小鼠脂肪组织中免疫细胞分离方法的优化及亚群在肥胖小鼠中的作用

doi:10.3969/j.issn.1006-7795.2021.04.009

首都医科大学学报 ›› 2021, Vol. 42 ›› Issue (4): 559-567.doi: 10.3969/j.issn.1006-7795.2021.04.009

小鼠脂肪组织中免疫细胞分离方法的优化及亚群在肥胖小鼠中的作用

武永乐¹, 尚宏伟², 孙广永³, 张栋^3*, 丁惠国^1*

1.首都医科大学附属北京佑安医院肝病消化中心,北京 100069;
2.首都医科大学基础医学院实验教学中心,北京100069;
3.首都医科大学附属北京友谊医院科研实验中心,北京 100050

收稿日期:2021-04-13 出版日期:2021-08-21 发布日期:2021-07-29
基金资助:
国家自然科学基金(81870399,81970503,81970525),北京市自然科学基金(7192046)。

Optimization of immune cell isolation method from mouse adipose tissue and the role of subgroups in obese mice

Wu Yongle¹, Shang Hongwei², Sun Guangyong³, Zhang Dong^3*, Ding Huiguo^1*

1. Center of Hepatic and Digestive Disease, BeijingYouan Hospital, Capital Medical University, Beijing 100069, China;
2. Experimental Teaching Center, School of Basic Medical Sciences,Capital Medical University, Beijing 100069, China;
3. Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China

Received:2021-04-13 Online:2021-08-21 Published:2021-07-29
Contact: * E-mail:zhangd@ccmu.edu.cn; dinghuiguo@ccmu.edu.cn
Supported by:
National Natural Science Foundation of China (81870399, 81970503, 81970525), Natural Science Foundation of Beijing (7192046).

摘要/Abstract

摘要： 目的尝试提供一种高效的、低损伤的分离脂肪组织免疫细胞的方式,以期为肥胖过程中免疫学机制的研究提供技术支持。方法首先分离正常小鼠附睾周围的脂肪组织,并采用机械研磨或酶消化(包括Ⅱ型/Ⅳ胶原酶、Liberase酶)的方式分离其中的免疫细胞,并结合流式细胞检测技术筛选最高效的免疫细胞分离方式。再通过高脂饮食(high fat diet,HFD)建立肥胖小鼠模型,并进一步分析肥胖过程中,小鼠脂肪组织中巨噬细胞及T淋巴细胞的比例及亚群变化。结果比较不同酶消化及机械研磨方式发现,通过高纯度的Liberase酶消化的方式获得CD45⁺免疫细胞死亡率最低,活细胞数最多;而通过机械研磨方式获得免疫细胞死亡率最高,活细胞数最低。与正常饮食小鼠相比,肥胖小鼠脂肪组织中固有免疫细胞如中性粒细胞、巨噬细胞比例增加,尤其是M1型巨噬细胞比例增加,M2型降低,同时巨噬细胞分泌肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)增加,髓样细胞表达激发受体1(triggering receptor expressed on myeloid cells 1,TREM1)表达增加。而适应性免疫细胞中,T淋巴细胞比例增加,特别是Th1/Treg细胞的比例增加最为明显。结论建立了一种从小鼠脂肪组织中高效获取有活力的免疫细胞的方法,并证实肥胖小鼠脂肪组织中巨噬细胞、T淋巴细胞比例增加,促炎作用增强,它们在诱发脂肪组织炎症反应中发挥重要作用。

关键词: 脂肪组织, 酶消化, 机械法, 巨噬细胞, T淋巴细胞

Abstract: Objective To provide an efficient and low-damage method to isolate immune cells from adipose tissue to provide technical support for the study of immunological mechanisms in obesity. Methods We separated the adipose tissue around the epididymis of normal mice, and separated the immune cells by mechanical grinding or enzymatic digestion (including type Ⅱ/Ⅳ collagenase, Liberase enzyme), and screened the most efficient immune cell separation method by flow cytometry technology. Then, an obese mouse model was established through high fat diet (HFD), and the proportion and subgroup changes of macrophages and T lymphocytes in mouse adipose tissue were further analyzed. Results We compared different enzymatic digestion and mechanical grinding methods and found that CD45⁺ immune cells obtained through high-purity Liberase enzyme digestion had the lowest mortality rate and the highest number of live cells. The death rate of immune cells obtained by mechanical grinding was the highest, and the number of living cells was the least.Compared with mice fed a normal diet, the proportion of innate immune cells such as neutrophils and macrophages in the adipose tissue of obese mice increased, especially the proportion of M1 macrophages, while the M2 macrophages decreased. The secretion of tumor necrosis factor-α(TNF-α) by macrophages increased, and the expression of triggering receptor expressed on myeloid cells 1(TREM1) enhanced. Among the adaptive immune cells, the proportion of T lymphocytes increased, especially the ratio of Th1/Treg cells increased the most. Conclusion We have established a method to efficiently obtain viable immune cells from mouse adipose tissue, and confirmed that the proportion of macrophages and T lymphocytes in the adipose tissue of obese mice increased, and their pro-inflammatory effect were enhanced. They played an important role in inducing inflammation in adipose tissue.

