合体滋养细胞微泡对树突状细胞免疫功能影响实验研究

Effects of syncytiotrophoblast extracellular vesicle on immunologic function of dendritic cells

目的研究小鼠合体滋养细胞微泡(STBM)对骨髓源性树突状细胞(BMDCs)免疫功能的影响。方法 2015年于第三军医大学大坪医院野战外科研究所妇产科中心,机械分离法将小鼠胎盘剪碎,采用差速离心法制备合体滋养细胞微泡。添加重组粒细胞巨噬细胞集落刺激因子(rm GM-csf)、重组白细胞介素-4(IL-4)培养骨髓源性树突状细胞,6 d后将其分为阴性对照组和实验组1、2、3、4以及阳性对照组。实验组1、2、3、4分别加入终浓度为1.0、1.5、2.0、2.5 mg/L的STBM,阴性对照组加等量的磷酸盐缓冲液(PBS),阳性对照组加入2.0 mg/L的脂多糖(LPS),继续培养2 d。流式细胞学技术(FCM)检测树突状细胞表面标志物CD11c及CD86的表达情况。同时ELISA法检测不同浓度STBM诱导下树突状细胞(dendritic cells,DCs)分泌肿瘤坏死因子-α(TNF-α),干扰素-γ(IFN-γ)及IL-12的含量。结果不同浓度STBM处理后的BMDCs明显上调了CD11c、CD86的表达,其中1.0、1.5、2.0、2.5 mg/L的STBM诱导DCs的CD11c/CD86双阳性表达率分别为46.1%、56.9%、60.5%、88.9%,与PBS组(18.2%)相比,差异具有统计学意义(P<0.05)。不同浓度的STBM刺激DCs后产生的TNF-α[(6.91±1.37)、(7.98±1.55)、(9.40±0.93)、(12.65±1.62)ng/L],IFN-γ[(12.14±1.39)、(17.52±1.67)ng/L]及IL-12[(6.98±1.19)、(7.88±0.10)、(9.18±1.69)ng/L]分别与各PBS组[(3.61±0.64)、(3.15±0.90)、(3.51±1.09)ng/L]相比,差异有统计学意义(P<0.05)。结论 STBM促进DCs的成熟,激活母体系统性炎症反应,可能是引起子痫前期发生重要原因。

dObjective To explore the effects of syncytiotrophoblast extracellular vesicle（STBM） on immunologic func- tion of murine derived dendritic cells（BMDCs）. Methods After breaking up the placenta mechanically, STBM were sep- arated by differential centrifugation in Obsterics and Gynecology Center, Daping Hospital, Research Institute of Field Surgery, Third Military Medical University in 2015. Mononuclear cells（MNCs） isolated from murine bone marrow were cultured in RPMI1640 medium containing rmGM-CSF and rmIL-4 for 6 d, and were divided into six groups. MNCs were stimulated for 2 d with 1.0, 1.5, 2.0, 2.5 mg/L STBM, 2 mg/L LPS（positive control） or PBS（negative control）. The expres- sion of CD1 lc and CD86 on DCs surface was analyzed by the fluorescence activated cell sorting（FCM）. ELISA was used to detect the concentrations of TNF-cdFN-7 and IL-12. Results The STBM-treated BMCs expressed high levels of CDllc and CD86. The positive expression rates of CDllc/CD86 induced by DCs in 1, 1.5, 2 and 2.5 STBM were 46.1%, 56.9%, 60.5% and 88.9%, and compared with the PBS group （18.2%）, the difference was statistically significant （P 〈0.05）.Different concentrations of STBM stimulated TNF- a（6.91 ± 1.37,7.98 ± 1.55,9.40 ± 0.93,12.65 ± 1.62）ng/L, IFN- a（12.14± 1.39,17.52 ±1.67）ng/L and IL- 12（6.98 ± 1.19,7.88±0.10,9.18 ± 1.69）ng/L produced by DCs, and compared with the PBS group（3.61 ±0.64 ,3.15 ± 0.90, 3.51 ± 1.09）ng/L, the differentce was statistically signifi- cant （P〈0.05）. Conclusions STBM can promote the maturation of DCs, increase its immune activity and enhance the systemic inflammatory response, which may be an important cause of PE.