小鼠骨髓源成熟树突状细胞的分离培养、纯化及鉴定
|
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。
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 experiment
We 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 marrow
Excute 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 beads
2.1Mark 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 moretime
2.2Separated 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 marrow
3.1Cell morphology observation
3.11Observe the morphologyofDC with convert fluorescence microscopy
3.12Observed DC form with scanning electron microscopy (sem) andtake photos
3.13Observed DC form with transmission electron microscope and takephotos
3.2The 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 observation
1.1Observe 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.
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。
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 experiment
We 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 marrow
Excute 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 beads
2.1Mark 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 moretime
2.2Separated 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 marrow
3.1Cell morphology observation
3.11Observe the morphologyofDC with convert fluorescence microscopy
3.12Observed DC form with scanning electron microscopy (sem) andtake photos
3.13Observed DC form with transmission electron microscope and takephotos
3.2The 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 observation
1.1Observe 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.
引文
1Walunas TL, Lenschow DC, Baker CY, et al. CTLA4can function asnegative regulator of T cell activation[J]. Immunity,1994,1(5):405-413
2Ralph M, Steinman, Ianvil A, Cohn. Pillars article: Identification of anovel cell type in peripheral lymphoid organs of mice. I Morpholog Q-uantitation, Tissue distribution[J].J Immunol,2007,178(1):5-25
3Chauvin C, Josien R. Dendritic cells as killers: mechanistic aspects andpo-tential roles[J]. J Immunol,2008,181(1):11-16
4Compston A, Coles A. Multiple sclerosis [J]. Lancet,2008,372(96-
48):1502-1517
5Pashenkov M, Teleshova N, Kouw enhoven M, et al. Elevted expressionof ccr5by myeloid (CD11c)blood dendritic cells in multiple sclerosis andacute optic neuritis[J]. Clin Exp. Immunol,2002,127(3):519-526
6Malerod L, Sporstol M, Juwet LK, et al. Hepatic scavenger receptor cl-ass B, type I is stimulated by peroxisome proliferator activated receptor-or gamma and hepatocyte nuclear factor4alpha[J]. Biochem Biophys ResComm-un,2003,305(3):557-565
7LuttonJD,Winston R,RodmartTC.Muitiple sclerosis:etio-logical mechani-sms and future directions[J].Fxp Biol Med(Maywood),2004,229(1):12-20
8Stinissen P,Raus J,Zhang J.Autoimmune pathogenesis of multiple scle-rosis:role of autoreactive T lymphocytes and new immunotherapeuticstrate-gies [J].Crit Rev Immunol,1997,17(1):33-75
9Medzhitov R,Janeway CA,Innate immunity:impact on the adaptive immu-ne response[J].Curr Opin Immunol,1997,9(1):49
10Kivisakk P, Mahad DJ, Callahan MK. et al. Experession of ccr7in mul-tiple sclerosis in plications for CNS immunity [J]. Ann Neurol,2004,55(5):627-638
11Inaba K, Inaba M, Romani N, et al. Generation of large numbers ofdendritic cells from mouse bone marrow cultures supplemented withgranu-locyte macrophage colony-stimulating factor [J]. Exp Med1992,
176:1693
12Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen bycultured human dendritic cells is maintained by granulocyte macrophagecolony-stimulating factor plus inter-leukin-4and downregulated by tumornecrosis factora [J]. Exp Med,1994,179:1109
