恒河猴骨髓来源未成熟树突状细胞的培养及鉴定

Culture and identification of immature dendritic cells from rhesus monkey myeloid

目的建立一种体外诱导恒河猴骨髓CD34+细胞分化为树突状细胞(DC)的方法,并对其细胞形态及表面标志进行鉴定。方法用免疫磁珠分选系统(MACS)分选恒河猴骨髓细胞,收集高纯度的CD34+造血干细胞;加入重组粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)及肿瘤坏死因子-α(TNF-α),诱导其分化为骨髓来源的树突状细胞,并采用电镜观察细胞形态。用树突状细胞表面标志CD1a及成熟树突状细胞(mDC)表面标志CD83流式抗体染色后分析。结果细胞因子诱导6d后成功培养出表面呈绒毛状和树枝状突起的典型DC样细胞,流式细胞术分析结果显示92.35%的细胞为DC,27.61%的细胞为m DC。结论用此方法可在体外培养出高纯度且典型的恒河猴骨髓源性未成熟树突状细胞(im DC),为进一步研究其生物学特性及功能奠定了基础。

Objective To develop a method for culturing dendritic cells （DC） from rhesus monkey bone marrow CD34 ＋ cells, and identify the cells by morphological observation and cell surface marker detection. Methods To screen rhesus monkey bone marrow cells using a magnetic activated cell sorting （MACS） system then to collect highly purified CD34 ＋ hematopoietic stem cells. Recombinant granulocyte macrophage colony stimulating factor （GM-CSF）, interleukin-4 （IL-4） and tumor necrosis factor-ct （TNF-ct） were applied to isolate CD34 ~ hematopoietic stem cells to induce bone marrow-derived DCs. Morphological change of cells was observed under an electron microscope, and cell surface molecules were detected using flow cytometry after cells were stained with DC surface marker CDla and ma- ture DC surface marker CD83. Results Typical DCs with surface villous and dendritic protrusions were identified 6 days after cytokine treatment. 92. 35% of the cultured cells were DC and 27.61% of the cultured cells were mature DCs （mDC）. Conclusions We have established a method for culturing highly purified rhesus monkey bone marrow- derived immature DCs （imDC）, which may function as a technine plateforrn to support research in future.