小鼠骨髓源树突状细胞体外诱导培养及初步鉴定

用重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）和重组小鼠白细胞介素4（rmIL-4）体外诱导小鼠骨髓细胞分化为树突状细胞,进行形态学变化观察,分析细胞表面分子,刺激T细胞增殖,探讨小鼠骨髓源树突状细胞（BMDC）体外诱导培养并进行初步鉴定。体外培养9d后BMDC可达80%以上,光镜下可见典型的树突状细胞形态。清楚表达成熟期主要表面标志物,可显著刺激同种异体混合淋巴细胞增殖。获得了较高纯度的BMDC,避免了使用传统磁珠分离方法所带来的成本高,操作复杂,产出率低的弊端,为研究BMDC功能以及运用开展下游实验提供材料。     

不同剂量LPS诱导肝Kupffer细胞释放TNF的体外研究

本实验观察了不同剂量LPS诱导大鼠肝Kupffer细胞释放TNF的作用。加入LPS后1小时,三种剂量LPS组Kupffer细胞培养上清中均可测到TNF活性,3小时达到峰值。100和150ng/ml LPS组TNF活性高于50ng/ml组(P<0.01),而100和150ng/ml两组之间无明显差异(P>0.05),再次加入LPS(终浓度100ng/ml),只有50ng/ml LPS组培养上清液中有TNF活性检出,但幅度明显下降(P<0.01)。上述结果提示LPS在体外诱导肝Kupffer细胞释放TNF在一定范围内具有剂量依赖关系,且呈一定的时间反应性。     

慢病毒介导的稳定表达人GM-CSF、IL-4的EA.hy926细胞株的建立

目的:获得能够稳定表达人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)的内皮细胞株。方法:构建重组慢病毒表达载体,在293FT细胞中进行病毒的包装,用获得的高滴度慢病毒感染EA.hy926细胞株,建立能够稳定、高效表达hGM-CSF、hIL-4的EA.hy926细胞株。结果:EA.hy926感染效率达90%以上,长期传代后阳性率仍维持在90%以上;实时定量PCR和ELISA方法证实感染后细胞hGM-CSF、hIL-4在mRNA水平的表达分别是未感染细胞的25倍和9946倍,在蛋白水平的表达分别是未感染细胞的145倍和23倍。结论:构建的细胞系可以稳定表达细胞因子GM-CSF、IL-4。     

小鼠骨髓源性树突状细胞的体外扩增、鉴定及生物学特性研究

目的:对树突状细胞体外大量扩增及树突状细胞的临床应用提供理论基础.方法:以重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)、重组小鼠白介素-4(rmIL-4)和重组小鼠肿瘤坏死因子-α(rm TNF-α)体外诱导小鼠骨髓细胞分化为DC,倒置显微镜动态观察细胞形态学变化,流式细胞术分析细胞表面分子,并应用混合淋巴细胞反应检测其刺激T淋巴细胞的增殖能力.结果:经体外诱导培养第2天即可见大量细胞集落形成;培养至第9天,DC成熟,具有典型的形态,同时DC可以显著刺激同种异体混合淋巴细胞增殖.结论:体外诱导培养可以获得大量小鼠骨髓来源的DC,可广泛应用于临床实验及实验研究.     

