荷瘤小鼠肿瘤组织髓系抑制性细胞的比例变化及意义

研究荷瘤小鼠肿瘤组织中髓系抑制性细胞(myeloid-derived suppressor cell,MDSC)的比例变化及分布特点,探讨其在机体抗肿瘤免疫中发挥的作用。采用肝癌细胞原位移植法建立小鼠原位肝癌模型,流式细胞仪检测荷瘤小鼠不同时间肿瘤组织中MDSC的比例;免疫组织化学法检测MDSC在肿瘤组织中的分布情况,以正常小鼠作对照。MDSC的比例在荷瘤小鼠肿瘤组织中比正常小鼠肝组织中明显升高,差别具有统计学意义(P0.05),且随着荷瘤时间的延长,MDSC的比例逐渐增加。正常小鼠肝组织中仅有极少量MDSC,而荷瘤后大量增加的MDSC主要分布于肿瘤组织与正常组织交界处。可见荷瘤小鼠肿瘤组织中MDSC的比例与肿瘤进展密切相关,提示荷瘤小鼠体内MDSC增多可能参与肿瘤的免疫逃逸,促进肿瘤细胞的生长。     

髓源性抑制细胞在鼻咽癌患者外周血及荷瘤小鼠体内的水平及作用初探

目的探讨髓源性抑制细胞(MDSCs)在鼻咽癌患者外周血中的表达水平及其临床意义,并在荷瘤小鼠体内探讨MDSCs可能的作用。方法采用流式细胞术分析49例鼻咽癌患者外周血及荷瘤小鼠脾脏MDSCs的比例,采用ELISA分析鼻咽癌患者及荷瘤小鼠中MDSCs对T细胞分泌细胞因子的影响。结果鼻咽癌患者外周血中MDSCs的比例显著高于健康对照组,并且鼻咽癌患者外周血中MDSCs的比例与肿瘤的分期有关,晚期患者外周血中MDSCs的比例高于早期患者;鼻咽癌荷瘤小鼠脾脏内MDSCs的数量明显高于对照组;研究表明MDSCs可明显抑制T细胞分泌细胞因子。结论鼻咽癌患者外周血中MDSCs的比例较健康人高,且与鼻咽癌分期密切相关,荷瘤小鼠实验表明肿瘤组小鼠脾脏MDSCs的数量明显高于对照组,并且MDSCs可影响荷瘤小鼠细胞因子的分泌。MDSCs可能作为鼻咽癌生物治疗的1个靶点。     

HDAC抑制剂丙戊酸通过减少MDSCs蓄积抑制肝癌的生长

目的研究组蛋白去乙酰化酶抑制剂丙戊酸(VPA)抑制肝癌发生发展的机制。方法将16只Balb/c小鼠注射小鼠肝癌细胞H22建立肝癌原位模型,随机分成2组:对照组和VPA组。术后第2天VPA组小鼠每天腹腔注射VPA(200 mg/kg),对照组每天腹腔注射等量的生理盐水。10 d后将小鼠处死,观察肿瘤大小及小鼠肝脏质量,利用流式分析小鼠脾脏部位CD4、CD8、NK细胞、MDSCs水平及肿瘤部位MDSCs的比例。建立小鼠肝癌原位模型,利用小动物活体成像分析MDSCs经1 mmol/L VPA处理后在体内迁移情况。结果 VPA抑制肝癌原位模型中肝癌生长,上调脾脏CD4、CD8水平,抑制肿瘤和脾脏部位MDSCs蓄积。MDSCs经VPA处理后向肿瘤和脾脏迁移的能力减弱,而MDSCs向骨髓的迁移的能力没有受影响。结论在肝癌微环境中,VPA能够抑制MDSCs向肿瘤和脾脏部位迁移、改善肝癌微环境,从而抑制肝癌生长。本研究为肝癌的治疗提供了一定的实验基础。     

