TET1基因对小鼠uNK细胞增殖及IFN-γ、VEGF-C和TGF-β1转录水平的影响

doi:10.11843/j.issn.0366-6964.2023.03.033

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (3): 1221-1228.doi: 10.11843/j.issn.0366-6964.2023.03.033

TET1基因对小鼠uNK细胞增殖及IFN-γ、VEGF-C和TGF-β1转录水平的影响

杨晓伟^1,2,3, 赵自亮¹, 付雨¹, 于子肖¹, 赵永聚^2,3*

1. 西南大学动物医学院, 重庆 402460;
2. 西南大学动物科学技术学院, 重庆 400715;
3. 草食动物科学重庆市重点实验室, 重庆 400715

收稿日期:2022-08-08 出版日期:2023-03-23 发布日期:2023-03-21
通讯作者: 赵永聚,主要从事草食牲畜遗传改良等领域的教学和研究工作,E-mail:zyongju@163.com
作者简介:杨晓伟(1980-)，女，吉林通化人，讲师，主要从事动物免疫学研究，E-mail: yangxiaowei396@163.com；赵自亮(1997-)，男，云南普洱人，硕士生，主要从事动物免疫学研究，E-mail: 1948840202@qq.com。
基金资助:
国家自然科学基金(32002356)；重庆市自然科学基金(cstc2019jcyj-msxmX0140)；2021年国家级大学生创新创业项目(202110635014)；重庆市科企联合体种质资源收集利用与品种试验项目(草食牲畜)

Effects of TET1 Gene on the Proliferation of Mouse uNK Cells and the Transcriptional Level of IFN-γ, VEGF-C and TGF-β1

YANG Xiaowei^1,2,3, ZHAO Ziliang¹, FU Yu¹, YU Zixiao¹, ZHAO Yongju^2,3*

1. College of Veterinary Medicine, Southwest University, Chongqing 402460, China;
2. College of Animal Science and Technology, Southwest University, Chongqing, 400715, China;
3. Chongqing Key Laboratory of Herbivore Science, Chongqing 400715, China

Received:2022-08-08 Online:2023-03-23 Published:2023-03-21

摘要/Abstract

摘要： 本研究旨在了解去甲基化酶1(ten eleven translocation，TET1)对小鼠子宫内自然杀伤细胞(uterine natural killer, uNK)的增殖及其细胞因子分泌表达的影响。采集妊娠9~12 d小鼠子宫内膜，分离淋巴细胞，使用链霉亲和素磁珠法进一步分选uNK细胞进行培养；针对TET1基因合成RNA干扰序列，并将其连接入RNA干扰载体GM-19167，构建干扰表达质粒，包装成慢病毒(lentivirus)并进行病毒滴度的测定；按照慢病毒与细胞数(10～200)∶1的比例转染uNK细胞，168 h后荧光显微镜检测转染效果，收集uNK细胞，以β-actin为内参进行荧光定量PCR检测以验证TET1基因的干扰效果，利用Cell Counting Kit-8 (CCK8)对uNK细胞的增殖情况进行检测，分别合成γ干扰素(interferon-γ，IFN-γ)、血管内皮生长因子(vascular endothelial growth factor C，VEGF-C)和β1转化因子(transforming growth factor-β1，TGF-β1)3种细胞因子的检测引物，通过荧光定量PCR方法进行检测。结果发现，慢病毒感染168 h后uNK细胞中TET1的表达量显著下降(P<0.05)；CCK8试验结果显示，与空载体病毒感染和空白对照组相比较，TET1干扰后其细胞增殖不受影响(P>0.05)；荧光定量PCR检测结果显示，细胞因子TGF-β1的表达水平显著上升(P<0.01)，而IFN-γ和VEGF-C两种细胞因子均显著下降(P<0.05)。下调TET1基因的表达不影响uNK细胞的正常增殖，但会影响其细胞因子的转录，进而对动物子宫内的天然免疫发挥重要作用。

