Qian Xue-Qian, Chen Li-Li, Cheng Qi, Tian Yang, Luo Xiao-Feng, Wan Xiao-Yun
Women's Hospital, School of Medicine, Zhejiang University, Xueshi Road 1#, Hangzhou, China.
Department of Gynecologic Oncology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
BMC Immunol. 2016 Jun 4;17(1):14. doi: 10.1186/s12865-016-0150-3.
Previous evidence suggested that the differentiation of Lin-CD45RA-DC precursors were prior to plasmcytoid dendritic cells (pDCs) than myeloid dendritic cells (mDCs) within ovarian cancer microenvironment. However, the mechanism is still unclear. Therefore, we investigated the function of Notch 1 signal pathway in the differentiation of Lin-CD45RA-DC precursors.
The CD34+ hematopoietic stem cells were extracted from umbilical cord blood in term parturition, and Lin-CD45RA-DC precusors were separated and induced mature. Expression of Notch1 receptor and ligands in Lin-CD45RA-DC precusors was detected by Real-time PCR and was down-regulated by shRNA or γ-secretase inhibitor (GSI). Flow cytometry was used to analyze the subset of DCs with or without SKOV3 culture supernatants. IL-12 level was detected by ELISA.
Expression of Notch1 receptors and ligands were detected in Lin-CD45RA-DC precursor cells. The Notch1 mRNA in Lin-CD45RA-DC precursors can be down-regulated by shRNA-Notch1 lentivirus transfection and GSI. ShRNA mediated Notch 1 knock-down significantly differentiated less plasmcytoid dendritic cells (pDCs), but generated more myeloid dendritic cells (mDCs), and this would not be influenced by the supernatant of the ovarian carcinoma cell line. GSI had the same effect in the differentiation of pDC. The secretion of IL-12 significantly increased after Notch1 knock-down with or without SKOV3 culture supernatants.
Notch1 is an important signaling pathway in the differentiation of Lin-CD45RA-DC precursor cells to plasmcytoid dendritic cells (pDCs). And this would not be affected by the supernatant of the ovarian carcinoma cell line.
先前的证据表明，在卵巢癌微环境中，Lin-CD45RA-DC前体细胞向浆细胞样树突状细胞（pDC）的分化早于向髓样树突状细胞（mDC）的分化。然而，其机制仍不清楚。因此，我们研究了Notch 1信号通路在Lin-CD45RA-DC前体细胞分化中的作用。
从足月分娩的脐带血中提取CD34+造血干细胞，分离并诱导Lin-CD45RA-DC前体细胞成熟。通过实时PCR检测Lin-CD45RA-DC前体细胞中Notch1受体和配体的表达，并用短发夹RNA（shRNA）或γ-分泌酶抑制剂（GSI）下调其表达。采用流式细胞术分析有无SKOV3培养上清液时DC的亚群。通过酶联免疫吸附测定（ELISA）检测白细胞介素-12（IL-12）水平。
在Lin-CD45RA-DC前体细胞中检测到Notch1受体和配体的表达。shRNA-Notch1慢病毒转染和GSI可下调Lin-CD45RA-DC前体细胞中的Notch1 mRNA。shRNA介导的Notch 1基因敲低显著减少了浆细胞样树突状细胞（pDC）的分化，但产生了更多的髓样树突状细胞（mDC），且这不受卵巢癌细胞系上清液的影响。GSI在pDC的分化中具有相同的作用。无论有无SKOV3培养上清液，Notch1基因敲低后IL-12的分泌均显著增加。
Notch1是Lin-CD45RA-DC前体细胞向浆细胞样树突状细胞（pDC）分化的重要信号通路。且这不受卵巢癌细胞系上清液的影响。
