[1]余静,唐婉琦,杨雪,等.Musculin KO-Foxp3-GFPKI小鼠模型的制备及在严重创伤介导Treg-Th17失衡中的初步应用[J].第三军医大学学报,2018,40(16):1435-1443.
 YU Jing,TANG Wanqi,YANG Xue,et al.Establishment of a transgenic mouse model with musculin knockout and Foxp3-GFP knockin and role of musculin in severe trauma-induced Treg-Th17 imbalance[J].J Third Mil Med Univ,2018,40(16):1435-1443.
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Musculin KO-Foxp3-GFPKI小鼠模型的制备及在严重创伤介导Treg-Th17失衡中的初步应用(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第16期
页码:
1435-1443
栏目:
基础医学
出版日期:
2018-08-30

文章信息/Info

Title:
Establishment of a transgenic mouse model with musculin knockout and Foxp3-GFP knockin and role of musculin in severe trauma-induced Treg-Th17 imbalance
作者:
余静唐婉琦杨雪马娓严军梁华平
陆军军医大学(第三军医大学)第三附属医院(野战外科研究所)第一研究室,创伤、烧伤与复合伤国家重点实验室
Author(s):
YU Jing TANG Wanqi YANG Xue MA Wei YAN Jun LIANG Huaping

State Key Laboratory of Trauma, Burns and Combined Injury, Department 1, Research Institute of Surgery, Third Affiliated Hospital, Army Military Medical University (Third Military Medical University), Chongqing, 400042, China

关键词:
MusculinFoxp3小鼠模型严重创伤Treg-Th17失衡
Keywords:
musculin Foxp3 transgenic mice severe trauma Treg-Th17 imbalance
分类号:
R-332; R392.3; R394.3
文献标志码:
A
摘要:

目的    制备转录因子Musculin(MSC)敲除(knockout, KO)-叉状头转录因子家族成员3(forkhead box P3, Foxp3)-绿色荧光蛋白(green fluorescent protein, GFP)敲入(knockin, KI)小鼠模型,并初步应用于严重创伤介导调节性T细胞(regulatory T cell, Treg)Th17失衡研究。方法    通过MSCKO纯合子(homozygote, -/-)小鼠和Foxp3GFPKI纯合子(homozygote,+/+)小鼠杂交,获取MSCKO(heterozygote,+/-)-Foxp3-GFPKI(+/-)杂合子小鼠,再按同笼兄妹近亲繁殖方法进行杂合子配对,直到获取MSCKO-Foxp3-GFPKI纯合子小鼠。将获得MSCKO-Foxp3-GFPKI小鼠连续交配5代以上,观察各代小鼠的基因型、表现型和遗传稳定性。对获取的MSCKO-Foxp3-GFPKI小鼠和Foxp3-GFPKI小鼠实施失血/骨折,3 h后取周围淋巴结(lymph nodes, LN)、肠系膜淋巴结(mesenteric lymph nodes, MLN)、肠道集合淋巴结(Peyer’s patches, PP)、脾(spleen, SP)、骨髓(bone marrow, BM)和胸腺(thymus, Thy)。流式细胞术检测机体免疫器官和组织Treg的分布变化,qPCR检测MSC、Foxp3、孤核受体-γt( orphan nuclear receptor-γt, ROR-γt)的表达水平,进一步验证MSCKO-Foxp3-GFPKI小鼠的实用性,并初步探讨MSC对严重创伤介导Treg-Th17失衡的影响。结果    杂交获得的各代MSCKO-Foxp3GFPKI纯合子小鼠均能表达各分子标志物,活力、体质量和繁育能力正常。流式细胞术检测结果显示,各免疫器官和组织中均存在不同强度的GFP信号;严重创伤3 h后,MSCKO-Foxp3-GFPKI小鼠Treg在LN、MLN、PP和SP中的百分比分别为5.81%、4.81%、1.41%和3.1%,较创伤前差异有统计学意义。qPCR检测结果显示,相对正常组而言,野生型创伤组MSC在外周淋巴器官MLN(6.154 9)和PP(7.789 7)中的相对表达量显著升高(P<0.05,P<0.01);Foxp3在MSCKO创伤组MLN(2.591 2)和PP(1.506 4)中的相对表达量上升(P<0.01),表明MSC缺失可使创伤后TregTh17平衡左移;而RORγt在同组MLN(0.5395)和PP(0.544 9)中的相对表达量下降(P<0.01),同样证实了Treg-Th17平衡发生了偏移。结论   成功制备MSCKO-Foxp3-GFPKI小鼠,遗传性状稳定,分子标志物和表现型明确。严重创伤后MSC可以影响Treg的分布,并使TregTh17平衡发生偏移,提示MSC可能作为严重创伤后恢复机体内环境免疫稳态的潜在调节靶点。

Abstract:

Objective    To establish a transgenic mouse model with musculin knockout (MSCKO) and Foxp3 and green fluorescent protein knockin (Foxp3-GFPKI) and investigate the role of musculin in regulatory T cell (Treg)-Th17 imbalance induced by severe trauma.  Method    sMSCKO homozygote (-/-) mice and Foxp3-GFPKI homozygote (+/+) mice were crossed to obtain the heterozygote (MSCKO+/--Foxp3-GFPKI+/-) mice. The offspring mice were bred continuously until the homozygote mice (MSCKO-Foxp3-GFPKI) were acquired. The MSCKO-Foxp3-GFPKI mice were crossed for at least 5 generations, and the mice in each generation were assessed for genotypes, phenotypes and genetic stability. Severe trauma (fracture with blood loss) models were then established in the MSCKO-Foxp3-GFPKI and Foxp3-GFPKI mice, and 3 h after the modeling, peripheral lymph nodes (LN), mesenteric lymph nodes (MLN), Peyer’s patch (PP), spleen, bone marrow (BM) and thymus samples were collected. The distribution of Treg in different immune organs and tissues was detected by flow cytometry, and the expression levels of musculin, Foxp3 and retinoic acid-related orphan receptor γt (ROR-γt) were measured using quantitative RTPCR to explore the role of musculin in Treg-Th17 imbalance mediated by severe trauma.  Results    All the homozygote (MSCKOFoxp3-GFPKI) mice in different generations expressed the molecular markers with normal activities, weight and breeding capacity. Fluorescence-activated cell sorting revealed GFP signals in the immune organs and tissues of the homozygote mice. MSCKO-Foxp3-GFPKI mice with severe trauma showed obvious changes in Treg percentages in the LN (5.81%), MLN (4.81%), PP (1.41%) and SP (3.1%) and exhibited significantly increased mRNA levels of musculin and Foxp3 and decreased RORγt mRNA expression in the MLN and PP (P<0.05 or 0.01), indicating a left shift of Treg-Th17 balance after trauma. Conclusion    We successfully obtain MSCKO-Foxp3-GFPKI mice with stable phenotypes. Musculin affects the distribution of Treg and causes shift of Treg/Th17 balance after severe trauma, suggesting its role as a potential regulatory target in the maintenance of the internal environment and immune homeostasis after severe trauma.

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更新日期/Last Update: 2018-09-01