类风湿性关节炎患者血清GM-CSF, IL-6, TNF-α水平的变化及其临床意义

目的:通过检测类风湿性关节炎(RA)患者血清粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-6(IL-6)及肿瘤坏死因子-α(TNF-α)水平,探讨其用于RA诊断与预后判断的临床意义。方法:选取我院2013年6月~2016年12月收治的126例RA患者为RA组,并根据病情严重程度分为活动期RA组(69例)和缓解期RA组(57例)两个亚组;另选取同期于我院体检中心接受体检的55例健康体检者为对照组。采用酶联免疫吸附法(ELISA)检测所有对象的血清GM-CSF、IL-6、TNF-α水平。记录比较各组血清细胞因子水平,并分析RA组患者各细胞因子水平及DAS28间的相关性。结果:活动期RA组的血清GM-CSF、IL-6、TNF-α水平均显著高于缓解期RA组和对照组(P0.01),缓解期RA组的血清GM-CSF、IL-6、TNF-α水平亦均显著高于对照组(P0.01)。血清GM-CSF、IL-6及TNF-α水平三者间均呈显著正相关(P0.01);DAS28与血清GM-CSF、IL-6、TNF-α水平亦均呈显著正相关(r=0.473、0.584、0.675,P均0.01)。结论:类风湿性关节炎患者的血清GM-CSF、IL-6、TNF-α水平均显著上调,且与类风湿性关节炎的活动性呈显著正相关,通过检测此类细胞因子对类风湿性关节炎的早期诊断、病情评估及判断预后均具有重要临床意义。     

肿瘤抗原致敏的GM-CSF基因转染巨噬细胞对实验性肺转移肿瘤的治疗作用

研究将对巨噬细胞双重功能均具有激活作用的细胞因子GM-CSF的基因转染小鼠腹腔巨噬细胞,再经肿瘤抗原致敏后通过静脉注射用于实验性CT26结肠癌肺转移小鼠的治疗.结果表明,腺病毒介导的GM-CSF基因转染小鼠腹腔巨噬细胞在转染后4h即可分泌较高水平的GM-CSF.转染后10d仍可有效表达;转染后16h左右小鼠腹腔巨噬细胞MHCⅡ类分子的表达明显增强,其抗原提呈能力也达最高水平;对肿瘤细胞的杀伤活性显著增高;荷瘤3d的实验性CT26结肠癌肺转移小鼠经肿瘤抗原致敏的GM-CSF基因转染巨噬细胞治疗后第16天肺部转移结节数明显减少;经治疗小鼠脾细胞经诱导的CTL杀伤活性也明显升高.以上结果提示,GM-CSF基因转染及表达能有效增强小鼠腹腔巨噬细胞的抗原提呈能力及效应功能;经肿瘤抗原刺激后其对转移性肿瘤也有明显的治疗作用.     

注射紫杉醇的小鼠骨髓细胞体外分化巨噬细胞增强

目的研究小鼠腹腔注射紫杉醇对体外骨髓细胞诱导分化巨噬细胞的影响。方法小鼠连续5d腹腔注射紫杉醇,无菌制备骨髓细胞,用含巨噬细胞集落刺激因子（M-CSF）的RPMI1640培养液培养骨髓细胞,通过流式细胞仪对其诱导分化的巨噬细胞表面分子、吞噬功能进行分析。结果紫杉醇明显降低小鼠骨髓细胞数量,但骨髓细胞体外诱导分化成巨噬细胞的数量明显增加;F4/80^＋巨噬细胞中CD80、CD14表面分子表达升高,而I-A^d表达降低;紫杉醇处理组诱导分化的巨噬细胞吞噬鸡红细胞的能力提高。结论结果提示紫杉醇可能具有调节巨噬细胞表面分子的表达和吞噬功能。     

TNF-α与G-CSF在脑缺血-再灌注损伤中的研究

再灌注损伤是由多种原因引起的复杂的病理生理过程,而级联的炎症反应是导致脑细胞损伤的重要病理环节。脑缺血再灌注后,浸润的炎性细胞产生的大量炎性介质,在再灌注损伤中占有重要地位。肿瘤坏死因子α(TNF-α)是一种具有广泛生物学功能的细胞因子,参与机体免疫应答和炎症反应TNF-α是细胞间粘附分子-1(ICAM-1)表达的强诱导剂,抑制细胞粘附分子(ICAM-1)表达可显著减低再灌注时白细胞粘附活化,减少损伤脑面积起保护作用。粒细胞集落刺激因子(G-CSF)能通过STAT途径减少缺血区肿瘤坏死因子-α等的释放,引起人们对其在脑缺血-再灌注损伤中的作用的极大关注。     

