines. SAA-treated BMDC that were serum starved for 48 h remained capable of presenting antigen and induced OTII CD4⁺ T cells to secrete IL-17A, IL-17F, IL-21, IL-22, and IFNγ in the presence of ovalbumin. IL-17A, IL-17F, IL-21, and IFNγ production occurred even when the CD4⁺ T cells were treated with dexamethasone (Dex), whereas glucocorticoid treatment abolished cytokine secretion by T cells cocultured with untreated serum-starved BMDC. Measurement of Dex-responsive gene expression demonstrated CD4⁺ T cells as the target of glucocorticoid hyperresponsiveness manifest as a consequence of BMDC stimulation by SAA. Finally, allergic airway disease induced by SAA and antigen inhalation was unresponsive to Dex treatment. Our results indicate that apo-SAA affects DC to both prolong their viability and increase their inflammatory potential under apoptosis-inducing conditions. These findings reveal mechanisms through which SAA enhances the CD4⁺ T-cell-stimulating capacity of antigen-presenting cells that may actively participate in the pathogenicity of glucocorticoid-resistant lung disease.     

Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

Background

Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs) stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs) and plasmacytoid (pDCs) are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown.

Methods

Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2^d) prior to transplanting into C57BL/6 mice (H-2^b), followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+).

Results

Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production.

Conclusion

Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.     

Dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can initiate antigen-specific humoral immunity independent of CD4+ T cells

We have investigated whether dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can trigger B cells to produce antigen-specific antibodies without CD4+ T-cell help. Dendritic cells modified with a recombinant adenovirus vector to express CD40 ligand and pulsed with heat-killed Pseudomonas induced naive B cells to produce antibodies against Pseudomonas in the absence of CD4+ T cells in vitro, initiated Pseudomonas-specific humoral immune responses in vivo in wild-type and CD4-/- mice, and protected immunized wild-type and CD4-/-, but not B-cell -/- mice, from lethal intrapulmonary challenge with Pseudomonas. Thus, genetic modification of dendritic cells with CD40 ligand enables them to present a complex mixture of microbial antigens and establish CD4+ T cell-independent, B cell-mediated protective immunity against a specific microbe.