来源骨髓的树突状细胞培养方法特征及其合理时间

Culture method and reasonable culture time of dendritic cells obtained from bone marrow

目的:观察从骨髓来源的免疫辅佐细胞树突状细胞的培养操作技术及合理的培养时间。方法:实验于2003-10/2004-12在卫生部细胞移植重点实验室进行。树突细胞来源为健康SD大鼠的骨髓细胞,分别加入4种细胞因子:重组大鼠粒细胞-巨噬细胞集落刺激因子5μg/L,重组大鼠白细胞介素45μg/L,重组大鼠肿瘤坏死因子α10μg/L,重组大鼠γ干扰素20μg/L,共培养2周。每隔2d加细胞因子1次,保持各细胞因子的浓度不变,分离树突细胞采用“培养黏附法”。在培养的第3天吸去上清后重新加入培养液,在培养2周后收获悬浮的树突状细胞,分离树突细胞采用“培养黏附法”。在培养的第1,3,5,7,9,11,13,15天用光学倒置显微镜观察细胞的形态;取一部分细胞在培养的第7,11,13,15天行流式细胞仪检查,并用PE标记的抗大鼠CD86单克隆抗体检测它的成熟度(CD86单抗阳性为成熟)。结果:①树突状细胞的形态:从培养第7天开始细胞周围开始逐渐伸出突起,第13天以后突起五六支左右,且长度逐渐缩短,成熟的树突状细胞伸出长短不等的突起,类似神经细胞的树突。②培养所得的树突状细胞数量:1只大鼠平均得到(1.18±0.21)×107。③树突状细胞的成熟度:培养第7,11,13,15天CD86单抗的阳性率分别为30%,80%,92%,94%。结论:应用骨髓来源的树突状细胞,在两种常规的细胞因子粒细胞-巨噬细胞集落刺激因子和白细胞介素4基础上加入肿瘤坏死因子α和γ干扰素,培养2周,应用培养黏附法分离树突状细胞,可得到成熟的树突状细胞,且数量较多。

AIM: To observe the culture operation technique and reasonable culture time of immunity accessory cell dendritic cells (DCs) obtained from bone marrow. METHODS: The experiment was conducted in Cell Transplantation Key Laboratory of Ministry of Health from October 2003 to December 2004. DC was isolated from myelocyte of health SD rats, and added with four kinds of cytokine: recombination rat granulocytic cell-histocyte colony stimulating factor (CSF) 5 μg/L, recombination rat interleukin 45 μg/L,recombination rat tumor necrosis factor alpha 10 μg/L and recombination rat gamma interferon 20 μg/L cultured for 2 weeks. Every 3 days cell factors were added once, and concentration of cell factors were unchanged. DC was separated by cultured sticky method. At the third day of culture added in culture medium after sucking supernatant fluid. Suspending DC was gained 2 weeks later by cultured sticky method. Form of cells was photolog invert microscope at the 1st,3rd,5th,7th,9th,11th,13th and 15th days. Some cells were detected by flow cytometer at the 7th ,11th ,13th ,15th days. Maturity degree was detected by rat CD86 monoclonal antibody tagged PE (CD86 mono anti-positive was maturity.). RESULTS: ① Form of dendrite cells: Apophysis began to stretch out around cells at the 7th day during culturing. Apophysis stretched out about five or six and the length became shorter gradually. The mature DC stretched out different length dendrites, and it was similar to the dendrite of nerve cell. ② Quantity of DC gained from culture: The quantity of DC of one rat was averagely(1.18±0.21)×107. ③ Maturity degree of DC: CD86 mono anti-positive rate at culturing 7th,11th,13th and 15th days was respectively 30%,80%,92% and 94%. CONCLUSION: DC obtained from bone marrow added with tumor necrosis factor alpha and gamma interferon on the basis of two kinds of regular cell factors granulocytic cell-histocyte CSF and interleukin 4 is cultured for 2 weeks and isolated by using cultured sticky method to obtain mature DC with a large amount.