13Lutz MB, Kukutsch N, Ogilvie AL, et al. An advanced culture methodfor generating large quantities of highly pure dendritic cells from mousebone marrow [J]. J Immunol Methods,1999,223:77-92
14Son YI, Egawa S, Tatsumi T, et al. A novel bulk-cuture method for gene-rating mature dendritic cells from mouse bone marrow cells [J]. ImmunolMethods,2002,262:145-157
15Van Voorhis WC, Hair LS, Steinman RM, et al. Human dendritic cells.Enrichment and characteriztation from peripheral blood [J]. Exp Med
1982,155(4):1172-1187
16Li L, Liu D, Hutt-Fletcher L, et al. Epstein-Barr virus inhibits the deve-lopment of dendritic cells by promoting apoptosis of their monocyteprecursors in the presence of granulocyte macrophage colony stimulatingfactor and interleukin-4. Blood,2002,99(10):3725-3734
17Lin CL, Suri RM, Rahdon RA, et al. Dendritic cell chemotaxis andtransendothelial migration are induced by distinct chemokines and are re-gulated on maturation. Eur J Immunol,1998,28(12):4114-4122
18Lutz MB, Schnare M, Menges M, et al. Differential functions of IL-4receptor type I and II for dendritic cell maturation and IL-2production andtheir dependency on GM-CSF [J]. Immunol,2002,169(7):3574-3580
19童华生．细胞因子对树突状细胞体外培养的影响．江西医学检验，2002，20（２）：102-104
20Oki M, Ando K, Hagihara M, et al. Efficient Ientiviral transduction ofhuman cord blood CD34(+) cells followed by their expansion anddifferenti-ation into dendritic cells. Exp Hematol,2001,29(10):1210-1217
21Curti A, Fogli M, Ratta M, et al. Stem cell factor and FLT3-ligand arestrictly required to sustain the long-term expansion of primitive CD34(+)DR-dendritic cell precursors [J]. Immunol,2001,166(2):848-854
22Pang L, Ye W, Che XM, et al. Reduction of inflammatory response in themouse brain with adenoviral-mediated transforming growth factor-sslexpression. Stroke,2001,32(2):544-552
23宗传龙，周白云，李亚鲁．小鼠骨髓树突状细胞体外扩增方法的探索[J]．中国临床康复，2004，8（19）：3900-3901
24Jacobsen SE, Ruscetti FW, Dubois CM, et al. Tumor necrosis actora-directly and indirectly regulates hematopoietic progenitor cell prolifer-ation: role of colony-stimulating factor receptor modulation[J].Exp Med,
1992,175(6):1795-1772
25Chen X, Regn S, Raffegerst S, et al. Interferonal pha in combecationwith GM-CSF induces the differentnation of leukaentic antigen presentingcells that have the capacity to stimulate a special anti-leukaemic eytotoxicT-cell response from pauents with chroic myeloid leukaemia. Br JHaematol,2000,111(2):596-607
26田垒，陈栋炜，季业伟，等．抗原浓度和DC数量对OT-I小鼠CD8+T细胞分化与增殖的影响[J]．西北农林科技大学学报（自然科学版），2009，37（9）：8-12
27曹雪涛．免疫学前沿进展[M]．北京：人民卫生出版社，2009：68-69
28Liu Q, Shu X, Sun A, et al. Plant-derived small molecule albacondsuppresses LPS triggered proinflammatory cytokine production andantigen presentation of dendritic cells by impairing NF-kB activation[J].Int Immuno-pharmacol,2008,8:1103-1111
29Xu H, Chen T, Wang HQ, et al. Combination treatment with donorinter-leukin-12P35silenced dendritic cells and cyclosporine induceslong-term survival of intestinal allografts in rats[J].TransplantProc,2007,39(1):286-288
30Ardavin C. Origin, precursors and differentiation of mouse dendriticcells[J]. Nat Rev Immunol,2003,3(7):582-590
31Coventry BJ, Austyn JM, Chryssidis S, et al. Indentification and isolationof CD1a positive putative tumour infitrating deneritic cells in humanbreast cancer. Adv Exp Med Biol,1997,417(1):571-577
32熊建文，肖化，张镇西． MTT法与CCK-8法检测细胞活性之测试条件比较[J]．激光生物学报，2007，16（5）:12-15
33曹维，季玟君，邢春燕．胰岛素B9-23多肽对树突状细胞表型及功能的影响[J]．南京医科大学学报（自然科学版），2010，30（7）：914-918
1贾建平．神经疾病诊疗学．[M]．北京：人民军医出版社，2006，237
2王维治．神经病学．[M]．北京：人民卫生出版社，2006，1136-1140
3Bettelli E, Pagany M， Weiner HL, et al. Myelin oligodendrocyteglycoprotein-specific T cell receptor transgenic mice develop spontaneousautommune optic neuritis[J].JExp Med,2003,197(9):1073-1081
