遗传 ›› 2024, Vol. 46 ›› Issue (11): 947-957.doi: 10.16288/j.yczz.24-181
收稿日期:
2024-06-18
修回日期:
2024-08-12
出版日期:
2024-11-20
发布日期:
2024-09-23
通讯作者:
黄志斌,博士,副教授,研究方向:人类疾病斑马鱼动物模型的构建、造血发育以及调控。E-mail: huangzhb1986@scut.edu.cn作者简介:
杨晓君,硕士,专业方向:生物学。E-mail: 2948225063@qq.com
基金资助:
Xiaojun Yang(), Zhenhan Huang, Wei Liu, Wenqing Zhang, Zhibin Huang(
)
Received:
2024-06-18
Revised:
2024-08-12
Published:
2024-11-20
Online:
2024-09-23
Supported by:
摘要:
先天免疫反应在维持机体稳态中扮演关键角色,其启动与先天免疫细胞表面的模式识别受体或损伤相关分子密切相关。CD209作为巨噬细胞或树突状细胞表面的模式识别受体,在免疫功能中具有重要作用。然而,目前尚不清楚CD209在体内对先天免疫细胞如巨噬细胞、中性粒细胞的影响。本研究通过多序列比对和系统进化树构建,发现斑马鱼(Danio rerio)中存在3个与人CD209同源的基因,分别是cd209(Ensembl ID:ENSDARG00000029461)、zgc:174904(Ensembl ID:ENSDARG00000059049)和si:dkey-187i8.2(Ensembl ID:ENSDARG00000096624)。与Ensembl数据库中的cd209和si:dkey-187i8.2基因相比,zgc:174904在序列上与人CD209更为相似。通过整体原位杂交和荧光共定位实验,本研究发现zgc:174904主要在巨噬细胞表达。进一步通过morpholino敲低实验,结果显示,敲低zgc:174904会导致M1型巨噬细胞相关基因的上调以及成熟中性粒细胞数量的减少,表明zgc:174904在功能上与CD209更为相似。本研究结果不仅揭示了CD209在调控巨噬细胞功能和中性粒细胞发育中的潜在作用,还为先天免疫机制的研究提供了重要的线索。
杨晓君, 黄振瀚, 刘伟, 张文清, 黄志斌. CD209同源基因在斑马鱼中的鉴定及功能表征[J]. 遗传, 2024, 46(11): 947-957.
Xiaojun Yang, Zhenhan Huang, Wei Liu, Wenqing Zhang, Zhibin Huang. Identification and functional characterization of CD209 homologous genes in zebrafish[J]. Hereditas(Beijing), 2024, 46(11): 947-957.
表1
不同物种CD209的氨基酸序列信息"
物种 | 氨基酸序列号 |
---|---|
人(Homo sapiens) | NP_066978.1 |
黑猩猩(Pan troglodytes) | XP_063656223.1 |
小鼠(Mus musculus) | NP_573501.1 |
家牛(Bos taurus) | NP_001139228.1 |
孔雀鱼(Poecilia reticulata) | XP_017157862.1 |
鲤鱼(Cyprinus carpio) | XP_042588436.1 |
草鱼(Ctenopharyngodon idella) | QYY47516.1 |
斑马鱼zgc:174094(Danio rerio) | NP_001170922 |
斑马鱼cd209(Danio rerio) | NP_001186302.2 |
斑马鱼si:dkey-187i8.2(Danio rerio) | XP_021335897.1 |
表2
基因及引物信息"
基因名称 | 引物序列(5'→3') |
---|---|
β-actin | F:GAGAGGTTTAGGTTGGTCGT R:GAGAGGTTTAGGTTGGTCGT |
ef1α | F:TACTTCTCAGGCTGACTGTG R:ATCTTCTTGATGTATGCGCT |
cd209l1 | F:GTTGCAGCTCAAGGATAAAGTCAC R:GTTCCTCAGCTGTGAGGATGATGG |
mfap4 | F:GTTTACACCATCTATCCAGCC R:GTTCTCTAGTCCCAGCCA |
il-1β | F:GTACTCAAGGAGATCAGCGG R:CTCGGTGTCTTTCCTGTCCA |
tnfα | F:GCGCTTTTCTGAATCCTACG R:TGCCCAGTCTGTCTCCTTCT |
il-8 | F:ATGACCAGCAAAATCATTTCAGTGTG R:TCATGGTTTTCTGTTGACAATGATCC |
irg1 | F:TGCTAGAAGAGCAGGACATCGC R:CAGTCCTTCAGCAGAGCTCAGA |
il-10 | F:TGTGCTCAGAGCAGGAGAGTCG R:GAGCTGTTGGCAGAATGGTCTCC |
tgfβ1b | F:ACTGGCTCTTGCTCCTATGTCTGG R:GGGTCTCAAGCACCTCAGTGTG |
arg2 | F:ATGGCGATGAGAGGACCACTG R:GATTGGCACGTCCAACTGTGC |
图2
cd209l1在斑马鱼幼鱼和成鱼中的表达 A:cd209l1、cd209、cd209l2在巨噬细胞中的表达水平;B~D:从单细胞至36 hpf斑马鱼cd209的时空表达;E~G从单细胞至36 hpf斑马鱼cd209l1的时空表达;H:48 hpf斑马鱼cd209的时空表达;I:48 hpf斑马鱼cd209l1的时空表达,红色箭头指示cd209l1在肝脏中表达;J:3 dpf斑马鱼cd209的时空表达,绿色箭头指示cd209在肝脏中的表达;K:3 dpf斑马鱼cd209l1的时空表达,红色箭头指示cd209l1在肝脏中表达,红色方框指示cd209l1在尾部造血组织中表达;L、M:4 dpf和6 dpf斑马鱼cd209的时空表达,绿色箭头指示cd209在肝脏中的表达;N、O:4 dpf和6 dpf斑马鱼cd209l1的时空表达,红色箭头指示cd209l1在肝脏中的表达,红色方框指示cd209l1在尾部造血组织中表达;P:cd209l1+细胞与mfap4+细胞共定位结果,红色cy3荧光标记内源性cd209l1 mRNA,绿色荧光为Tg(mfap4:GFP)标记的巨噬细胞,白色箭头指示cd209l1与mfap4荧光共定位的细胞,黄色箭头指示cd209l1不与mfap4荧光共定位的细胞;Q:与mfap4+细胞共定位的cd209l1+细胞比例统计结果;R:通过不同转基因品系斑马鱼分选成鱼骨髓血巨噬细胞、粒细胞、红系细胞、淋巴细胞后检测cd209l1的表达。"
图3
cd209l1敲低促进M1型巨噬细胞极化 A:cd209l1 MO效率检测结果;B:mfap4标记的巨噬细胞原位杂交结果(右下角为红色方框的放大图);C:cd209l1敲低后mfap4原位杂交结果信号统计;D:RT-qPCR检测M1型巨噬细胞表达基因(il-1β、tnfα、il-8、irg1、nos2a)以及M2型巨噬细胞表达基因(il-10、tgfβ1b、arg2)表达量统计结果;E:cd209l1敲低后il-1β的原位杂交结果;F:cd209l1敲低后il-1β原位杂交统计结果;G:cd209l1敲低后tnfα原位杂交结果;H:cd209l1敲低后tnfα原位杂交统计结果;I:cd209l1敲低后irg1原位杂交结果;J:cd209l1敲低后irg1原位杂交统计结果。"
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