Key words: adipose tissue, enzyme digestion, mechanical method, macrophages, T lymphocytes

中图分类号:

R965

武永乐, 尚宏伟, 孙广永, 张栋, 丁惠国. 小鼠脂肪组织中免疫细胞分离方法的优化及亚群在肥胖小鼠中的作用[J]. 首都医科大学学报, 2021, 42(4): 559-567.

Wu Yongle, Shang Hongwei, Sun Guangyong, Zhang Dong, Ding Huiguo. Optimization of immune cell isolation method from mouse adipose tissue and the role of subgroups in obese mice[J]. Journal of Capital Medical University, 2021, 42(4): 559-567.

参考文献

[1] Mathis D. Immunological goings-on in visceral adipose tissue [J]. Cell Metab, 2013,17(6):851-859.
[2] Liu B, Yu H, Sun G, et al. OX40 promotes obesity-induced adipose inflammation and insulin resistance[J]. Cell Mol Life Sci, 2017,74(20):3827-3840.
[3] Senesi L, De Francesco F, Farinelli L, et al. Mechanical and enzymatic procedures to isolate the stromal vascular fraction from adipose tissue: preliminary results[J]. Front Cell Dev Biol, 2019,7:88.
[4] 崔梦莹,郭澍,佟爽,等.脂肪干细胞来源外泌体通过Wnt/β-catenin信号通路促进脂肪干细胞成骨分化[J].中国医科大学学报,2019,48(2):114-118.
[5] Wetzels S, Bijnen M, Wijnands E, et al. Characterization of immune cells in human adipose tissue by using flow cytometry[J]. J Vis Exp,2018,(133):57319.
[6] Zuk P A, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies[J]. Tissue Eng,2001,7(2):211-228.
[7] Gentile P, Calabrese C, De Angelis, et al. Impact of the different preparation methods to obtain human adipose-derived stromal vascular fraction cells(AD-SVFs) and human adipose-derived mesenchymalstem cells (AD-MSCs): enzymatic digestion versus mechanical centrifugation[J].Int J Mol Sci,2019, 20(21):5471.
[8] Alstrup T, Eijken M, Brunbjerg M E, et al. Measured levels of human adipose tissue-derived stem cellsinadipose tissue is strongly dependent on harvesting method and stem cell isolationtechnique[J].Plast Reconstr Surg, 2020,145(1):142-150.
[9] Jaitin D A, Adlung L, Thaiss C A, et al. Lipid-associated macrophages control metabolic homeostasis in a Trem2-dependent manner[J]. Cell, 2019,178(3):686-698.
[10] Abidi A, Laurent T, Bériou G, et al. Characterization of rat ILCs reveals ILC2 as the dominant intestinal subset[J]. Front Immunol, 2020,11:255.
[11] Duan W, Lopez· M J. Effects of enzyme and cryoprotectant concentrations on yield of equine adipose-derived multipotent stromal cells[J].Am J Vet Res, 2018,79(10):1100-1112.
[12] 何津,张毅,江小霞,等.人胎盘贴壁细胞的分离培养及造血相关因子表达[J].中华血液学杂志,2003,24(12):652-654.
[13] 王剑明,邹声泉.GCDC诱发体外培养大鼠肝细胞凋亡的实验研究[J].世界华人消化杂志,1999,7(10): 890.
[14] Weinstock A, Brown E J,Garabedian M L, et al. Single-cell rna sequencing of visceral adipose tissue leukocytes reveals that caloric restriction following obesity promotes the accumulation of a distinct macrophage population with features of phagocytic cells[J]. Immunometabolism,2019,1:e190008.
[15] 张伟平,聂占国,代忠明,等.人结肠上皮细胞的原代培养[J].现代肿瘤医学, 2012,20(8):1546-1548.
[16] 于华,陈金平,吴咏静,等.2型糖尿病合并冠心病患者PCT、hs-CRP及血脂水平与冠状动脉病变的相关性研究[J].临床误诊误治,2019,32(6):62-66.
[17] Wang C, Zhang M, Wu J,et al.The Effect and mechanism of TLR9/KLF4 in FFA-induced adipocyte inflammation[J]. Mediators Inflamm, 2018, 2018: 6313484.
[18] Guilliams M, Mildner A, Yona S. Developmental and functional heterogeneity of monocytes[J]. Immunity, 2018,49(4):595-613.
[19] Liu Q K, Johnson E M, Lam R K, et al. Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity[J]. Nat Immunol, 2019,20(8):1023-1034.
[20] Leisegang K, Henhel R, Agarwal A. Obesity and metabolic syndrome associated with systemic inflammation and the impact on the male reproductive system[J].Am J Reprod Immunol, 2019, 82(5):e13178.
[21] Wang M, Chen F, Wang J, et al. Th17 and Treg lymphocytes in obesity and type 2 diabetic patients[J]. Clin Immunol, 2018, 197:77-85.