TNF-α与G—CSF在脑缺血-再灌注损伤中的研究

再灌注损伤是由多种原因引起的复杂的病理生理过程,而级联的炎症反应是导致脑细胞损伤的重要病理环节。脑缺血再灌注后,浸润的炎性细胞产生的大量炎性介质,在再灌注损伤中占有重要地位。肿瘤坏死因子α（TNF-α）是一种具有广泛生物学功能的细胞因子,参与机体免疫应答和炎症反应TNF-α是细胞间粘附分子-1（ICAM-1）表达的强诱导剂,抑制细胞粘附分子（ICAM-1）表达可显著减低再灌注时白细胞粘附活化,减少损伤脑面积起保护作用。粒细胞集落刺激因子（G-CSF）能通过STAT途径减少缺血区肿瘤坏死因子-α等的释放,引起人们对其在脑缺血-再灌注损伤中的作用的极大关注。     

HA纳米载体介导转hGM-CSF基因的HepG2疫苗诱导的 抗瘤效应研究

目的:观察HA纳米颗粒载体介导转染hGM-CSF基因的HepG2细胞疫苗体外抗肿瘤效应,为hGM-CSF基因修饰的HepG2细胞疫苗的临床应用提供依据。方法:HA纳米颗粒载体介导hGM-CSF基因转染HepG2细胞制备转GMCSF基因的HepG2细胞疫苗。密度梯度离心法分离人PBMC,体外诱导人PBMC。WST-1法测定PBMC的增殖活性及对HepG2细胞的杀伤效应,流式细胞术分析CD4+和CD8+的阳性表达率,ELISA法测定INF-γ的分泌。结果:WST-1结果显示,转基因HepG2组疫苗能诱导PBMC增殖,其增殖率优于野生型疫苗(p<0.05);其诱导的PBMC对HepG2的杀伤率高于各野生型疫苗组和各空白对照组(p<0.05)。FCM结果显示,转基因HepG2疫苗组PBMC中CD4+和CD8+阳性表达率均高于各野生型疫苗组和各空白对照组(p<0.05)。ELISA结果显示,转基因组PBMC培养上清中IFN-γ含量为1989.76±254.21pg/ml,高于各野生型疫苗组和各空白对照组(p<0.05)。结论:HA纳米颗粒载体介导转染hGM-CSF基因能增加HepG2细胞疫苗的免疫原性,转hGM-CSF基因HepG2细胞疫苗可有效诱导PBMC增殖、分化,增加INF-γ的分泌,提高其对HepG2细胞的杀伤作用。     

大鼠骨髓源树突状细胞吞噬功能的超微结构观察

目的:利用透射电镜对体外扩增并纯化的大鼠骨髓来源树突状细胞的吞噬过程进行超微结构观察。方法:利用粘附法诱导分离大鼠骨髓细胞,延长树突状细胞的培养时间,在透射电镜下观察树突状细胞对坏死细胞碎片的吞噬过程。结果:树突状细胞识别坏死的细胞碎片后,伸出粗大突起将坏死的细胞碎片环形包裹吞噬;吞噬坏死细胞后,树突状细胞呈不规则形,细胞表面可见明显突起,消化过程中细胞内形成大量大小不等、电子密度不均一的次级溶酶体及髓鞘样膜结构。结论:树突状细胞对凋亡或坏死细胞的提呈对于保持机体内环境的稳定非常重要;利用树突状细胞的吞噬作用进行肿瘤免疫、自身免疫研究,将为树突状细胞临床治疗提供新的途径。     

香菇多糖对小鼠骨髓树突状细胞表型和功能的影响

通过研究香菇多糖(Lentinan,LNT)对小鼠骨髓源树突状细胞(bone marrow dendritic cells,BMDCs)功能调节的机制,进一步阐明香菇多糖的免疫活性。本实验将BMDCs分成脂多糖(LPS)阳性对照组、LNT处理组和RPMI1640空白对照组,应用酸性磷酸酶活性检测、流式细胞仪检测技术、吞噬实验、酶联免疫吸附试验检测各组BMDCs表型和功能的各种指标。结果显示,LNT(50μg/mL)处理组BMDCs酸性磷酸酶活性降低;BMDCs吞噬能力比RPMI1640空白对照组明显下降;BMDCs表面MHCⅡ、CD40、CD83、CD80、CD86和DEC205的表达增加;BMDCs白细胞介素12(IL-12)、白细胞介素10(IL-10)和肿瘤坏死因子-α(TNF-α)的表达增加。证明了适宜浓度的LNT可以促进BMDCs表型及功能的成熟。     