4Mosmann TR, Cherwinski H, Bond MW, et al. Two types ofmurine helperT cell clone. I. Definition according to profiles of lymphokine activitiesand secreted proteins.J Immunol,1986,136(7):2348-2357
5Curti A, Pandolfi S, AluigiM, et al. I nterleukin-12p roduction byleukemia derived dendritic cells counteracts the inhibitory effect ofleukemic micro environment on T cells[J]. Exp Hematol,2005,33(12):1521-1530
6Comabella M, Montalban X, Münz C, Lünemann JD. Targeting dendriticcells to treat multiple sclerosis. Nat Rev Neurol.2010Sep;6(9):499-507
7Mahnke K, Johns on TS, Ring S, et al. Toler ogenic dendritic cells andregulatory T cells: A t wo way regulationship [J]. J Dermat ol Sci,2007,
46(3):159-167
8Xiao BG, ZhuWH, Lu CZ. The presence of GM-CSF and IL-4interfereswith effect of TGF-betal on antigen p resenting cells in patientswithmultip le sclerosis and in ratswith experimental autoi mmune encephalomyelitis[J]. Cell I mmunol,2007,249(1):30-36
9Veldhoen M, Hocking RJ, Atkins CJ, et al. BTGFbeta in the context of aninflammatory cytokine milieu supports denovo differentiation of IL-17-producing T cells [J]. Immunity,2006;24:179-189
10Ishizu T, Osoegawa M, Mei FJ, et al. Intrathecal activation of the IL-17/IL-8axis in opticospinal multiple sclerosis.Brain,2005,128(5):988-1002
11Brucklacher-Waldert V, Stuerner K, KolsterM, et al. Phenotypical andfunctional characterization of T helper17cells in multiple sclerosis. Brain,2009,132(12):3329-3341
12Piconese S, Costanza M, Tripodo C, Sangaletti S, Musio S, Pittoni P,Poliani PL, Burocchi A, Passafaro AL, Gorzanelli A, Vitali C, Chiodoni C,Barnaba V, Pedotti R, Colombo MP.J Autoimmun.2011Sep28
13Banchereau J, Steinman RM. Dendritic cells and the control of immunity[J].Nature,1998,392:245-252
14袁婷婷，刘艳荣．树突状细胞生物学研究进展[J]．中国实验血液学杂志，2010，8(4)：1074-1078
15Park H，Li Z, Yang XO, et al. A distinct lineage of CD4T cells regulatestissue inflammation by producing interleukin17[J]. NatImmunol.2005:6(11):1133-1141
16Torres-Aguilar H, Blank M, Kivity S, Misgav M, Luboshitz J, PierangeliSS, Shoenfeld Y.Ann Rheum Dis.2011Sep12
17Prechtel AT, Steinkasser A. CD83: an update on function and prospects ofthe maturation maker of dendritic cells[J]. Arch Dermatol Res,2007，
299(2):59-69
18Cools N, Van Tendeloo VF, Smits EL, et al. Immunosuppressioninduced by immature dendritic cells ismediated by TGF-beta/IL-10doublepositive CD4+regulatory T cells [J]. Cell Mol Med,2008,12(2):690-700
19Varol C，Vallon-Eberhard A，Elinav E，et al．Intestinal lamina propriadendritic cell subsets have different origin and functions［J］．Immunity，2009，31(3):502-512
20Isaksson M，Ardesj B，Rnnblom L，et al．Plasmacytoid DC promotepriming of autoimmune Th17cells and EAE［J］．Eur J Immunol，2009,39(10):2925-2935
21郭晓强，周强，冯志霞．树突细胞的发现者—斯坦曼[J]．生物学通报，2009，44(2)：60-62
22Weninger W, von Andrian U H. Chemokine regulation ofnative T celltraffic in healt h and disease [J]. Semin Immunol,2003,15(5):2572270.
23Banchereau J,Ppasual V,Palucka AK. Autoimmunity through cytokine-induced dendritic cell activation[J].Immunity,2004,26(4):539-550
24Jonuleit H, Schmitt E, Schuler G, et al. Induction of interleukin10producing, nonproliferating CD4(+) T cells with regulatory properties byrepetitive stimulation with allogeneic immature human dendritic cells. JExp-Med,2000,192(9):1213-1222
25Sharma MD，Hou DY，Liu Y，et al.Indoleamine2,3dioxygenasecontrolsconversion of Foxp3+Tregs to TH17like cells in tumordraining lymphnodes［J］.Blood,2009,113(24):6102-6111
26Morelli AE, Thomson AW. Tolerogenic dendritic cells and the questfortransplant tolerance [J]. Nat. Rev. Immunol.2007,7:610-621
