摘要
目的:探讨在体外无需预先分离CD34+造血祖细胞即可有效诱导脐带血单个核细胞而获得脐带血树突状细胞的方法,并对人脐带血树突状细胞的生物学功能进行鉴定。方法:实验于2003-12/2004-10在吉林省肿瘤防治研究所完成。健康孕妇正常胎儿脐带血由吉林省妇幼保健院提供,孕妇签署知情同意书。无菌采集健康孕妇正常胎儿脐带血50mL左右,肝素抗凝。生理盐水稀释后加入甲基纤维素使终浓度为1g/L,室温沉降45min,取上层富含细胞的血浆层,1500r/min离心10min,弃上清,沉淀加两倍体积的生理盐水稀释,以1∶1体积叠加于淋巴细胞分层液上,分离出脐带血中的单个核细胞,体外培养24h后去除悬浮细胞,加入粒细胞-巨噬集落刺激因子、白细胞介素4联合刺激脐带血单个核细胞分化为脐带血树突状细胞。在光镜和透射电镜下观察培养的脐带血树突状细胞的形态学特征。碱性磷酸酶-抗碱性磷酸酶桥联法测量脐带血树突状细胞表面分子CD1α、人类白细胞抗原-DR的表达水平。四甲基偶氮唑蓝法测定不同细胞密度的树突状细胞刺激同种T淋巴细胞增殖的能力。结果:①不同诱导时间体外培养的脐带血树突状细胞形态学观察:倒置显微镜下,淋巴细胞经细胞因子诱导后第3d聚集成均匀散布的细胞聚体,部分细胞变形,胞体拉长;第7d可见不明显突起,细胞形态不规则,呈疏松贴壁生长或悬浮生长;14d集落样生长,具有典型的树突状细胞形态。透射电镜下可见树突状细胞呈不规则形,细胞表面粗糙,突起明显;细胞核呈肾形或马蹄形,核浆比增大;胞浆中富含线粒体,较少溶酶体。②细胞因子诱导前后脐血淋巴细胞表型的变化:诱导前脐带血淋巴细胞表面不表达CD1α,诱导后CD1α的表达上升为(22.00±4.36)%;人类白细胞抗原-DR由诱导前的(3.67±2.08)%表达上升为(61.67±7.02)%。③脐带血树突状细胞对T淋巴细胞增殖能力的影响:脐带血树突状细胞具有明显刺激同种T淋巴细胞增殖的功能,且其数量的不同对T淋巴细胞的刺激能力亦不同。脐带血树突状细胞与淋巴细胞比值为1∶10时平均刺激指数为3.95,比值为1∶100时平均刺激指数为2.16。结论:采取粒细胞-巨噬集落刺激因子、白细胞介素-4联合刺激可成功诱导出形态典型、纯度较高的脐带血树突状细胞,且体外培养后具有明显的刺激同种T淋巴细胞增殖的能力。
AIM： To investigate a method to effectively induce dendritic cells from human umbilical cord blood in vitro without isolating CD34^＋ hemopoietic progenitor beforehand, and identify the biological function. METHODS： The experiment was carried out in Jilin Research Institute of Tumor Prevention and Cure between December 2003 and October 2004. The normal fetal umbilical cord blood of healthy pregnant women was provided by Jilin Hospital of Women and Children＇s Health with the permission of the pregnant women. Normal fetal umbilical cord blood （about 50 mL） of healthy pregnant women was collected, and anticoagulated with heparin. The diluted saline was added by methylcellulose till the terminal concentration reached 1 g/L, sedimentated at room temperature for 45 minutes, and the plasma layer containing rich cells in the supematant was obtained, and centrifugated for 10 minutes （1 500 r per minute）, and then the supernatant was abandoned, deposited and then diluted with saline of double volume, and then overlapped to the lymphocyte demixing according to the volume of 1：1, menonuclear cells were isolated from umbilical cord blood, the suspended cells were eliminated after in vitro culture for 24 hours, and then granulecytemacrophage colony stimulating factor （GM-CSF） and interleukin-4 were added to together stimulate the umbilical cord blood mononuclear cells differentiating into dendritic ceils. The morphological characters of the cultured dendritic cells were observed under light microscope and transmission election microscope. The expressions of dendritic cells surface molecule CD1α and human leucocyte antigen-DR （HLA-DR） were detected with alkaline phosphatase-anti-alkaline phosphatase bridge method. The ability of dendritic cells of different density in stimulating proliferation of isotype T lymphocyte was detected with methyl thiazolyl tetrazolium （MTT） assay. RESULTS： ① Morphological observation of in vitro cultured dendritic cells from human umbilical cord blood at different induction time： Under inverted microscope, the lymphocyte aggregated into evenly scattered cell polymers at 3 days after induction, part of the cells were deformed, and the cell bodies were stretched; At 7 days, unobvious processes were obstrved, cell forms were irregular, and showed loose adherent or suspending growth; At 14 days, the cells grew like colony, and had typical forms as dendritic cells. Under transmission election microscope, the forms of dendritic cells were irregular, the cell surface was rough, and the progresses were obvious; The nuclei were kidney-like or horseshoe shape, and the ratio of karyoplasms was increased. There were rich mitochondria but less lysosome in cytoplasm. ② Changes of the phenotype of lymphoeyres from umbilical cord blood before and after cytokine induction： Before cytokine induction, there was no expression of CD1α on the surface of lymphocytes from umbilical cord blood; After induction, the expression of CD1α increased to （22.00±4.36）%, HLA-DR increased from （3.67±2.08）% to （61.67±7.02）%. ③Influence of dendritic cells from umbilical cord blood on the proliferative ability of T lymphocyte： Dendritic cells from umbilical cord blood had the obvious function of stimulating the proliferation of isotype T lymphocyte, and the different number had different stimulative ability. The average stimulative index was 3.95 when the ratio of dendritic cells to T lymphocyte from umbilical cord blood was 1：10, and it was 2.16 when the ratio was 1：100. CONCLUSION： The stimulation of GM-CSF together with interleukin-4 can successfully induce dendritic cells from umbilical cord blood of typical form and high purity, and have the obvious ability in stimulating the proliferation of isotype T lymphocyte after culture in vitro.
出处
《中国临床康复》
CSCD
北大核心
2006年第9期40-42,i0002,共4页
Chinese Journal of Clinical Rehabilitation