小鼠骨髓间充质干细胞的分离、培养及鉴定

目的建立一种从小鼠骨髓中分离培养间充质干细胞（MSCs）的高效方法。方法采取贴壁细胞分离法分离和纯化小鼠骨髓间充质干细胞（mMSCs）,检测mMSCs在不同诱导条件下向成骨细胞及脂肪细胞分化能力,用流式细胞术及显微镜分别检测mMSCs纯度和形态特征。结果mMSCs贴壁生长后形态较均一,细胞形态呈成纤维细胞样,流式细胞术检测：CD45、CD11b、CD44及CD29分别为（3.34）%、（2.41）%、（98.46）%及（99.36）%。第4代mMSCs经诱导后可向成骨细胞和脂肪细胞分化。结论通过贴壁培养可以从小鼠骨髓中分离培养出高纯度mMSCs,该方法效率高,稳定性好。     

Functional Analysis of Neuron-like Cells Differentiated from Neural Stem Cells Derived from Bone Marrow Stroma Cells in vitro

The transversal differentiation of bone marrow stroma cell (BMSCs) into neural stem cells (NSCs) has attracted much attention in recent years because of their therapeutic potential. However, the problem in therapeutic application of NSCs was how to confirm whether neuron-like cells differentiated from bone marrow stroma cell-derived neural stem cells (BMSCs-D-NSCs) possess corresponding functions of neurochemistry and electrophysiology. In the present study, we tried to affirm the function of neuron-like cells differentiated from BMSCs-D-NSCs in vitro. The BMSCs were harvested by gradient centrifugation in Ficoll-Paque and cultured in “NSCs medium”. Immunocytochemistry was used to detect positive expression of neuron-specific nuclear protein (NeuN) in neuron-like cells derived from the BMSCs-D-NSCs. High-pressure liquid chromatography (HPLC) was used to identify neuron-like cells by detecting excitable amino acids [aspartic acid (Asp), glutamic acid (Glu)], inhibited amino acids [glycine (Gly), gamma (γ) -aminobutyric acid (GABA), alanine (Ala)] or monoamines [noradrenaline (NE), 5-hydroxytryptamine (5-HT), dopamine (DA)]. Electrophysiological properties of the neuron-like cells were also examined using patch clamp analysis to verify their neuron-like functions. It was found that the neuron-like cells differentiated from the BMSCs-D-NSCs could express positive NeuN, synthesize and excrete amino acids, and show some typical electrophysiological properties including the typical Na⁺ and K⁺ ion channel membrane current under the voltage patch clamp condition, the typical static electrical membrane potential under the current patch clamp condition, and the differential membrane capacitance and resistance values in series between undifferentiated BMSCs-D-NSCs and differentiated neuron-like cells under the whole-cell patch clamp condition. The neuron-like cells differentiated from BMSCs-D-NSCs exhibit both neuron-like biochemical function and some corresponding electrophysiological properties.     

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction^{1, 2}. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells^{1, 2}. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells^{1, 2}. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide^{1, 2}. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.Download video file.^{(152M, mp4)}     

Dendritic Cells and Regulatory T Cells in Atherosclerosis

Although macrophages and other immune system cells, especially T cells, have been shown to play disease-promoting roles in atherosclerosis, less is known about the role of antigen presenting cells. Functional, immune stimulating dendritic cells (DCs) have recently been detected in aortic intima, the site of origin of atherosclerosis. We had compared DCs with macrophages in mice with experimental atherosclerosis, to clearly define cell types by developmental and functional criteria. This review summarizes recent advances in studies of DCs in humans and in mouse models of atherosclerosis, as well as providing a simple strategy to measure regulatory T (Treg) cells in the mouse aorta.     