关键词: TET1基因, uNK细胞, 细胞增殖, 细胞因子, 小鼠

Abstract: The purpose of this study was to evaluate the effect of ten eleven translocation 1 (TET1) on the proliferation and cytokine secretion and expression of mouse uterine natural killer (uNK) cells. The endometrium of pregnant mice was collected on gestational day 9-12, and lymphocytes were isolated. uNK cells were further sorted by streptavidin magnetic bead method for culture. The RNA interference sequence of TET1 gene was synthesized and connected to RNA interference vector GM-19167 to construct interference expression plasmid, then lentivirus was packaged and virus titer was determined. uNK cells were transfected according to the ratio of lentivirus to cell number of (10-200)∶1. After 168 h, the transfection effect was detected by fluorescence microscope. uNK cells were collected and fluorescent quantitative PCR (qPCR) method was used to verify the interference effect of TET1 gene while β- actin was used as an internal reference. Cell counting kit-8 (CCK8) was used to detect the proliferation of uNK cells. Primers of interferon-γ (IFN-γ), vascular endothelial growth factor (VEGF-C) and transforming growth factor β1 (TGF-β1) were synthesized respectively and were used to detect the expression of these three cytokines by qPCR method. The results showed that the expression of TET1 in uNK cells decreased significantly after 168 h of lentivirus infection (P<0.05), indicating that the expression of TET1 gene was significantly down regulated under RNA interference. When compared with the blank control groups, the uNK cells proliferation was not affected after TET1 interference (P>0.05) which shown by CCK8 detection results. Fluorescent qPCR results showed that after TET1 interference, the expression level of TGF-β1 was increased significantly (P<0.01), while IFN-γand VEGF-C were both decreased significantly (P<0.05). These findings revealed that interference with down-regulation of TET1 gene expression did not affect the proliferation of uNK cells, but had an effect on the transcriptional level of cytokines, thereby playing an important role in the innate immunity of animals in the uterus.

Key words: TET1, uNK cell, cellular proliferation, cytokine, mouse

中图分类号:

S857.2

杨晓伟, 赵自亮, 付雨, 于子肖, 赵永聚. TET1基因对小鼠uNK细胞增殖及IFN-γ、VEGF-C和TGF-β1转录水平的影响[J]. 畜牧兽医学报, 2023, 54(3): 1221-1228.

YANG Xiaowei, ZHAO Ziliang, FU Yu, YU Zixiao, ZHAO Yongju. Effects of TET1 Gene on the Proliferation of Mouse uNK Cells and the Transcriptional Level of IFN-γ, VEGF-C and TGF-β1[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1221-1228.