LPS诱导CD11b~＋Gr-1~＋髓源抑制性细胞在小鼠脾脏募集的研究

目的观察脂多糖（LPS）诱导的髓源抑制性细胞（MDSCs）在小鼠脾脏中的募集情况,以探讨诱导MDSCs募集的新方法。方法采用LPS反复腹腔注射的方法使小鼠产生内毒素耐受。流式细胞术检测正常小鼠和LPS注射小鼠第5、10天脾脏中CD11b＋Gr-1＋双阳性细胞,即MDSCs的比例。结果经LPS处理后第5、10天脾脏MDSCs的比例分别为（17.99±1.66）%和（27.37±6.62）%,较正常小鼠（4.53±3.00）%显著增高,差异有统计学意义（P〈0.05）。结论 LPS可在短期内诱导大量MDSCs在小鼠脾脏中聚集。     

荷瘤小鼠脾脏髓源抑制细胞的分选及鉴定

目的研究荷瘤小鼠脾脏中髓源抑制细胞(myeloid derived suppresser-cell,MDSCs)的分选与鉴定方法。方法常规培养小鼠肾癌细胞(Renca细胞),于Balb/c小鼠皮下建立小鼠肾癌模型;分离小鼠脾脏制成单细胞悬液,磁珠分选Gr-1+CD11b+双阳性的MDSCs;台酚蓝染色检测细胞存活率;流式细胞仪(flow cytometry,FCM)测其细胞纯度;显微镜下观察细胞形态;免疫荧光测其表面Gr-1、CD11b荧光表达情况;RT-PCR检测Cox2和Arg-1的m RNA的表达情况;小鼠皮下成瘤实验观察MDSCs促肿瘤生长。结果成功建立小鼠肾癌模型,磁珠分选后经FCM检测Gr-1+CD11b+双阳性的MDSCs细胞可达92.3%,显著高于分选前(P0.01);免疫荧光鉴定结果显示:分选后的细胞形态完整,Gr-1和CD11b的荧光表达于细胞膜且2种荧光可以融合;RT-PCR检测分选后的MDSCs细胞群的Cox2和Arg-1的m RNA相对表达量显著高于分选前(P0.05);小鼠皮下成瘤观察到MDSCs组与实验对照组有显著差异(P0.05)。结论用免疫磁珠从荷瘤小鼠脾脏中成功分选得到MDSCs,具有较高纯度和良好的生物活性,为后续实验提供了理想的细胞来源。     

髓源抑制性细胞与肝癌

髓源抑制性细胞（MDSCs）作为肿瘤免疫微环境中的一种异质性细胞群,可在肿瘤患者和荷瘤动物体内大量扩增及聚集,并发挥免疫抑制活性。近年来的一些研究表明,MDSCs在肝癌的发生、发展和转移过程中也发挥重要作用。本文将综述MDSCs与肝癌的相关研究进展,探讨其在肝癌中的作用及潜在治疗策略。     

髓源性抑制细胞在慢性骨髓炎中的作用研究北大核心CSCD

目的 研究髓系来源的抑制性细胞（myeloid-derived suppressor cells,MDSCs）在大鼠慢性骨髓炎疾病中的免疫作用，为骨髓炎的治疗探索新方法。方法 首先建立大鼠慢性骨髓炎模型，并使用吉西他滨抑制MDSCs增长，通过流式细胞术及免疫荧光检测大鼠骨髓及脾脏中的MDSCs比例，ELISA检测外周血中炎性因子变化，分析正常大鼠、骨髓炎大鼠以及吉西他滨抑制后的大鼠外周血中炎性因子水平（TNF-α、PCT、IL-4、IL-10、IL-17、IFN-γ、TGF-β）与骨髓中MDSCs的相关性，分析MDSCs在慢性骨髓炎疾病中的免疫作用。结果 骨髓炎模型大鼠骨髓及脾脏中的MDSCs细胞比例增高，使用吉西他滨组MDSCs细胞比例明显降低（P<0.05），炎性因子水平（TNF-α、PCT、IL-4、IL-10、IL-17、IFN-γ、TGF-β）与MDSCs的细胞比例变化呈正相关（P<0.05）。结论 大鼠骨髓炎中MDSCs细胞比例变化与炎性因子水平呈正相关，吉西他滨抑制MDSCs可能通过抑制MDSCs而降低炎性反应，促进机体修复。     

