中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (26): 4101-4106.doi: 10.12307/2022.809

• 组织构建细胞学实验 cytology experiments in tissue construction •    下一篇

维生素D缺乏模型小鼠子宫转录组的测序分析

陈  霞1，商宇伟1，王林晓2，史轶超1，刘慧君1，孙慧婷1   

  1. 南京医科大学附属常州市第二人民医院，1生殖中心，2神经疾病研究室，江苏省常州市   213003
  • 收稿日期:2021-05-12 接受日期:2021-07-14 出版日期:2022-09-18 发布日期:2022-03-08
  • 通讯作者: 孙慧婷，硕士，主治医师，南京医科大学附属常州市第二人民医院生殖中心，江苏省常州市 213003
  • 作者简介:陈霞，女，1988年生，江苏省常州市人，汉族，2014年南京医科大学毕业，硕士，助理研究员，主要从事生殖医学方面的研究。
  • 基金资助:
    常州市科技局应用基础研究计划 (CJ20200103)，项目负责人：孙慧婷；常州市科技计划资助项目(CJ20210110)，项目负责人：陈霞；国家自然科学基金青年基金项目(81803498)，项目负责人：王林晓

Transcriptome sequencing of the uterine in a mouse model of vitamin D deficiency

Chen Xia1, Shang Yuwei1, Wang Linxiao2, Shi Yichao1, Liu Huijun1, Sun Huiting1   

  1. 1Department of Reproductive Center, 2Laboratory of Neurological Diseases, Changzhou Second People’s Hospital Affiliated to Nanjing Medical University, Changzhou 213003, Jiangsu Province, China
  • Received:2021-05-12 Accepted:2021-07-14 Online:2022-09-18 Published:2022-03-08
  • Contact: Sun Huiting, Master, Attending physician, Department of Reproductive Center, Changzhou Second People’s Hospital Affiliated to Nanjing Medical University, Changzhou 213003, Jiangsu Province, China
  • About author:Chen Xia, Master, Assistant researcher, Department of Reproductive Center, Changzhou Second People’s Hospital Affiliated to Nanjing Medical University, Changzhou 213003, Jiangsu Province, China
  • Supported by:
    Changzhou Sci & Tech Program, No. CJ20200103 (to SHT); Changzhou Sci & Tech Program, No. CJ20210110 (to CX); the National Natural Science Foundation of China (Youth Fund), No. 81803498 (to WLX)

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摘要：

文题释义：
胰岛素抵抗：是指各种原因使胰岛素促进葡萄糖摄取和利用的效率下降，机体代偿性的分泌过多胰岛素产生高胰岛素血症，以维持血糖的稳定。胰岛素抵抗易导致代谢综合征和2型糖尿病，这也是多囊卵巢综合征患者常见的内分泌紊乱疾病，多种研究证实，胰岛素抵抗女性患者可引起高雄激素血症。
转录组测序：是以基因功能及结构研究的基础和出发点，通过新一代高通量测序，全面快速地获得某一物种特定组织或器官在某一状态下转录出来的所有RNA的总和，主要包括mRNA和非编码RNA。转录组研究能够从整体水平研究基因功能以及基因结构，揭示特定的生物学过程。

