【作者】 王金丽；

【导师】 郭力；

【作者基本信息】 河北医科大学 ， 神经病学， 2012， 硕士

【摘要】 目的：树突状细胞（dendritic cells,DC）是迄今为止人类发现的唯一能够刺激初始T细胞的活化增殖和功能最强大的抗原提呈细胞（antigenpresenting cell,APC），其可介导抗原转移，在体内激活静息T细胞，启动特异性免疫应答。多发性硬化（muitiple sclerosis,MS）是主要以T细胞介导的中枢神经系统慢性炎症性脱髓鞘疾病，它的发病机制虽然仍未得到完全明确，但众多研究表明，MS的发生与T细胞异常活化有关，并且，MS的病理免疫应答与DC的参与有直接关系。基于DC的特性，近年来，将DC用于自身免疫性疾病的研究已得到深入开展，特别是将其应用于多发性硬化的治疗已成为目前的研究热点。因此，能够正确、高效的分离培养出DC并对其进行鉴定是运用DC开展下游实验的基础与前提。我们设计了本实验，旨在用rmGM-CSF、rmIL-4及TNF-α在体外诱导培养小鼠骨髓源成熟树突状细胞与免疫磁珠分离纯化相结合的方法获得DC并对DC从形态学观察、细胞表面分子的检测及刺激T细胞增值能力三方面进行鉴定，探讨及优化小鼠骨髓源成熟树突状细胞的分离培养、纯化及鉴定方法，为进一步研究DC在多发性硬化治疗方法中的应用提供实验基础。方法:1.骨髓源性成熟DC的分离及培养将Ｃ57BL/6小鼠用拉颈法处死后浸泡在75%乙醇中3-5分钟，无菌取出下肢骨，用注射器针头在长骨两端扎眼使髓腔联通，用注射器吸少量RPMI-1640培养液将骨髓冲出，用200目滤网过滤后加入红细胞裂解液溶解红细胞，RPMI-1640培养液洗涤细胞，然后用RPMI1640完全培养液（含10%胎牛血清、rmGM-CSF（10ng/ml）、rmIL-4(10ng/ml)）调整细胞浓度至106/ml，接种于6孔培养板中，将细胞培养板放入37oC、含5%CO2的培养箱中培养，第6天加入rmTNF-α(15ng/ml)至细胞培养板中并继续培养，至第7天收集细胞培养板中所有的悬浮细胞，此即为小鼠骨髓源性的成熟DC。2．CD11c免疫磁珠分离纯化DC：2.1使用CD11c免疫磁珠标记DC：对DC进行计数，重悬于缓冲液中。加入CD11c免疫磁珠，于2--8oC孵育15分钟后重悬于缓冲液中。2.2使用磁珠分选器分离DC：将LS柱放置在MiDi MACS磁珠分选器中，加入适量缓冲液润洗LS柱。将磁珠标记的细胞悬液放入分选柱中，收集流出物，这是未标记的阴性细胞。将分选柱从分选器上移下，加入5ml缓冲液，用配套活塞将标记细胞推出来，此即为纯化后的成熟DC。3.小鼠骨髓源成熟树突状细胞的鉴定3.1细胞形态学观察3.1.1在倒置荧光显微镜下对DC形态进行观察。3.1.2扫描电镜对DC形态进行观察并拍照。3.1.3透射电镜观察并拍照。3.2流式细胞仪检测细胞表面分子：收集细胞培养板中的悬浮细胞，经离心、洗涤、计数、定容、标记后分别加入抗体，室温避光孵育20分钟后离心、洗涤后使用流式细胞仪检测DC表面CD11c、CD80、CD86、MHCII分子的表达情况。3.3采用细胞增殖检测试剂（CCK-8）法测定单向混合淋巴细胞反应（one-way MLR）中的DC对同种异基因T细胞刺激增殖的能力：（1）取Ｃ57BL/6小鼠骨髓中的成熟DC作为刺激细胞，方法同前。（2）提取BALB/c小鼠脾脏中的T细胞作为反应细胞，并取部分T细胞做分选前流式检测（3）采用CD90免疫磁珠分离纯化T细胞。（4）流式细胞仪检测T细胞纯度。（5）DC与T细胞共培养：DC与T细胞的比例分别设定为1：5、1:10、1:20、1:40，共培养72小时，于培养结束前4小时加入CCK-8，每孔20μL，继续培养4小时并于加入CCK-8后的第2、3、4小时分别上酶联检测仪检测一次，记录吸光度值。结果:1.成熟DC的形态观察1.1倒置荧光显微镜观察：小鼠骨髓细胞经GM-CSF、rmIL-4诱导培养48小时可见大部分细胞贴壁生长，细胞形态大小不等，培养72小时后悬浮细胞较前增多，部分细胞形成集落。培养第6天可见悬浮细胞继续增多，并可见少量树突样突起。加入rmTNF-α培养24小时后细胞表面出现典型的树枝样突起，完全符合典型成熟DC的形态。1.2扫描电镜下观察：DC形态多样，结构完整，表面粗糙，好似覆盖着一层薄纱，细胞表面粗糙，有大量褶皱及树枝样突起，符合典型的成熟DC的形态。1.3透射电镜观察：DC胞体较大，形状不规则，细胞表面有较长的树枝状突起，长短不一，粗细不均；胞核形态多样，可见清晰的核膜；胞浆内细胞器丰富，可见大量的高尔基体、内质网、线粒体及核糖体，但囊泡状结构及溶酶体减少。2.流式细胞仪检测DC表面分子：小鼠骨髓源成熟树突状细胞高表达CD11c，纯度大于90%。细胞表面CD80（94.73%±1.45%）、CD86（96.43%±2.00%）、MHCII（94.13%±5.86%），均呈高表达，符合成熟DC的表达特征。3.DC刺激同种异基因T细胞增殖的能力：磁珠分选后的小鼠T细胞经流式检测其纯度大于90%，各小时组、各DC：T细胞比例的刺激指数均能说明DC有刺激同种异基因T细胞增殖的能力，但在加入CCK-8后的第2、3小时分别检测其各相邻DC：T细胞比例的刺激指数无统计学差异；第4小时DC刺激T细胞增殖的能力与DC所占比例呈正比，各组间差异有统计学意义（P0.05）。结论:1.本实验利用GM-CSF、IL-4及TNF-α体外诱导培养与免疫磁珠分离提纯相结合的方法成功培养并扩增出高纯度的成熟DC，利用此方法培养出来的成熟DC具有典型的树突状形态，数量大，纯度高。2.通过形态学观察、细胞表面因子流式学检测及单向混合淋巴细胞反应三方面对细胞进行了鉴定，证明其为成熟的DC。在测定单向混合淋巴细胞反应中DC对T细胞刺激增殖能力时，我们用CCK-8法替代了传统的MTT法，使得实验结果更加稳定、可靠，实验结果还证明利用CCK-8法最佳测定时间是在加入CCK-84小时后，且DC刺激T细胞增殖的能力与DC所占比例成正比。3.本实验对小鼠骨髓源成熟树突状细胞的体外分离培养、纯化及鉴定进行了探讨，为研究DC的功能及其在多发性硬化等免疫疾病治疗中的应用提供实验基础。更多还原

【Abstract】 Objective: Dendritic cells is the most powerful antigen presenting cells,it is so far the only one that can stimulate initial T cell activation andproliferation. It can be mediated antigen transfer, activated resting T cells inthe body, started the specific immune response. Multiple sclerosis is a chronicinflammatory disease of the central nervous system,it be mediated by T cells.Its pathogenesis has not yet had a clear,,but many researches show that MS isconnected with the abnormal activation of T cells, and,the pathologicalimmune response of MS and the participation of DC has a direct relation.Based on the characteristics of DC, in recent years, the research that used DCin autoimmune disease has been thorough development, especially itsapplication in the treatment of multiple sclerosis has become the hotspot.Therefore, to correct