mRNA 仅通过使用脂质膜包封在细胞内递送来诱导滋养细胞中 RANTES 的产生，而 TLR3 则是必需的。

mRNA induces RANTES production in trophoblast cells via TLR3 only when delivered intracellularly using lipid membrane encapsulation.

机构信息

University of British Columbia, Critical Care Research Laboratories, Heart + Lung Institute, St. Paul's Hospital, Vancouver, BC, Canada.

出版信息

Placenta. 2011 Jul;32(7):500-5. doi: 10.1016/j.placenta.2011.04.011. Epub 2011 May 4.

DOI:10.1016/j.placenta.2011.04.011

PMID:21546084

Abstract

BACKGROUND

Trophoblasts express Toll-like receptor 3 (TLR3). The artificial TLR3 ligand, PolyI:C, induces an inflammatory response in trophoblasts but an endogenous ligand has not been identified. Notably, inflammatory disorders of pregnancy are associated with increased circulating placenta-derived mRNA. Endogenous degraded, uncapped mRNA is recognized by TLR3 in other cell lines.

OBJECTIVE

We tested the hypothesis that plasma-derived mRNA induces an inflammatory response in a trophoblast cell line via TLR3.

METHODS

Experiments were performed in the human first trimester extravillous trophoblast cell line HTR-8/SV neo. Plasma-derived mRNA was amplified using modified template switching and final in vitro transcription. We compared free mRNA (which favors cell surface interaction) to liposomally encapsulated mRNA (which favors intracellular mRNA delivery). We tested for the specific requirement of TLR3 signaling using siRNA. We tested for involvement of the canonical signaling pathway downstream of TLR3 by measuring NF-κB and IFN regulatory factor transcriptional activity using firefly-luciferase constructs.

RESULTS

Free mRNA did not induce RANTES production. In contrast, liposomal mRNA resulted in marked induction of RANTES production (non-stimulated control 3.4 ± 0.6 pg/mL, liposomal mRNA 169.7 ± 26.2 pg/mL, p < 0.001), and this RANTES production was abolished by siRNA for TLR3. Downstream of TLR3, liposomal mRNA-induced dose-response NF-κB and IFN regulatory factor transcriptional activity, and IFN beta production.

CONCLUSION

Plasma-derived 5' uncapped mRNA delivered intracellularly signals to induce NF-κB activation and increase RANTES production via TLR3.

摘要

背景

滋养层细胞表达 Toll 样受体 3（TLR3）。人工 TLR3 配体聚肌苷酸：聚胞苷酸（PolyI:C）可诱导滋养层细胞发生炎症反应，但尚未鉴定出内源性配体。值得注意的是，妊娠炎症性疾病与循环胎盘来源的 mRNA 增加有关。在其他细胞系中，内源性降解、无帽 mRNA 可被 TLR3 识别。

目的

我们通过 TLR3 检测了血浆来源的 mRNA 是否会诱导滋养层细胞系发生炎症反应的假说。

方法

在人早孕绒毛外滋养层细胞系 HTR-8/SV neo 中进行实验。使用改良模板转换和最终体外转录扩增血浆来源的 mRNA。我们比较了游离 mRNA（有利于细胞表面相互作用）和脂质体包裹的 mRNA（有利于细胞内 mRNA 传递）。我们使用 siRNA 检测 TLR3 信号的特异性要求。我们通过使用萤火虫荧光素酶构建体测量 NF-κB 和 IFN 调节因子转录活性，检测 TLR3 下游的经典信号通路参与情况。

结果

游离 mRNA 不会诱导 RANTES 产生。相比之下，脂质体 mRNA 导致 RANTES 产生明显增加（非刺激对照 3.4±0.6 pg/mL，脂质体 mRNA 169.7±26.2 pg/mL，p<0.001），且 TLR3 的 siRNA 可消除 RANTES 产生。TLR3 下游，脂质体 mRNA 诱导 NF-κB 和 IFN 调节因子转录活性及 IFNβ产生的剂量反应。