27Increased frequency of Regulatory T cells and T lymphocyte activation inpersons with previously treated extrapulmonary Tuberculosis.de AlmeidaAS, Fiske CT, Sterling TR, Kalams SA.Clin Vaccine Immunol.2011Oct28
28Wing JB, Sakaguchi S.Eur J Immunol.2011Nov;41(11):3097-100
29Andersson J, Stefanova I, Stephens GL, et al. CD4+CD25+regulatory Tcellsare activated in vivo by recognition of self [J]. Int Immuno,2007,19(4):557-566
30MiyaraM, Sakaguchi S. Natural regulatory T cells:mechanis of supper-ssionn [J]. Trends Mol Med,2007,13(3):108-116
31Keller AM，Schildknecht A，Xiao Y，et al． Expression of costimulatoryligand CD70on steady-state dendritic cells breaks CD8+T cell toleranceand permits effective immunity［J],Immunity,2008,29(6):934-946
32孙景武，孙世波．树突状细胞与移植免疫耐受[J]．实用医学杂志，2005，
21(5)：546-548
33Wang L，Pino-Lagos K，de Vries VC，et al．programmed death l ligandSignaling regulates the generation of adaptive FOXP3-CD4-regulatory Tcells.Proc Natl Acad Sci USA,2008,105(27):9331-9336
34Wiesemann E，Deb M，Trebst C，et al．Effects of interferon-beta on co-signaling molecules： upregulation of CD40， CD86and PD-L2onmonocytes in relation to clinical response to interferon-beta treatment inpatients with multiple sclero sis［J］.Mult Scler,2008,14(2):166-176
35Pashenkov M, Teleshova N, Link H. Inflammation in the central nervoussystem: the role for dendritic cells. Brain Pathol,2003,13(1):38-51
36Serafini B, Rosicarelli B, Magliozzi R, et al. Dendritic cell inmultiplesclerosis lesions: maturation stage, myelinuptake, and interaction with pro-liferating T cells[J]. J Neuropathol Exp Neuro,l2006,65(2):124-141
37Ifergan I, Kebir H, Dodelet Devillers A, et al. The blood brain barrierinduces differentiation of migrating monocytes into Th17-polarizingdendritic cells[J]. Brain,2008,131(3):785-799
38罗秋云，许南燕，邹琼辉．多发性硬化患者血清TNF-α、IFN-γ、IL-10检测及临床意义[J]．实用临床医学，2008，9(3)；10-11
39阎晓波，凌云阳，孟若娟，等．树突状细胞在多发性硬化免疫机制治疗中作用的研究[J]．哈尔滨医科大学学报，2010，44(2)：121-125
40梁军利，唐玉兰．多发性硬化树突状细胞分泌细胞因子的检测与意义[J]．实用医学杂志，2011，27(2)：218-220
41Yao C，Sakata D，Esaki Y，et al．Prostaglandin E2-EP4signaling promotesimmune inflammation through TH1cell differentiation and TH17cell.Expan-sion［J］.Nat Medicine,2009,15(6):633-640
42Li Y，Chu N，Hu A，et al．Increased IL-23p19expression inmultiplesclerosis lesions and its induction in microglia［J］．Brain,2007,130(2):4-490-501
43Vlad G, Cortesini R, Suciu Foca N. License to heal: bidirectionalinteraction of antigens pecific regulatory T cells and toler ogenic APC [J].J I mmunol,2005,174(10):5907-5914
44Hirata S, Matsuyoshi H, Fukuma D, et al. Involvement of regulat ory Tcells in the experimental autoimmune encephalomyelitis preventive effectof dendritic cells exp ressing myelin oligodendrocyte glycop rotein plusTRA IL [J]. J I mmunol,2007,178(2):918-925
45Rossi M, Young JW. Human dendritic cells: potent antigen presenting cellsat the crossroads of innate and adaptive immunity [J]. Immunol,2005，175:1373-1381
46Mellergard J，Edstrm M，Vrethem M，et al．Natalizumab treatment inmultiple sclerosis:marked decline of chemokines and cytokines incerebro-spinal fluid［J］.Multiple Sclerosis,2010,16(2):208-217
47Langrish CL, Chen Y, Blumenschein WM, et al. IL-23drives a patho-genicT cell population that induces autoimmune inflammation [J]. ExpMe-d,2005,201(2):233-240
48Li J, Menzel C, Meier DA, et al. Comparative study of different vectordesigns for the mammalian expression of recombinant IgG antibodies[J].Immunol Methods.2007Jan10,318(1-2):113-124
49Liu Z, Xu X, Hsu HC, et al. CII-DC-AdTRAIL cell gene therapy inhibit sinfilt ration of CII-reactive T cells and CII-induced arthritis[J]. J ClinInvest,2003,112(9):1332-1341
50Forster R, Davalos-MisslitzA C, RotA. CCR7and its ligands: balancingimmunity and tolerance [J]. Nat Rev I mmunol,2008,8(5):362-371.