and efficient separate cultivate DC and carries on theappraisal is the foundation and premise of the DC downstream experimentWe have designed this experiment, aims to use the method that cultivatemature dendritic cells from mice bone marrow induced by external combiningseparation and purification with immune magnetic beads to obtain DC andfrom morphological observation, the cell surface molecular detection andstimulate the T cells appreciation ability to identify DC, explore and optimisethe method of separation、purification and appraisal of mice mature dendriticcells from bone marrow,for the further study for the application of DC inmultiple sclerosis treatment provide experimental basis.Methods:1The separation and cultivation of mature DC from mice bone marrowExcute C57BL/6mice by pull the neck, bathed in75%ethanol in3to5minutes, remove the femur sterile,prick both ends of the femoral medullarycavity with needles and unicom them, suck RPMI1640cultures with a syringe to rush out the bone marrow, filter and add the red blood cells cracking liquidto dissolve red blood cells, adjust cell concentration to106/ml by RPMI1640completely cultures（contain10%tire bovine serum, rmGM-CSFS (10ng/ml),rmIL-4(10ng/ml)）, vaccinate in six holes training board, train the cellculture plate in the incubator that contains37o C、5%of of CO2,add rmTNFalpha (15ng/ml) in the fixth day and continue to cultivate to the seventh day,collect all the cells suspended, that is the mice mature DC from bone marrow.2. Purification DC with CD11c immune magnetic beads2.1Mark DC with CD11c immune magnetic beads: Count to DC,suspended it with buffer liquid. Add CD11c immune magnetic beads to it,incubate15minutes in2-8o C and suspended it with buffer liquid one moretime2.2Separated DC by magnetic beads sorting device: placed the LScolumn in the MiDi MACS sorting device. Add buffer liquid into the LScolumn wash embellish LS column. Add the magnetic beads labeled cellssuspension liquid into sorting device, collect effluents, this is not labeled cells.Move up column from sorting device,add5ml buffer liquid, push the markcells out with the matching pistons.3. Identification of the mice mature dendritic cells from bone marrow3.1Cell morphology observation3.11Observe the morphologyofDC with convert fluorescence microscopy3.12Observed DC form with scanning electron microscopy (sem) andtake photos3.13Observed DC form with transmission electron microscope and takephotos3.2The detection of cell surface molecules by flow cytometric: Collectsuspended cell in the culture plate, after the centrifugal, washing, counting, setthe capacity and mark, add the antibody to the cells, incubate avoid light inroom temperature20minutes and centrifugal, washing, detect the molecularexpression of CD11c, CD80, CD86, MHCII in DC surface.3.3Detect the ability of proliferation that DC stimulate the linebreed gene T in one-way mixed lymphocyte responses (one-way MLR)with themethod of cell proliferation detection reagents (CCK-8):(1) Take mature DCfrom C57BL/6mice bone marrow as the stimulation cells.(2) Take T cellsfrom BALB/c mice spleen as the reaction cells, and detect a few T cells byflow cytometric before detection (3)Purify T cells with CD90magnetic beads.(4) Detect the purity of T cells by flow cytometric.