菊粉对多囊卵巢综合征小鼠外周血、脾脏、肝脏髓源抑制性细胞及血浆炎症因子的影响

目的分析菊粉对多囊卵巢综合征(polycystic ovary syndrome,PCOS)小鼠髓源抑制性细胞(myeloid-derived suppressor cells,MDSCs)、单核细胞样MDSCs(monocytic-MDSCs,M-MDSCs)比例的变化和意义及其与血浆炎症因子的相关关系。方法采用60%高脂饮食联合颈背部皮下注射脱氢表雄酮6 mg/100 g+0.1 ml芝麻油制备PCOS小鼠模型,选取21～27日龄的雌性C57BL/6J小鼠30只,随机分为正常对照组、模型组、菊粉干预组,每组10只。流式细胞仪检测各组小鼠外周血、脾脏、肝脏中MDSCs和M-MDSCs的比例变化以及炎症因子TNF-α、IL-17A和IL-10的水平。结果对MDSCs和M-MDSCs分析发现:与正常对照组小鼠相比,模型组小鼠外周血、脾脏、肝脏中MDSCs和M-MDSCs显著增加,与模型组小鼠相比,菊粉干预组小鼠外周血、脾脏和肝脏MDSCs及M-MDSCs也显著增加。对血浆炎症因子分析发现:TNF-α、IL-17A在模型组与正常对照组小鼠中相比显著增加,而在模型组与菊粉干预组中相比显著降低,IL-10在模型组与正常对照组小鼠中相比显著降低,而在模型组与菊粉干预组中相比增加,但是没有统计学意义。对MDSCs和M-MDSCs与血浆炎症因子TNF-α, IL-17A相关性分析发现:PCOS小鼠外周血、脾脏、肝脏中MDSCs和M-MDSCs与血浆炎症因子TNF-α、IL-17A均呈负相关。结论菊粉能够诱导MDSCs、M-MDSCs在外周血、脾脏和肝脏中募集,减少促炎因子TNF-α、IL-17A的生成,从而延缓疾病的进展。     

前列腺癌源性外泌体通过MMP家族蛋白酶调控骨髓源性免疫抑制细胞的迁移能力

目的探讨前列腺癌来源的外泌体(exosomes)是否通过上调骨髓源性免疫抑制细胞(MDSCs)的金属基质蛋白酶(matrix metalloproteinase)MMP9、MMP2的分泌,进而增强MDSCs向外周迁移的能力。方法通过超速离心法提取鼠源性前列腺癌RM-1细胞上清中的外泌体,电镜观察外泌体的典型形态,Western blot鉴定外泌体的表面标记蛋白。流式细胞术检测荷瘤小鼠不同时间段肿瘤组织中MDSCs的比例,以及外泌体注射至正常小鼠体内后骨髓、脾脏中MDSCs的比例变化。免疫磁珠提取正常小鼠骨髓中的MDSCs,将MDSCs与RM-1-exosomes(50μg)共培养24 h后,免疫荧光观察MDSCs对外泌体的摄取情况。Western blot检测MDSCs表达MMP9、MMP2蛋白的情况,并用侵袭迁移实验检测MDSC与外泌体共培养前后的穿透能力。结果电镜下观察RM-1来源的外泌体呈典型椭圆碟形结构,直径约为30～100 nm。其表面标志蛋白CD63、HSP70、TSG101高表达验证了提取物质为外泌体。MDSCs与外泌体共培养后,免疫荧光可观察到MDSCs能大量摄取RM-...     

紫草萘醌组分制剂逆转可移植肿瘤的免疫损伤效应

目的:观察紫草萘醌组分可溶性提取物的抗肿瘤和免疫调节作用.方法:紫草萘醌醇提物制备成可溶于水的微乳液(LE)制剂,给荷瘤模型小鼠灌胃;皮下接种HepA22肝癌细胞或S180肉瘤细胞制作荷瘤模型;观察其生存时间、瘤重和免疫学指标.结果:荷瘤小鼠胸腺和脾脏指数以及NK细胞毒、ConA诱导淋转和IL-2生成能力明显降低,胸腺皮质和脾脏生发中心严重萎缩;应用LE阻止荷瘤动物免疫器官形态改变的发生;提高其脾细胞免疫功能.结论:紫草萘醌组分制剂抑制可移植肿瘤原位增殖,逆转荷瘤对小鼠免疫系统的损害.     