51辛海明，彭毅志，郭浩，等．第三军医大学学报[J]．2009，31(17)，1629-1632
52Littman DR, Rudensky A. Th17and regulatory T Cells in mediating andrestraining inflammation. Cell,2010,140(6):845-58
53Mucida D, Park Y, Kim G, et al. Reciprocal TH17and regulatory T celldifferentiation mediated by retinoic acid. Science,2007,317(5835):256-60
54Laurence A, Tato CM, Davidson TS, et al. Interleukin-2signaling viaSTAT5constrainsT helper17cell generation. Immunity,2007,26(3):371-81
2Ralph M, Steinman, Ianvil A, Cohn. Pillars article: Identification of anovel cell type in peripheral lymphoid organs of mice. I Morpholog Q-uantitation, Tissue distribution[J].J Immunol,2007,178(1):5-25
3Chauvin C, Josien R. Dendritic cells as killers: mechanistic aspects andpo-tential roles[J]. J Immunol,2008,181(1):11-16
4Compston A, Coles A. Multiple sclerosis [J]. Lancet,2008,372(96-
48):1502-1517
5Pashenkov M, Teleshova N, Kouw enhoven M, et al. Elevted expressionof ccr5by myeloid (CD11c)blood dendritic cells in multiple sclerosis andacute optic neuritis[J]. Clin Exp. Immunol,2002,127(3):519-526
6Malerod L, Sporstol M, Juwet LK, et al. Hepatic scavenger receptor cl-ass B, type I is stimulated by peroxisome proliferator activated receptor-or gamma and hepatocyte nuclear factor4alpha[J]. Biochem Biophys ResComm-un,2003,305(3):557-565
7LuttonJD,Winston R,RodmartTC.Muitiple sclerosis:etio-logical mechani-sms and future directions[J].Fxp Biol Med(Maywood),2004,229(1):12-20
8Stinissen P,Raus J,Zhang J.Autoimmune pathogenesis of multiple scle-rosis:role of autoreactive T lymphocytes and new immunotherapeuticstrate-gies [J].Crit Rev Immunol,1997,17(1):33-75
9Medzhitov R,Janeway CA,Innate immunity:impact on the adaptive immu-ne response[J].Curr Opin Immunol,1997,9(1):49
10Kivisakk P, Mahad DJ, Callahan MK. et al. Experession of ccr7in mul-tiple sclerosis in plications for CNS immunity [J]. Ann Neurol,2004,55(5):627-638
11Inaba K, Inaba M, Romani N, et al. Generation of large numbers ofdendritic cells from mouse bone marrow cultures supplemented withgranu-locyte macrophage colony-stimulating factor [J]. Exp Med1992,
176:1693
12Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen bycultured human dendritic cells is maintained by granulocyte macrophagecolony-stimulating factor plus inter-leukin-4and downregulated by tumornecrosis factora [J]. Exp Med,1994,179:1109
13Lutz MB, Kukutsch N, Ogilvie AL, et al. An advanced culture methodfor generating large quantities of highly pure dendritic cells from mousebone marrow [J]. J Immunol Methods,1999,223:77-92
14Son YI, Egawa S, Tatsumi T, et al. A novel bulk-cuture method for gene-rating mature dendritic cells from mouse bone marrow cells [J]. ImmunolMethods,2002,262:145-157
15Van Voorhis WC, Hair LS, Steinman RM, et al. Human dendritic cells.Enrichment and characteriztation from peripheral blood [J]. Exp Med
1982,155(4):1172-1187
16Li L, Liu D, Hutt-Fletcher L, et al. Epstein-Barr virus inhibits the deve-lopment of dendritic cells by promoting apoptosis of their monocyteprecursors in the presence of granulocyte macrophage colony stimulatingfactor and interleukin-4. Blood,2002,99(10):3725-3734
17Lin CL, Suri RM, Rahdon RA, et al. Dendritic cell chemotaxis andtransendothelial migration are induced by distinct chemokines and are re-gulated on maturation. Eur J Immunol,1998,28(12):4114-4122
18Lutz MB, Schnare M, Menges M, et al. Differential functions of IL-4receptor type I and II for dendritic cell maturation and IL-2production andtheir dependency on GM-CSF [J]. Immunol,2002,169(7):3574-3580