参考文献 21

[1]	FAAS M M, DE VOS P. Uterine NK cells and macrophages in pregnancy[J]. Placenta, 2017, 56:44-52.
[2]	REDLINE R W. Role of uterine natural killer cells and interferon γ in placental development[J]. J Exp Med, 2000, 192(2):F1-F4.
[3]	丁培阳,刘艳利,曹阳坡,等.妊娠山羊子宫uNK细胞与VEGF动态分布研究[J].畜牧兽医学报, 2014, 45(5):821-826.DING P Y, LIU Y L, CAO Y P, et al. Study of the dynamic distribution of uNK cells and VEGF in the pregnant goat[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(5):821-826.(in Chinese)
[4]	RAJAGOPALAN S. HLA-G-mediated NK cell senescence promotes vascular remodeling:implications for reproduction[J]. Cell Mol Immunol, 2014, 11(5):460-466.
[5]	HUANG G A, LIU L L, WANG H S, et al. Tet1 deficiency leads to premature reproductive aging by reducing spermatogonia stem cells and germ cell differentiation[J]. iScience, 2020, 23(3):100908.
[6]	KHOUEIRY R, SOHNI A, THIENPONT B, et al. Lineage-specific functions of TET1 in the postimplantation mouse embryo[J]. Nat Genet, 2017, 49(7):1061-1072.
[7]	YAMAGUCHI S, SHEN L, LIU Y T, et al. Role of Tet1 in erasure of genomic imprinting[J]. Nature, 2013, 504(7480):460-464.
[8]	RAKOCZY J, PADMANABHAN N, KRZAK A M, et al. Dynamic expression of TET1, TET2, and TET3 dioxygenases in mouse and human placentas throughout gestation[J]. Placenta, 2017, 59:46-56.
[9]	JAFARPOUR F, HOSSEINI S M, OSTADHOSSEINI S, et al. Comparative dynamics of 5-methylcytosine reprogramming and TET family expression during preimplantation mammalian development in mouse and sheep[J]. Theriogenology, 2017, 89:86-96.
[10]	谭强,罗南剑,张艳丽,等.山羊早期胎儿组织TET1与Wnt通路基因的表达变化及其相关性[J].中国农业科学, 2017, 50(14):2816-2825.TAN Q, LUO N J, ZHANG Y L et al. Expression patterns and correlation of Wnts and TET1 genes in early fetal tissues of Dazu black goat[J]. Scientia Agricultura Sinica, 2017, 50(14):2816-2825.(in Chinese)
[11]	ZHANG H K, ZHANG X, CLARK E, et al. TET1 is a DNA-binding protein that modulates DNA methylation and gene transcription via hydroxylation of 5-methylcytosine[J]. Cell Res, 2010, 20(12):1390-1393.
[12]	YANG R L, QU C Y, ZHOU Y, et al. Hydrogen sulfide promotes Tet1-and Tet2-mediated Foxp3 demethylation to drive regulatory T cell differentiation and maintain immune homeostasis[J]. Immunity, 2015, 43(2):251-263.
[13]	杨晓伟,赵永聚.哺乳动物子宫自然杀伤(uNK)细胞对妊娠的调控作用[J].畜牧兽医学报, 2020, 51(5):899-906.YANG X W, ZHAO Y J. The regulation role of uterine natural killer (uNK) cells during pregnancy in mammals[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5):899-906.(in Chinese)
[14]	KAUFMANN P, BLACK S, HUPPERTZ B. Endovascular trophoblast invasion:implications for the pathogenesis of intrauterine growth retardation and preeclampsia[J]. Biol Reprod, 2003, 69(1):1-7.
[15]	MOR G, ALDO P, ALVERO A B. The unique immunological and microbial aspects of pregnancy[J]. Nat Rev Immunol, 2017, 17(8):469-482.
[16]	LASH G E, SCHIESSL B, KIRKLEY M, et al. Expression of angiogenic growth factors by uterine natural killer cells during early pregnancy[J]. J Leukoc Biol, 2006, 80(3):572-580.
[17]	STOIKOS C J, HARRISON C A, SALAMONSEN L A, et al. A distinct cohort of the TGFβ superfamily members expressed in human endometrium regulate decidualization[J]. Hum Reprod, 2008, 23(6):1447-1456.
[18]	张玉杰,彭景楩,卿素珠. TGF-β1对人蜕膜基质细胞中趋化因子及其受体表达的影响[J].动物学杂志, 2012, 47(2):36-45.ZHANG Y J, PENG J P, QING S Z. The Effect of TGF-β1 on expression of chemokine and its receptor in human decidual stromal cells[J]. Chinese Journal of Zoology, 2012, 47(2):36-45.(in Chinese)
[19]	LIU W F, LUO M L, ZOU L, et al. uNK cell-derived TGF-β1 regulates the long noncoding RNA MEG3 to control vascular smooth muscle cell migration and apoptosis in spiral artery remodeling[J]. J Cell Biochem, 2019, 120(9):15997-16007.
[20]	DONG Z J, WEI H M, SUN R, et al. Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1. 1+ cells[J]. World J Gastroenterol, 2004, 10(13):1928-1933.
[21]	BURLESON J D, SINIARD D, YADAGIRI V K, et al. TET1 contributes to allergic airway inflammation and regulates interferon and aryl hydrocarbon receptor signaling pathways in bronchial epithelial cells[J]. Sci Rep, 2019, 9(1):7361.