Anti-Gr-1去除新生小鼠髓源性抑制细胞的动态变化

目的 分析anti-Gr-1去除新生小鼠髓源性抑制细胞(MDSCs)的动态变化,探讨其去除MDSCs的效率和反应时间.方法 将50只BALB/c新生小鼠随机分成5组,每组10只,根据不同时间点腹腔注射anti-Gr-1(15 μg);于出生后第9天收取肝脏、脾脏标本,并将各自充分研磨后的组织悬液通过Percoll液进行梯度离心,分离出其中的单个核细胞;利用流式细胞仪分别检测肝、脾组织的髓源性抑制细胞(CD11b+Gr-1+)数量.结果 ①经anti-Gr-1注射4h后,实验组脾脏中的MDSCs百分比明显低于正常对照组[(9.19±1.40)％比(26.01±2.26)％,t=20.266,P＜0.05],并能有效维持24～48 h,去除率达到64.6％,当48 h后MDSCs百分比(16.87±3.49)％,去除率下降至35.1％;②经anti-Gr-1注射24h后,实验组肝脏中的MDSCs百分比与正常对照组比较[(8.98±1.03)％比(15.75±1.10)％,t=11.12,P＜0.05],具有统计学意义,去除率为42.9％,至48 h后MDSCs百分比(4.57±0.95)％,去除率达到70.9％;③同一实验组,脾脏和肝脏各自的M-MDSCs/G-MDSCs比值区别较大.结论 Anti-Gr-1对肝脏、脾脏的MDSCs去除效率和反应时间不同:经anti-Gr-1注射后4～48 h,脾脏的MDSCs去除率为35.1％ ～64.6％,而24 ～48 h后肝脏的MDSCs去除率为42.9％～70.9％.     

髓样抑制细胞免疫抑制机理的研究

目的:研究荷瘤小鼠来源的髓样抑制细胞(Myeloid derived suppressor cell,MDSC)在肿瘤免疫抑制中的作用机理。方法:用Percoll分离法从荷瘤小鼠的脾脏和骨髓中分离Gr-1+CD11b+MDSC;用流式细胞术检测MDSC对T细胞增殖的抑制作用;分别用生化法和ELISA技术检测MDSC体外培养上清中抑制性因子NO、ROS和IL-10、TGF-β的含量。结果:MDSC在荷瘤小鼠的脾脏和骨髓中聚集增多,且其在骨髓中所占的比例显著高于脾脏;MDSC可以明显抑制脾脏细胞的增殖,体外培养6小时的MDSC可以分泌大量NO、ROS和IL-10、TGF-β。结论:本实验进一步证实MDSC可以通过分泌大量NO、ROS和IL-10、TGF-β抑制T细胞增殖。     

A copper chelate selectively triggers apoptosis in myeloid-derived suppressor cells in a drug-resistant tumor model and enhances antitumor immune response

Myeloid-derived suppressor cells (MDSCs), one of the major orchestrators of immunosuppressive network are present in the tumor microenvironment suppress antitumor immunity by subverting Th1 response in tumor site and considered as a great obstacle for advancement of different cancer immunotherapeutic protocols. Till date, various pharmacological approaches have been explored to modulate the suppressive functions of MDSCs in vivo. The present study describes our endeavor to explore a possibility of eradicating MDSCs by the application of a copper chelate, namely copper N-(2-hydroxy acetophenone) glycinate (CuNG), previously found to be a potential immunomodulator that can elicit antitumorogenic Th1 response in doxorubicin-resistant EAC (EAC/Dox) bearing mice. Herein, we demonstrated that CuNG treatment could reduce Gr-1+CD11b+ MDSC accumulation in ascitic fluid and spleen of EAC/Dox tumor model. Furthermore, we found that CuNG mediated reduction in MDSCs is associated with induction of Th1 response and reduction in Treg cells. Moreover, we observed that CuNG could deplete MDSCs by inducing Fas-FasL mediated apoptotic cell death where death receptor Fas expression is enhanced in MDSCs and FasL is provided by activated T cells. However, MDSC expansion from bone marrow cells and their differentiation was not affected by CuNG. Altogether, these findings suggest that the immunomodulatory property of CuNG is attributed to, at least in part, by its selective cytotoxic action on MDSCs. So, this preclinical study unveils a new mechanism of regulating MDSC levels in drug-resistant cancer model and holds promise of translating the findings into clinical settings.     