19童华生．细胞因子对树突状细胞体外培养的影响．江西医学检验，2002，20（２）：102-104
20Oki M, Ando K, Hagihara M, et al. Efficient Ientiviral transduction ofhuman cord blood CD34(+) cells followed by their expansion anddifferenti-ation into dendritic cells. Exp Hematol,2001,29(10):1210-1217
21Curti A, Fogli M, Ratta M, et al. Stem cell factor and FLT3-ligand arestrictly required to sustain the long-term expansion of primitive CD34(+)DR-dendritic cell precursors [J]. Immunol,2001,166(2):848-854
22Pang L, Ye W, Che XM, et al. Reduction of inflammatory response in themouse brain with adenoviral-mediated transforming growth factor-sslexpression. Stroke,2001,32(2):544-552
23宗传龙，周白云，李亚鲁．小鼠骨髓树突状细胞体外扩增方法的探索[J]．中国临床康复，2004，8（19）：3900-3901
24Jacobsen SE, Ruscetti FW, Dubois CM, et al. Tumor necrosis actora-directly and indirectly regulates hematopoietic progenitor cell prolifer-ation: role of colony-stimulating factor receptor modulation[J].Exp Med,
1992,175(6):1795-1772
25Chen X, Regn S, Raffegerst S, et al. Interferonal pha in combecationwith GM-CSF induces the differentnation of leukaentic antigen presentingcells that have the capacity to stimulate a special anti-leukaemic eytotoxicT-cell response from pauents with chroic myeloid leukaemia. Br JHaematol,2000,111(2):596-607
26田垒，陈栋炜，季业伟，等．抗原浓度和DC数量对OT-I小鼠CD8+T细胞分化与增殖的影响[J]．西北农林科技大学学报（自然科学版），2009，37（9）：8-12
27曹雪涛．免疫学前沿进展[M]．北京：人民卫生出版社，2009：68-69
28Liu Q, Shu X, Sun A, et al. Plant-derived small molecule albacondsuppresses LPS triggered proinflammatory cytokine production andantigen presentation of dendritic cells by impairing NF-kB activation[J].Int Immuno-pharmacol,2008,8:1103-1111
29Xu H, Chen T, Wang HQ, et al. Combination treatment with donorinter-leukin-12P35silenced dendritic cells and cyclosporine induceslong-term survival of intestinal allografts in rats[J].TransplantProc,2007,39(1):286-288
30Ardavin C. Origin, precursors and differentiation of mouse dendriticcells[J]. Nat Rev Immunol,2003,3(7):582-590
31Coventry BJ, Austyn JM, Chryssidis S, et al. Indentification and isolationof CD1a positive putative tumour infitrating deneritic cells in humanbreast cancer. Adv Exp Med Biol,1997,417(1):571-577
32熊建文，肖化，张镇西． MTT法与CCK-8法检测细胞活性之测试条件比较[J]．激光生物学报，2007，16（5）:12-15
33曹维，季玟君，邢春燕．胰岛素B9-23多肽对树突状细胞表型及功能的影响[J]．南京医科大学学报（自然科学版），2010，30（7）：914-918
1贾建平．神经疾病诊疗学．[M]．北京：人民军医出版社，2006，237
2王维治．神经病学．[M]．北京：人民卫生出版社，2006，1136-1140
3Bettelli E, Pagany M， Weiner HL, et al. Myelin oligodendrocyteglycoprotein-specific T cell receptor transgenic mice develop spontaneousautommune optic neuritis[J].JExp Med,2003,197(9):1073-1081
4Mosmann TR, Cherwinski H, Bond MW, et al. Two types ofmurine helperT cell clone. I. Definition according to profiles of lymphokine activitiesand secreted proteins.J Immunol,1986,136(7):2348-2357
5Curti A, Pandolfi S, AluigiM, et al. I nterleukin-12p roduction byleukemia derived dendritic cells counteracts the inhibitory effect ofleukemic micro environment on T cells[J]. Exp Hematol,2005,33(12):1521-1530
6Comabella M, Montalban X, Münz C, Lünemann JD. Targeting dendriticcells to treat multiple sclerosis. Nat Rev Neurol.2010Sep;6(9):499-507
7Mahnke K, Johns on TS, Ring S, et al. Toler ogenic dendritic cells andregulatory T cells: A t wo way regulationship [J]. J Dermat ol Sci,2007,
46(3):159-167
8Xiao BG, ZhuWH, Lu CZ. The presence of GM-CSF and IL-4interfereswith effect of TGF-betal on antigen p resenting cells in patientswithmultip le sclerosis and in ratswith experimental autoi mmune encephalomyelitis[J]. Cell I mmunol,2007,249(1):30-36