TET1基因对小鼠uNK细胞增殖及IFN-γ、VEGF-C和TGF-β1转录水平的影响

Effects of TET1 Gene on the Proliferation of Mouse uNK Cells and the Transcriptional Level of IFN-γ, VEGF-C and TGF-β1

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 21

相关文章 15

编辑推荐

Metrics

本文评价

[1]	张晨俭, 李隐侠, 丁强, 刘伟佳, 王慧利, 何南, 吴家顺, 曹少先. CRISPR/Cas9技术高效制备山羊SOCS2基因编辑胚胎[J]. 畜牧兽医学报, 2024, 55(1): 129-141.
[2]	苗舒, 安济山, 王祚, 肖定福, 兰欣怡, 刘磊, 沈维军, 万发春. 亮氨酸通过PI3K-AKT信号通路促进牛成肌细胞的增殖[J]. 畜牧兽医学报, 2024, 55(1): 142-152.
[3]	梁睿, 范小瑞, 张晋强, 庞全海. 小鼠黑色素细胞沉默及过表达色素上皮衍生因子对黑色素合成的影响[J]. 畜牧兽医学报, 2023, 54(9): 3916-3930.
[4]	陈喜宏, 路桂聪, 王浩磊, 苟少校, 玉永雄, 林涛, 蒋曹德. 利用牛乳腺细胞和小鼠乳腺组织分析异绿原酸C通过NF-κB信号通路对乳腺炎症反应的抑制效应[J]. 畜牧兽医学报, 2023, 54(9): 3931-3940.
[5]	张万锋, 赵天枝, 李娇, 尤紫薇, 杨阳, 蔡春波, 高鹏飞, 曹果清, 郭晓红, 李步高. NR2F2基因调控猪PK15细胞增殖和凋亡的研究[J]. 畜牧兽医学报, 2023, 54(8): 3242-3251.
[6]	王婉洁, 陈南珠, 邹惠影, 周心仪, 郝海生, 庞云渭, 朱化彬, 赵学明, 余大为, 杜卫华. 过表达组蛋白甲基转移酶ASH1L对牛卵丘细胞增殖和凋亡的影响[J]. 畜牧兽医学报, 2023, 54(8): 3358-3368.
[7]	黄江, 李闯, 崔月琦, 袁雪莹, 赵志诚, 刘宇, 周玉龙, 朱战波, 张泽财. 基于小鼠模型研究肠道菌群紊乱对BVDV易感性的影响[J]. 畜牧兽医学报, 2023, 54(8): 3466-3473.
[8]	张鹏, 王明秀, 敬科民, 彭巍, 田园, 李雨谦, 付长其, 舒适, 钟金城, 蔡欣. FGFs/FGFRs及其介导信号通路基因的异常表达影响犏牛未分化精原细胞增殖活性[J]. 畜牧兽医学报, 2023, 54(7): 2886-2897.
[9]	邢宝瑞, 刘振, 赵海平, 马泽芳, 李勋胜, 周珏, 孙红梅. 鹿茸逆向成骨的研究进展[J]. 畜牧兽医学报, 2023, 54(6): 2231-2240.
[10]	曹西月, 纪晓霞, 张崇昊, 张亚峰, 陈雨涛, 张源淑. 二脒那秦通过内源性激活ACE2抑制AngⅡ-TGF-β1通路缓解小鼠肺纤维化[J]. 畜牧兽医学报, 2023, 54(6): 2631-2640.
[11]	许甜甜, 张彤彤, 王蒙, 王昕. 转录因子Foxq1通过WNT/β-catenin信号通路影响绒山羊毛囊干细胞增殖的研究[J]. 畜牧兽医学报, 2023, 54(6): 2653-2661.
[12]	朱家桥, 程来洋, 曹江琴, 朱闽, 李军伟, 鞠辉明, 刘宗平. XRCC1在卵子和早期胚胎中的定位与功能的初步分析[J]. 畜牧兽医学报, 2023, 54(5): 2126-2133.
[13]	王崇年, 于嘉霖, 宫照乾, 吴晓玲, 邓光存. 脂肪分化相关蛋白2对BCG诱导小鼠传代巨噬细胞自噬的调控作用[J]. 畜牧兽医学报, 2023, 54(5): 2134-2146.
[14]	任晓丽, 范玉营, 皇甫和平, 刘云, 石冬梅. GSK126对犬乳腺肿瘤细胞上皮间质转化的影响[J]. 畜牧兽医学报, 2023, 54(4): 1721-1729.
[15]	杨光, 徐景, 李新, 胡德宝, 郭益文, 丁向彬, 郭宏, 张林林. 干扰lncbMD对牛骨骼肌卫星细胞增殖分化的影响[J]. 畜牧兽医学报, 2023, 54(3): 1015-1025.