Polyunsaturated fatty acids promote the expansion of myeloid‐derived suppressor cells by activating the JAK/STAT3 pathway

Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects that could prove beneficial in clinical therapies for certain autoimmune and inflammatory disorders. However, the mechanism of PUFA‐mediated immunosuppression is far from understood. Here, we provide evidence that PUFAs enhance the accumulation of myeloid‐derived suppressor cells (MDSCs), a negative immune regulator. PUFA‐induced MDSCs have a more potent suppressive effect on T‐cell responses than do control MDSCs. These observations were found both in cultured mouse bone marrow cells in vitro and in vivo in mice fed diets enriched in PUFAs. The enhanced suppressive activity of MDSCs by PUFAs administration was coupled with a dramatic induction of nicotinamide adenine dinucleo‐ tide phosphate oxidase subunit p47^phox and was dependent on reactive oxygen species (ROS) production. Mechanistic studies revealed that PUFAs mediate its effects through JAK‐STAT3 signaling. Inhibition of STAT3 phosphorylation by JAK inhibitor JSI‐124 almost completely abrogated the effects of PUFAs on MDSCs. Moreover, the effects of PUFAs on MDSCs and the underlying mechanisms were confirmed in tumor‐bearing mice. In summary, this study sheds new light on the immune modulatory role of PUFAs, and demonstrates that MDSCs expansion may mediate the effects of PUFAs on the immune system.     

Tumor conditions induce bone marrow expansion of granulocytic,but not monocytic,immunosuppressive leukocytes with increased CXCR2 expression in mice

Myeloid‐derived suppressor cells (MDSCs) promote tumor growth through, in part, inhibiting T‐cell immunity. However, mechanisms underlying MDSC expansion and guidance of MDSCs toward the tumor microenvironment remain unclear. Employing Percoll density gradients, we separate bone marrow (BM) leukocytes from tumor‐bearing mice into four density‐increasing bands with myeloid leukocytes enriched in bands III and IV. Band III comprises monocytes and low‐density granulocytes, both confirmed to be M‐MDSCs and G‐MDSCs, respectively, by displaying potent inhibition of T‐cell proliferation. However, monocytes act as M‐MDSCs not only under tumor conditions but also the healthy condition. In contrast, band IV contains non‐inhibitory, mature granulocytes. Only band III G‐MDSCs display significant expansion in mice bearing B16 melanoma, Lewis lung carcinoma, or MC38 colon carcinoma. The expanded G‐MDSCs also show increased CXCR2 expression, which guides egress out of BM, and produce arginase‐1 and ROS upon encountering antigen‐activated T cells. Adoptive transfer assays demonstrate that both G‐MDSCs and mature granulocytes infiltrate tumors, but only the former displays sustention and accumulation. Intratumoral administrations of granulocytes further demonstrate that G‐MDSCs promote tumor growth, whereas mature granulocytes exert minimal effects, or execute powerful anti‐tumor effects providing the presence of PMN activation mechanisms in the tumor microenvironment.     

