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目的:分析未成熟树突状细胞(immature dendritic cells,imDCs)的免疫学功能,诱导同种异体复合组织移植局部免疫耐受性。方法:采用细胞因子诱导培养黏附法,取大鼠骨髓前体细胞,粒细胞-巨噬细胞集落刺激因子联合白细胞介素-4细胞因子诱导下培养7 d,收集贴壁细胞光学显微镜观察imDCs的形态学特征,用质量分数2%锥虫蓝染色检测活性细胞率;应用流式细胞仪分析imDCs表面标志分子;MTT比色法测定imDCs与同种异体T淋巴细胞混合培养后T淋巴细胞的增殖能力。结果:制备诱导培养的imDCs具有形态不规则、表面粗糙层叠状皱褶、胞质突起短而多、树枝样分叉明显等典型形态特征;活性细胞率高达93%;表面较特异标志分子OX62为(61.08±4.86)%,OX62阳性细胞表达CD80、CD86、组织相容性复合体-Ⅱ表达率分别为(3.56±0.73)%、(5.38±1.09)%、(6.45±0.74)%,均为较低表达;imDCs对同种异体T淋巴细胞基本无刺激增殖能力。结论:用细胞因子诱导培养黏附法取骨髓前体细胞,可较快分选出活性和纯度较高、数量足够的imDCs,为同种异体复合组织移植诱导免疫耐受防止排斥反应的研究奠定良好的基础。
OBJECTIVE: To analyze the immunological function of immature dendritic cells (imDCs) and induce local immune tolerance in allograft tissues. Methods: Adopting cytokine-induced culture adhesion method, bone marrow progenitor cells, granulocyte-macrophage colony-stimulating factor and interleukin-4 cytokines were cultured for 7 days. The adherent cells were collected to observe the expression of imDCs Morphological characteristics, the percentage of active cells was detected by trypan blue staining with 2% mass fraction; surface marker molecules of imDCs were analyzed by flow cytometry; T lymphocyte proliferation was measured by MTT colorimetric method after mixed culture of imDCs with allogeneic T lymphocytes ability. RESULTS: The imDCs prepared by induction culture had typical morphological features such as irregular shape, superficial rugged folds, short and large cytoplasm, and obvious dendritic branches. The rate of active cells was as high as 93%. The specific surface marker OX62 was (61.08 ± 4.86)% respectively. The expression rates of CD80 and CD86 in OX62 positive cells were (3.56 ± 0.73)%, (5.38 ± 1.09)% and (6.45 ± 0.74)% Lower expression of imDCs on allogeneic T lymphocytes basically no stimulation of proliferation. CONCLUSIONS: Bone marrow progenitor cells can be sorted out by cytokine-induced culture adhesion method, and imDCs with high activity and high purity can be sorted out more quickly, and the research of immunological tolerance and rejection prevention induced by allogeneic composite tissue transplantation is well established Foundation.