小鼠脂肪组织中免疫细胞分离方法的优化及亚群在肥胖小鼠中的作用

Optimization of immune cell isolation method from mouse adipose tissue and the role of subgroups in obese mice

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王茜, 张家坤, 刘青, 孙琳, 李晶洁. M2巨噬细胞对新生乳鼠受损心脏组织的再生和修复机制[J]. 首都医科大学学报, 2022, 43(2): 178-186.
[2]	张兴华, 邢洁, 孙灿, 张希, 王拥军. 溃疡性结肠炎中B细胞对巨噬细胞趋化作用的初步研究[J]. 首都医科大学学报, 2022, 43(1): 42-46.
[3]	李沛霖, 崔磊. 真皮内脂肪组织调控皮肤生理病理过程与机制的研究进展[J]. 首都医科大学学报, 2020, 41(6): 896-900.
[4]	王田田, 高琛琛, 李利生, 徐敬东. 消化道巨噬细胞的功能与炎性肠病和肠道肿瘤的相关性研究进展[J]. 首都医科大学学报, 2019, 40(6): 972-981.
[5]	王凯丽, 陈世璋. 人类免疫缺陷病毒感染者拔牙围手术期的风险评估和管理[J]. 首都医科大学学报, 2019, 40(5): 754-757.
[6]	李妍, 徐薇, 程海艳, 孙晓丽, 李邻峰. 白介素4、10、12、13、IFN-γ、TGF-β在不同时期特应性皮炎病人血清中的变化[J]. 首都医科大学学报, 2017, 38(5): 635-639.
[7]	袁琳洁, 陈诗皓, 安高, 黄琼, 易达委, 李艳, 吕喆, 王晶晶, 黄克武, 王炜, 孙英. IL-33诱导的小鼠过敏原非依赖性哮喘样模型肺组织免疫细胞及亚群的改变[J]. 首都医科大学学报, 2016, 37(5): 561-567.
[8]	张媛媛, 李伟阳, 杨琳, 李丽英. 磷酸鞘胺醇调节小鼠单核巨噬细胞炎性细胞因子表达的机制研究[J]. 首都医科大学学报, 2015, 36(5): 729-733.
[9]	杨琳, 田蕾, 谢杰施, 李丽英. 一种简单的分离、培养及鉴定小鼠外周血单核巨噬细胞方法的建立[J]. 首都医科大学学报, 2015, 36(4): 610-613.
[10]	李玉琳, 吴依娜, 张聪聪, 阚晓玉, 阿希, 赵伟, 王绿娅, 杜杰. 小鼠骨髓巨噬细胞过继性回输的体内示踪及其在高血压心脏损伤中的应用研究[J]. 首都医科大学学报, 2013, 34(3): 391-397.
[11]	蔡永明;张春云;申文晋;李铭;姜凌;张宗鹏;. 重组人粒细胞巨噬细胞刺激因子栓在大鼠和Beagle犬中的免疫原性[J]. 首都医科大学学报, 2012, 33(3): 345-349.
[12]	穆珺;庄晓明;刘锐敏;曾静波. 血清巨噬细胞移动抑制因子、肿瘤坏死因子-α与糖尿病肾病的相关性研究[J]. 首都医科大学学报, 2012, 33(3): 385-388.
[13]	梁骑;焦艳梅;计云霞;李田;吴昊;张国元;唐中. 慢性丙型肝炎患者DNT细胞及T细胞亚群的研究[J]. 首都医科大学学报, 2012, 33(2): 214-217.
[14]	徐胜利;宣琪;周明. 共聚物-1致敏T淋巴细胞保护MPTP帕金森病模型小鼠黑质多巴胺能神经元[J]. 首都医科大学学报, 2009, 30(5): 611-615.
[15]	刘欣;李萍;赵京霞;梁代英;王燕. 凉血解毒方对JurkatT淋巴细胞增生、活化及细胞因子分泌的影响[J]. 首都医科大学学报, 2009, 30(4): 418-422.