不同浓度维生素D 刺激下体外滋养细胞的二代测序研究

  目的  利用二代测序技术筛选维生素D刺激后胎盘滋养细胞中差异表达基因（differential expression gene，DEG），分析其在维生素D缺乏引起的不良妊娠结局发病机制中可能发挥的潜在作用。  方法  通过二代测序分析不同浓度的维生素D（0.1、1、10、100 nmol/L）刺激细胞的mRNA表达情况，筛选出DEG，应用 GO 和 KEGG 进行富集分析，构建PPI网络筛选中枢基因。  结果  4种不同浓度维生素D刺激的细胞样本和未加维生素D刺激的正常对照样本的基因表达情况如下:VitD 0.1 vs Control组共获取了 354 个DEG，包括 242 个上调，112个下 调；VitD 1 vs Control组共获取了 320 个DEG，包括 216 个上调，104 个下调；VitD 10 vs Control组共获取了 374 个DEG，包括 277 个上调，97 个下调；VitD 100 vs Control组共获取了 300 个DEG，包括151个上调，149个下调。最后对4组DEG取交集，共获得 9个共同的DEG。GO分析显示，生物过程主要集中在胶原原纤维组织、细胞分化、细胞趋化、细胞分化的负调控、信号受体活性的调节、细胞外结构组织、细胞外基质的组织、蛋白质分泌，调节的细胞成分富集在胶原三聚体复合物、蛋白质的细胞外基质、细胞的顶端部分、胶原蛋白三聚物，调节的分子功能富集于受体配体活动、受体调节器活动、蛋白质桥接、细胞因子活性。KEGG分析显示在刺激神经组织的受体配体相互作用、细胞粘附分子、胃癌、细胞因子受体相互作用、产生IgA的肠道免疫网络、炎症性肠病、疟疾、阿米巴病和 PI3K-Akt 信号通路中显著富集。PPI网络确定17个中枢基因（COL1A2、ACTA2、S100A4、TAGLN、CSF1R、TLR4、TNFSF13B、FTCD、APOBEC3G、IL6、IGF1、PDGFRB、TGFB2、BGLAP、COL4A4、COL8A1和COL11A1）。  结论  COL1A2、ACTA2、S100A4、TAGLN、CSF1R、TLR4、TNFSF13B、FTCD、APOBEC3G、IL6、IGF1、PDGFRB、TGFB2、BGLAP、COL4A4、COL8A1、COL11A1可能与维生素D缺乏引起的不良妊娠结局的发生、发展相关，也可为维生素D缺乏引起的不良妊娠结局提供潜在的治疗靶点。

Second-generation Sequencing Study of Trophoblast Cells in vitro Stimulated by Different Concentrations of Vitamin D

  Objective  To screen differential expression gene （DEG） in placental trophoblasts stimulated by vitamin D by second-generation sequencing technology, and to analyze their potential role in the pathogenesis of adverse pregnancy outcomes caused by vitamin D deficiency.   Methods  The second-generation sequencing analysis was used to detect mRNA expression in placental trophoblasts stimulated by different concentrations of vitamin D （0.1, 1, 10, 100 nm） to screen out DEG, GO and KEGG enrichment analysis was applied, and PPI network gene screening center was constructed.   Results  Gene expression in four different concentrations of vitamin D stimulated cells of the samples and normal control samples without vitamin D are as follows: a total of 354 DEG were obtained in VitD 0.1 vs Control group, including 242 up-regulated and 112 down-regulated; a total of 320 DEG were obtained in VitD 1 vs Control group, including 216 up-regulated and 104 down-regulated; a total of 374 DEG were obtained in VitD 10 vs Control group, including 277 up-regulated and 97 down-regulated; a total of 300 DEG were obtained in VitD 100 vs Control group, including 151 up-regulated and 149 down-regulated. At last, the intersection of four groups of DEG was taken, and nine common DEGs were obtained. GO analysis showed that biological processes are mainly concentrated in the collagen fibrils tissue, cell differentiation, cell chemotaxis, cell differentiation, the regulation of the negative control, signal receptor activity, outside the cell structure, extracellular matrix organization, protein secretion, adjusting the enrichment of the cellular elements in collagen trimer compounds, proteins of the extracellular matrix, the top part of the cell and collagen trimer; Adjust the molecular function of enrichment in receptor ligands, receptor modulators, protein bridge, the activity of cytokines. KEGG analysis showed significant enrichment in stimulating nerve receptor-ligand interactions, cell adhesion molecules, gastric cancer, cell factor receptor interactions,produce IgA intestinal immune network, inflammatory bowel disease, malaria, amoebiasis and PI3K-Akt signaling pathway. The PPI network identified 17 hub genes, including COL1A2, ACTA2, S100A4, TAGLN, CSF1R, TLR4, TNFSF13B, FTCD, APOBEC3G, IL6, IGF1, PDGFRBTGFB2, BGLAP, COL4A4, COL8A1 and COL11A1.   Conclusions  COL1A2, ACTA2, S100A4, TAGLN, CSF1R, TLR4, TNFSF13B, FTCD, APOBEC3G, IL6, IGF1, PDGFRBTGFB2, BGLAP, COL4A4, COL8A1 and COL11A1 may be linked to vitamin D deficiency caused by the occurrence and development of adverse pregnancy outcomes, and provide potential therapeutic targets adverse pregnancy outcomes caused by vitamin D deficiency.