Dynamic Change and Impact of Myeloid-Derived Suppressor Cells in Allogeneic Bone Marrow Transplantation in Mice

Myeloid-derived suppressor cells (MDSCs) are a group of myeloid cells composed of hematopoietic progenitor cells, immature macrophages, dendritic cells, and granulocytes, which accumulate in inflammatory diseases and various cancers. Here, we investigated the dynamic changes and effects of MDSCs in graft-versus-host disease (GVHD) development and/or tumor relapse after syngeneic and allogeneic bone marrow transplantation (BMT). We found that adding functional MDSCs in donor graft alleviated GVHD, whereas removal of MDSCs in vivo exacerbated GVHD. After T cell-deplete BMT, MDSCs transiently accumulated in the blood and spleen of recipients without GVHD. In contrast, after T cell-replete BMT, the levels of blood MDSCs were constantly elevated in recipients with GVHD. MDSC accumulation positively correlated with the severity of GVHD. Additionally, MDSC accumulation was further increased upon tumor relapse. Although MDSCs isolated from both syngeneic and allogeneic BMT recipients inhibited T cell proliferation in response to alloantigen stimulation ex vivo, MDSCs from the recipients with GVHD showed much higher suppressive potency compared with those from recipients without GVHD. These results indicate that MDSCs can regulate the immune response in acute GVHD, and possibly tumor relapse, subsequent to allogeneic BMT.     

Activated NKT cells facilitated functional switch of myeloid-derived suppressor cells at inflammation sites in fulminant hepatitis mice

Myeloid-derived suppressor cells (MDSCs) confer immunosuppressive properties, but their roles in fulminant hepatitis have not been well defined. In this study, we systematically examined the distribution of MDSCs in bone marrow (BM), liver and spleen, and their functional and differentiation status in an acute fulminant hepatitis mouse model induced by lipopolysaccharide and D-galactosamine (LPS-GalN). Moreover, the interaction between NKT cells and MDSCs was determined. Our study revealed that BM contained the largest pool of MDSCs during pathogenesis of fulminant hepatitis compared with liver and spleen. MDSCs in liver/spleen expressed higher levels of chemokine receptors such as CCR2, CX3CR1 and CXCR2. At inflamed tissues such as liver or spleen, activated NKT cells induced differentiation of MDSCs through cell–cell interaction, which markedly dampened the immunosuppressive effects and promoted MDSCs to produce pro-inflammatory cytokines and activate inflammatory cells. Our findings thus demonstrated an unexpected pro-inflammatory state for MDSCs, which was mediated by the activated NKT cells that precipitated the differentiation and functional evolution of these MDSCs at sites of inflammation.     

Antigen specificity of immune suppression by myeloid-derived suppressor cells

Among the mechanisms set in motion by the tumor to escape the control of the immune system, MDSCs play a central role in inducing tolerance to a variety of anti-tumor effectors, including T lymphocytes. It has been demonstrated that MDSCs expand in tumor-bearing mice and in cancer patients, leading to an impairment of T cell reactivity against the tumor. However, as the presence of MDSCs is not correlated with a general immune suppression, it was advanced that a mechanism regulating the specificity of MDSC inhibition must be present. In this article, we review the literature showing that MDSCs exert their immune-suppressive function on Ag-specific T cell responses but at times, also on mitogen-activated T lymphocytes, therefore bypassing the Ag dependency. We propose that the features of MDSC-mediated immune suppression might be influenced not only by the specific microenvironment in which MDSCs expand and by the tumor characteristics but also by the levels of activation of the target lymphocytes.     

Endogenous glucocorticoids promote the expansion of myeloid-derived suppressor cells in a murine model of trauma

Stress-dose of glucocorticoid has been demonstrated to be beneficial for trauma patients in clinical studies. Recently, a heterogeneous population of myeloid cells with immunosuppressive activity named myeloid-derived suppressor cells (MDSCs) has been found to accumulate in the trauma host and can be induced by glucocorticoids in vitro. In order to explore the effect of endogenous glucocorticoids on MDSCs under trauma conditions, we blocked the glucocorticoid signal in a murine trauma model using the antagonist of the glucocorticoid receptor RU486 (mifepristone). We found for the first time that RU486 not only blunted MDSC expansion induced by trauma in the spleen, peripheral blood and bone marrow especially at 6 h after traumatic stress but also decreased the survival rate from 100 to 20% in traumatic mice within 7 days. Moreover, neither MDSCs producing arginase-1 nor the morphological characterization of trauma-induced MDSCs was affected by the blockage of the glucocorticoid receptor. Our results suggest that endogenous glucocorticoids may promote MDSCs expansion in a murine trauma model and MDSCs may be beneficial for the trauma host.