9Veldhoen M, Hocking RJ, Atkins CJ, et al. BTGFbeta in the context of aninflammatory cytokine milieu supports denovo differentiation of IL-17-producing T cells [J]. Immunity,2006;24:179-189
10Ishizu T, Osoegawa M, Mei FJ, et al. Intrathecal activation of the IL-17/IL-8axis in opticospinal multiple sclerosis.Brain,2005,128(5):988-1002
11Brucklacher-Waldert V, Stuerner K, KolsterM, et al. Phenotypical andfunctional characterization of T helper17cells in multiple sclerosis. Brain,2009,132(12):3329-3341
12Piconese S, Costanza M, Tripodo C, Sangaletti S, Musio S, Pittoni P,Poliani PL, Burocchi A, Passafaro AL, Gorzanelli A, Vitali C, Chiodoni C,Barnaba V, Pedotti R, Colombo MP.J Autoimmun.2011Sep28
13Banchereau J, Steinman RM. Dendritic cells and the control of immunity[J].Nature,1998,392:245-252
14袁婷婷，刘艳荣．树突状细胞生物学研究进展[J]．中国实验血液学杂志，2010，8(4)：1074-1078
15Park H，Li Z, Yang XO, et al. A distinct lineage of CD4T cells regulatestissue inflammation by producing interleukin17[J]. NatImmunol.2005:6(11):1133-1141
16Torres-Aguilar H, Blank M, Kivity S, Misgav M, Luboshitz J, PierangeliSS, Shoenfeld Y.Ann Rheum Dis.2011Sep12
17Prechtel AT, Steinkasser A. CD83: an update on function and prospects ofthe maturation maker of dendritic cells[J]. Arch Dermatol Res,2007，
299(2):59-69
18Cools N, Van Tendeloo VF, Smits EL, et al. Immunosuppressioninduced by immature dendritic cells ismediated by TGF-beta/IL-10doublepositive CD4+regulatory T cells [J]. Cell Mol Med,2008,12(2):690-700
19Varol C，Vallon-Eberhard A，Elinav E，et al．Intestinal lamina propriadendritic cell subsets have different origin and functions［J］．Immunity，2009，31(3):502-512
20Isaksson M，Ardesj B，Rnnblom L，et al．Plasmacytoid DC promotepriming of autoimmune Th17cells and EAE［J］．Eur J Immunol，2009,39(10):2925-2935
21郭晓强，周强，冯志霞．树突细胞的发现者—斯坦曼[J]．生物学通报，2009，44(2)：60-62
22Weninger W, von Andrian U H. Chemokine regulation ofnative T celltraffic in healt h and disease [J]. Semin Immunol,2003,15(5):2572270.
23Banchereau J,Ppasual V,Palucka AK. Autoimmunity through cytokine-induced dendritic cell activation[J].Immunity,2004,26(4):539-550
24Jonuleit H, Schmitt E, Schuler G, et al. Induction of interleukin10producing, nonproliferating CD4(+) T cells with regulatory properties byrepetitive stimulation with allogeneic immature human dendritic cells. JExp-Med,2000,192(9):1213-1222
25Sharma MD，Hou DY，Liu Y，et al.Indoleamine2,3dioxygenasecontrolsconversion of Foxp3+Tregs to TH17like cells in tumordraining lymphnodes［J］.Blood,2009,113(24):6102-6111
26Morelli AE, Thomson AW. Tolerogenic dendritic cells and the questfortransplant tolerance [J]. Nat. Rev. Immunol.2007,7:610-621
27Increased frequency of Regulatory T cells and T lymphocyte activation inpersons with previously treated extrapulmonary Tuberculosis.de AlmeidaAS, Fiske CT, Sterling TR, Kalams SA.Clin Vaccine Immunol.2011Oct28
28Wing JB, Sakaguchi S.Eur J Immunol.2011Nov;41(11):3097-100
29Andersson J, Stefanova I, Stephens GL, et al. CD4+CD25+regulatory Tcellsare activated in vivo by recognition of self [J]. Int Immuno,2007,19(4):557-566
30MiyaraM, Sakaguchi S. Natural regulatory T cells:mechanis of supper-ssionn [J]. Trends Mol Med,2007,13(3):108-116
31Keller AM，Schildknecht A，Xiao Y，et al． Expression of costimulatoryligand CD70on steady-state dendritic cells breaks CD8+T cell toleranceand permits effective immunity［J],Immunity,2008,29(6):934-946