Modeling Dynamics and Function of Bone Marrow Cells in Mouse Liver Regeneration

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大鼠骨髓来源未成熟与成熟树突状细胞的对比研究

目的:对比培养大鼠骨髓来源的未成熟树突状细胞与成熟树突状细胞,并从形态学、表型及功能检测等多方面进行对比研究,为后续的实验做出基础研究。方法:大鼠脱臼法处死后取两侧胫骨、股骨,PBS冲洗骨髓腔收集骨髓细胞,经GM-CSF和IL-4刺激培养六天后,对比研究经LPS刺激组与未经LPS刺激培养组细胞状况。结果:①成熟树突状细胞悬浮生长,集落分散,扫描电镜下见其突起数目明显多于未成熟树突状细胞。②成熟树突状细胞高表达表面标记分子CD80、CD86、MHCⅡ,而未成熟树突状细胞均低表达。③成熟树突状细胞培养基上清中IL-12水平高,而未成熟树突状细胞培养基上清中IL-12水平低。④成熟树突状细胞具有强的刺激T细胞增殖能力,而未成熟树突状细胞基本不具有诱导T细胞增殖能力。结论:未成熟状态的树突状细胞具备致耐受原性,可抑制T细胞的应答,而成熟状态的树突状细胞由于获得了免疫刺激潜能从而会对炎性刺激做出反应。     

氧化苦参碱对骨髓来源细胞生长的双向调节作用

目的:观察氧化苦参碱(oxymatrine,OMT)对骨髓来源细胞增殖的影响.方法:应用MTr比色法、流式细胞仪检测法、集落形成法和免疫细胞活性测定等方法,检测OMT对白血病细胞K562的抑制作用;骨髓造血干细胞的集落形成实验和脾细胞对肿瘤细胞的杀伤的生物活性试验,检测OMT对小鼠免疫和造血的影响.结果:(1)不同浓度的OMT可明显抑制白血病细胞K562细胞的增殖、集落形成,导致细胞凋亡(P＜0.05),且作用呈浓度依赖性.(2)OMT可以促进小鼠骨髓粒系造血,且在0.2475 mg/mL时达到峰值.(3)OMT可抑制小鼠的免疫功能.结论:OMT可抑制白血病K562细胞的增殖并诱导其凋亡,同时抑制小鼠的免疫功能,促进小鼠骨髓CFU-GM的形成,表现出对骨髓来源细胞生长的双向调节作用.     

Myogenic Reprogramming of Bone Marrow Derived Cells in a W41Dmdmdx Deficient Mouse Model

Lack of expression of dystrophin leads to degeneration of muscle fibers and infiltration of connective and adipose tissue. Cell transplantation therapy has been proposed as a treatment for intractable muscle degenerative disorders. Several reports have demonstrated the ability of bone-marrow derived cells (BMDC) to contribute to non-haematopoietic tissues including epithelium, heart, liver, skeletal muscle and brain following transplantation by means of fusion and reprogramming. A key issue is the extent to which fusion and reprogramming can occur in vivo, particularly under conditions of myogenic deterioration.To investigate the therapeutic potential of bone marrow transplantation in monogenetic myopathy, green fluorescent protein-positive (GFP⁺) bone marrow cells were transplanted into non-irradiated c-kit receptor – deficient (W⁴¹) mdx mice. This model allows BMDC reconstitution in the absence of irradiation induced myeloablation. We provide the first report of BMDC fusion in a W⁴¹/Dmd^mdx deficient mouse model.In the absence of irradiation induced injury, few GFP⁺ cardiomyocytes and muscle fibres were detected 24 weeks post BMT. It was expected that the frequency of fusion in the hearts of W⁴¹Dmd^mdx mice would be similar to frequencies observed in infarcted mice [1].Although, it is clear from this study that individual cardiomyocytes with monogenetic deficiencies can be rescued by fusion, it is as clear that in the absence of irradiation, the formation of stable and reprogrammed fusion hybrids occurs, with the current techniques, at very low levels in non-irradiated recipients.