(5) DC and T cells weretraining together:the percentage of DC and T cells were set to1：5、1:10、1:20、1:40, and training72hours, before the end of the training in four hours to addCCK-8, each hole20μ L, continue to cultivate4hours and to inspecte andrecord the absorbency value in each hour after add CCK-8by enzyme leaguedetector.Results:1The morphology of mDC observation1.1Observe the morphology of DC with convert fluorescencemicroscopy: Bone marrow cells in mice by gm-csf, rmIL-4induce and aftertraining48hours, most cell on the growth visible, the size and form of cellswere different, training after72hours cells suspended increased, some cellsform colony. The sixth day suspended cells continue to increase, and appear afew of dendritic sample bumps. Add rmTNF alpha into the cell and training24hours, cell surface appear typical branched bumps, accord with mature DCform.1.2Observe the morphologyof DC with scanning electron microscopy：the form of DC is various, and the structure is intact, the surface is rough, isseems to be covered with a layer of gauze，there are large branched bumps anddrape, accord with mature DC form.1.3Observe the morphology of DC with transmission electronmicroscope: DC body is large, cell shape is irregular, the cell surface has along dentrites, different length, uneven thickness., the surface has branchedbumps;nuclear membrane is clear, the organelles in cytoplasm are rich,obviously, a lot of golgi body, endoplasmic reticulum, mitochondria, andribosomes, but saclike structure and the lysosome reduced. 2. The detection of cell surface molecules by flow cytometric:In maturedendritic cells from mice bone marrow,CD11c is highly expressed, purity ismore than90%. The cell surface CD80(94.73%±1.45%), CD86(96.43%±2.00%), MHCII (94.13%±5.86%), are highly expressed, accord with matureDC’s express features.3. The proliferation of ability that DC stimulate linebreed gene T cell:After the separation of magnetic beads,T cells’s purity is more than90%bystreaming testing,each stimulus index of the proportion of DC: T cells canexplain that DC have the ability of proliferation which exciting linebreed geneT cells,but in2,3hours that after add CCK-8into cells,test the stimulationindex in different proportion of DC: T cells respectively,there was nostatistical difference; the proliferation of ability that DC stimulate T cell inproportion to the proportion of DC in the forth hour. The differences betweeneach group have statistical significance.(P0.05)Conclusion:1This experiment use the method that induce training by gm-csf、IL-4and TNF-αin vitro combine with purify DC by immune magnetic beads,Successful culture and the augment high purity mature DC, mature DCdeveloped using this method has typically dendritic form, quantity is big, highpurity.2Through three aspects cells appraisal:the morphological observation,the detection of cell surface molecules by flow cytometric and one-way mixedlymphocyte reaction, proved this DC is mature DC. In the determination ofproliferous ability that DC stimulate T cells by mixed lymphocyte responsesof one-way, we use CCK-8method has replaced traditional MTT method, andmakes the experimental results more stable, reliable, and the experimentalresults also show that best measured time use CCK-8is add CCK-84hourslater,and the proliferation of ability that DC stimulate T cell in proportion tothe proportion of DC in the forth hour.3This experiments discussed separation,training in vitro, purification andappraisal of mature dendritic cells from mice bone marrow,and for the study of DC and its application in treatment of multiple sclerosis to provideexperimental basis.更多还原