32孙景武，孙世波．树突状细胞与移植免疫耐受[J]．实用医学杂志，2005，
21(5)：546-548
33Wang L，Pino-Lagos K，de Vries VC，et al．programmed death l ligandSignaling regulates the generation of adaptive FOXP3-CD4-regulatory Tcells.Proc Natl Acad Sci USA,2008,105(27):9331-9336
34Wiesemann E，Deb M，Trebst C，et al．Effects of interferon-beta on co-signaling molecules： upregulation of CD40， CD86and PD-L2onmonocytes in relation to clinical response to interferon-beta treatment inpatients with multiple sclero sis［J］.Mult Scler,2008,14(2):166-176
35Pashenkov M, Teleshova N, Link H. Inflammation in the central nervoussystem: the role for dendritic cells. Brain Pathol,2003,13(1):38-51
36Serafini B, Rosicarelli B, Magliozzi R, et al. Dendritic cell inmultiplesclerosis lesions: maturation stage, myelinuptake, and interaction with pro-liferating T cells[J]. J Neuropathol Exp Neuro,l2006,65(2):124-141
37Ifergan I, Kebir H, Dodelet Devillers A, et al. The blood brain barrierinduces differentiation of migrating monocytes into Th17-polarizingdendritic cells[J]. Brain,2008,131(3):785-799
38罗秋云，许南燕，邹琼辉．多发性硬化患者血清TNF-α、IFN-γ、IL-10检测及临床意义[J]．实用临床医学，2008，9(3)；10-11
39阎晓波，凌云阳，孟若娟，等．树突状细胞在多发性硬化免疫机制治疗中作用的研究[J]．哈尔滨医科大学学报，2010，44(2)：121-125
40梁军利，唐玉兰．多发性硬化树突状细胞分泌细胞因子的检测与意义[J]．实用医学杂志，2011，27(2)：218-220
41Yao C，Sakata D，Esaki Y，et al．Prostaglandin E2-EP4signaling promotesimmune inflammation through TH1cell differentiation and TH17cell.Expan-sion［J］.Nat Medicine,2009,15(6):633-640
42Li Y，Chu N，Hu A，et al．Increased IL-23p19expression inmultiplesclerosis lesions and its induction in microglia［J］．Brain,2007,130(2):4-490-501
43Vlad G, Cortesini R, Suciu Foca N. License to heal: bidirectionalinteraction of antigens pecific regulatory T cells and toler ogenic APC [J].J I mmunol,2005,174(10):5907-5914
44Hirata S, Matsuyoshi H, Fukuma D, et al. Involvement of regulat ory Tcells in the experimental autoimmune encephalomyelitis preventive effectof dendritic cells exp ressing myelin oligodendrocyte glycop rotein plusTRA IL [J]. J I mmunol,2007,178(2):918-925
45Rossi M, Young JW. Human dendritic cells: potent antigen presenting cellsat the crossroads of innate and adaptive immunity [J]. Immunol,2005，175:1373-1381
46Mellergard J，Edstrm M，Vrethem M，et al．Natalizumab treatment inmultiple sclerosis:marked decline of chemokines and cytokines incerebro-spinal fluid［J］.Multiple Sclerosis,2010,16(2):208-217
47Langrish CL, Chen Y, Blumenschein WM, et al. IL-23drives a patho-genicT cell population that induces autoimmune inflammation [J]. ExpMe-d,2005,201(2):233-240
48Li J, Menzel C, Meier DA, et al. Comparative study of different vectordesigns for the mammalian expression of recombinant IgG antibodies[J].Immunol Methods.2007Jan10,318(1-2):113-124
49Liu Z, Xu X, Hsu HC, et al. CII-DC-AdTRAIL cell gene therapy inhibit sinfilt ration of CII-reactive T cells and CII-induced arthritis[J]. J ClinInvest,2003,112(9):1332-1341
50Forster R, Davalos-MisslitzA C, RotA. CCR7and its ligands: balancingimmunity and tolerance [J]. Nat Rev I mmunol,2008,8(5):362-371.
51辛海明，彭毅志，郭浩，等．第三军医大学学报[J]．2009，31(17)，1629-1632
52Littman DR, Rudensky A. Th17and regulatory T Cells in mediating andrestraining inflammation. Cell,2010,140(6):845-58
53Mucida D, Park Y, Kim G, et al. Reciprocal TH17and regulatory T celldifferentiation mediated by retinoic acid. Science,2007,317(5835):256-60
54Laurence A, Tato CM, Davidson TS, et al. Interleukin-2signaling viaSTAT5constrainsT helper17cell generation. Immunity,2007,26(3):371-81