Department of Obstetrics and Gynecology,Affiliated Hospital of Xuzhou Medical University.
Department of Obstetrics and Gynecology,Affiliated Hospital of Xuzhou Medical University.
Eur J Obstet Gynecol Reprod Biol. 2019 Jun;237:209-214. doi: 10.1016/j.ejogrb.2018.12.006. Epub 2018 Dec 14.
To investigate the expression of TLR4 in human early pregnancy decidual stromal cells (DSCs) induced by lipopolysaccharide (LPS) and its effect on the peripheral blood regulatory T (Treg) cells subgroup in women of childbearing age.
Isolating and cultivating normal human early pregnancy DSCs followed by treatment with 0, 25, 50, 100 and 200 ng/ml LPS, and the expression level of TLR4 mRNA was detected by RT-PCR. After 3 or 4 generation we divide the DSCs into 5 groups: ①Control group: Cultivation of peripheral blood lymphocyte (PBLC); ②Co-cultivation group: Co-cultivation of PBLC and DSCs; ③LPS stimulation group: PBLC + DSCs + LPS; ④PDTC blocking-up group: PBLC + DSCs + LPS + PDTC; ⑤TLR4 blocking-up group: PBLC + DSCs + LPS + TLR4mAb. In ①-④ groups, western blot was used to detect the expression of inhibitory factor-κB (IκB-α) protein and RT-PCR was used to detect the expression of FoxP3 mRNA. In ①-⑤ groups, flow cytometry was applied to detect the percentage of Treg cells subgroup.
The purity of primary cultured DSCs was more than 95%. RT-PCR results showed that the expression level of TLR4 mRNA increased gradually with the augment of LPS concentration. Western blot and RT-PCR showed that the expression of IκBα protein and FoxP3 mRNA in the other 3 groups was significantly higher than that in the control group (P < 0.05), and the expression of IκBα protein and FoxP3 mRNA in LPS stimulation group was lower than that in the co-cultivation group (P < 0.05). Compared with the LPS stimulation group, the expression of IκBα protein and FoxP3 mRNA in PDTC blocking-up group was higher than that in the LPS stimulation group (P < 0.05), but still lower than the co-cultivation group (P < 0.05). The proportion of Treg cells in the other 4 groups detected by flow cytometry was significantly higher than that in the control group (P < 0.05). Compared with the co-cultivation group, the Treg cells ratio of the LPS stimulation group was significantly decreased (P < 0.05). The proportions of Treg cells in PDTC blocking-up group and TLR4 blocking-up group were higher than that in the LPS stimulation group, but still lower than that in the co-cultivation group (P < 0.05). There was no significant difference between the PDTC blocking-up group and the TLR4 blocking-up group (P > 0.05).
Human early pregnancy DSCs can promote the differentiation of Treg cells. LPS can stimulate the expression of TLR4 in early pregnancy DSCs and decrease the proportion of Treg cells in PBLC, with NF-κB signaling pathway being the potential underlying mechanisms.
探讨脂多糖（LPS）诱导人早孕蜕膜基质细胞（DSC）中 TLR4 的表达及其对育龄妇女外周血调节性 T（Treg）细胞亚群的影响。
分离培养正常早孕人 DSC，分别用 0、25、50、100 和 200ng/ml LPS 处理，采用 RT-PCR 检测 TLR4 mRNA 表达水平。传代至第 3 或 4 代时，将 DSCs 分为 5 组：①对照组：培养外周血淋巴细胞（PBLC）；②共培养组：PBLC 和 DSCs 共培养；③LPS 刺激组：PBLC+DSCs+LPS；④PDTC 阻断组：PBLC+DSCs+LPS+PDTC；⑤TLR4 阻断组：PBLC+DSCs+LPS+TLR4mAb。在①-④组中，采用 Western blot 检测抑制因子-κB（IκB-α）蛋白的表达，采用 RT-PCR 检测 FoxP3 mRNA 的表达。在①-⑤组中，采用流式细胞术检测 Treg 细胞亚群的比例。
原代培养的 DSCs 纯度均大于 95%。RT-PCR 结果显示，TLR4 mRNA 的表达水平随 LPS 浓度的增加而逐渐升高。Western blot 和 RT-PCR 结果显示，与对照组相比，其他 3 组的 IκBα 蛋白和 FoxP3 mRNA 的表达均明显升高（P<0.05），LPS 刺激组的 IκBα 蛋白和 FoxP3 mRNA 表达均低于共培养组（P<0.05）。与 LPS 刺激组相比，PDTC 阻断组的 IκBα 蛋白和 FoxP3 mRNA 表达均升高（P<0.05），但仍低于共培养组（P<0.05）。流式细胞术检测的其他 4 组 Treg 细胞比例均明显高于对照组（P<0.05）。与共培养组相比，LPS 刺激组 Treg 细胞比例明显降低（P<0.05）。PDTC 阻断组和 TLR4 阻断组的 Treg 细胞比例均高于 LPS 刺激组，但仍低于共培养组（P<0.05）。PDTC 阻断组与 TLR4 阻断组之间差异无统计学意义（P>0.05）。
人早孕 DSCs 可促进 Treg 细胞的分化。LPS 可刺激早孕 DSCs 中 TLR4 的表达，降低 PBLC 中 Treg 细胞的比例，其潜在机制可能与 NF-κB 信号通路有关。
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