小鼠骨髓源性树突细胞的体外优化培养及其生物学特性鉴定

In vitro culture of bone marrow-derived dendritic cells and the identification of their biological characteristics in the mice

目的对小鼠骨髓源性树突状细胞(DCs)的体外培养方法进行优化。方法从小鼠获取骨髓细胞,用含因子的完全培养基于37℃5%CO2培养箱中培养,3 h后弃去上清,加入相同因子浓度的完全培养基继续培养,48 h收集上清细胞离心后种于6孔板中以上培养基继续培养,96 h后换液处理,第8天时添加LPS刺激作为LPS刺激组,未添加LPS作为对照组,结合流式细胞仪(FCM)、酶联免疫吸附技术(ELISA)等从细胞形态、表面分子[B7-2分子(CD86)]及主要组织相容性复合体Ⅱ(MHCⅡ)的特异性表达、相关因子[白细胞介素-12(IL-12)、白细胞介素-10(IL-10)]分泌及诱导T淋巴细胞的增殖能力等进行鉴定。结果镜下观察细胞有明显的不规则枝状突起,有典型DCs形态特征。流式结果显示细胞纯度为77.4%左右;经LPS刺激后树突细胞CD86、MHCⅡ的比例均明显增加(P<0.001);ELISA结果显示与对照组相比,LPS刺激组IL-12、IL-10的含量明显增加(P<0.01),IL-12的表达量增加约18倍,IL-10的表达量增加约2倍。MLR混合淋巴细胞反应结果显示经LPS刺激后LPS刺激组细胞诱导T淋巴增殖能力显著增强(P<0.05)。结论树突细胞体外原代培养48 h后,选用上清细胞继续培养可以获得纯度较高性能较好的DCs。

Objective To optimize the method of in vitro culture of bone marrow-derived dendritic cells(DCs）in the mice. Methods The bone marrow cells were collected from the mice and cultured in the 5% CO2 incubator at 37℃. The supernatant was removed 3 h later and the complete medium of the same factor concentration was added to keep on the culture. In 48 h,the supernatant was collected. After centrifugation,the cells were cultured in the 6-hole plate. The solution changed in 96 h. On the 8 thday,LPS stimulation group was set up by adding LPS and those without adding LPS were taken as the control group. The identification was conducted in light of the cellular morphology,surface molecule such as B7-2 molecule(CD86）,the specificity expressions of MHC II with FCM and ELISA,the secretion of relevant factors such as IL-12 and IL-10 as well as the proliferation capacity of the induced T cells. Results Under microscopic observation,the irregular dendritic protrusions were seen obviously,indicating the typical morphologic characteristics of DCs. The FCM showed that cell purity was about 77. 4%. After LPS stimulation,the proportions of CD86 and MHC II of DCs were increased remarkably(P 〈 0. 001）. ELISA showed that the levels of IL-12 and IL-10 in the LPS stimulation group were increased obviously as compared with the control group(P 〈 0. 01）. The expression of IL-12 was increased by about 18 times and IL-10 by about 2 times. The results of MLR mixed T cell reaction indicated that the proliferation capacity of the induced T cells was intensified significantly in the LPS stimulation group(P 〈 0. 05）. Conclusion After 48 h in vitro primary culture of DCs,the continuous culture with the supernatant obtains DCs of the high purity and performance.