背景：近来发现维生素D受体广泛分布在卵巢、子宫等女性生殖系统，与自然流产及妊娠期糖尿病等不良妊娠结局密切相关，但其具体机制尚不清楚。
目的：通过对维生素D缺乏小鼠子宫组织进行全转录组分析，寻找潜在差异表达miRNA及相关调控网络。
方法：取雌性C57BL/6J小鼠18只随机分为2组，维生素D缺乏组给予维生素D缺乏饲料喂养，正常对照组给予维生素D充足饲料喂养，每周记录小鼠体质量。8周后检测小鼠血清中25，(OH)D3和激素水平，对小鼠子宫组织进行苏木精-伊红染色并收集两组子宫组织进行转录组测序，进行基因本体、京都基因与基因组百科全书生物信息学分析。
结果与结论：①两组小鼠间体质量无明显差异；与正常对照组相比，维生素D缺乏组小鼠血清25，(OH)D3、雌二醇水平显著降低，睾酮水平显著升高；②子宫组织苏木精-伊红染色结果显示，维生素D缺乏组子宫内膜皱褶减少；③转录组测序结果共筛选出差异表达的miRNA 25个，其中上调9个(如miR-541-5p和miR-205-5p)，下调16个(如miR-378d和miR-708-5p)；④同时维生素D缺乏小鼠在多项基因本体富集类别中具有显著差异，如发育过程、细胞成分组织或生物发生等；此外，京都基因与基因组百科全书功能富集结果显示，差异表达基因多与生化代谢途径和信号转导途径相关，且些富集通路中的差异表达基因主要影响了PI3K-Akt信号通路、MAPK信号通路、胰岛素信号通路、促性腺激素释放激素信号通路及卵母细胞减数分裂通路，与生殖系统调控密切相关；⑤结果表明，维生素D缺乏可导致雌鼠子宫着床条件变差，并与调节小鼠生殖系统胰岛素等多个信号通路相关。
缩略语：京都基因与基因组百科全书：Kyoto encyclopedia of genes and genomes，KEGG

https://orcid.org/0000-0002-3854-7242 (陈霞) 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 维生素D缺乏, 子宫, 转录组测序, miRNA, 生殖系统

Abstract: BACKGROUND: It has been recently discovered that vitamin D receptor is widely distributed in the female reproductive system such as the ovary and uterus, which is closely related to adverse pregnancy outcomes such as spontaneous abortion and gestational diabetes mellitus, but its specific mechanism is still unclear.  
OBJECTIVE: To investigate potential differentially expressed miRNAs and related regulatory networks via a whole transcriptome analysis of the uterine tissue of vitamin D-deficient mice.
METHODS: Eighteen female C57BL/6J mice were randomly divided into two groups: a vitamin D deficiency group and a normal control group. Mice in the vitamin D deficiency group were fed with vitamin D deficiency diet, and mice in the normal control group were fed with vitamin D sufficiency diet. The body mass of the mice was recorded every week. After 8 weeks, the serum levels of 25,(OH)D3 and hormones in the mice were tested. Hematoxylin-eosin staining was used to detect mouse uterine tissue. Uterine tissue samples of two groups were collected for transcriptome sequencing, and bioinformatics analyses of gene ontology and Kyoto encyclopedia of genes and genomes.  
RESULTS AND CONCLUSION: There was no difference in body mass between the two groups. Compared with the normal control group, the levels of 25,(OH)D3 and estradiol in mouse serum were significantly reduced, and the testosterone level was significantly increased in the vitamin D deficiency group. Hematoxylin-eosin staining of uterine tissue showed a reduction in the endometrial folds of mice in the vitamin D deficiency group. Twenty-five differentially expressed miRNAs were screened out by transcriptome sequencing, of which 9 were up-regulated (such as miR-541-5p and miR-205-5p) and 16 were down-regulated (such as miR-378d and miR-708-5p). At the same time, vitamin D deficient mice showed significantly differences in multiple gene ontology enrichment categories, such as developmental process, cell composition, tissue or biogenesis. In addition, the functional enrichment results of Kyoto genes and genomes encyclopedia showed that differentially expressed genes were mostly related to biochemical metabolic pathways and signal transduction pathways. Differentially expressed genes in these enrichment pathways mainly affected PI3K-Akt signaling pathway, MAPK signaling pathway, insulin signaling pathway, gonadotropin releasing hormone signaling pathway, and oocyte meiosis pathway, all of which were closely related to the regulation of the reproductive system. To conclude, vitamin D deficiency can lead to poor uterine implantation conditions in female mice, which is related to multiple signaling pathways, such as insulin regulation, in mouse reproductive system.

Key words: vitamin D deficiency, uterus, transcriptome sequencing, miRNA, reproductive system

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