乌司他丁对脓毒症小鼠调节性T细胞凋亡及细胞因子分泌的影响

doi:10.3877/cma.j.issn.1674-6880.2017.03.002

目的

观察乌司他丁对脓毒症小鼠调节性T细胞（Treg）凋亡及细胞因子分泌的影响。

方法

将60只小鼠分为假手术组、模型组和乌司他丁组，每组20只。采用盲肠结扎穿孔方法制备脓毒症大鼠模型，假手术组除不结扎和穿刺盲肠外，其余手术步骤相同，乌司他丁组小鼠术后30 min尾静脉给药100 000 U/kg。采用流式细胞仪检测各组小鼠CD4⁺CD25⁺Treg的凋亡率，采用酶联免疫吸附试验检测各组小鼠白细胞介素2（IL-2）、IL-4、干扰素γ的水平。

结果

假手术组、模型组和乌司他丁组小鼠CD4⁺CD25⁺Treg细胞的凋亡率比较，差异有统计学意义[（20.1 ± 1.0）%、（10.9 ± 0.8）%、（13.7 ± 0.8）%，F = 10.236，P = 0.030]，且乌司他丁组小鼠显著低于假手术组，而高于模型组（P均< 0.05）。同时，与假手术组比较，模型组与乌司他丁组IL-2[（352 ± 76）、（172 ± 59）、（249 ± 64）ng/L]和干扰素γ[（177 ± 25）、（56 ± 14）、（109 ± 18）ng/L]均显著下降，而IL-4显著升高[（46 ± 15）、（328 ± 81）、（242 ± 62）ng/L]，且与模型组比较，乌司他丁组IL-2和干扰素γ均明显升高，而IL-4明显降低（P均< 0.05）。

结论

乌司他丁可诱导脓毒症小鼠Treg细胞凋亡，上调IL-2和干扰素γ，降低IL-4水平。

关键词: 脓毒症, 细胞凋亡, 细胞因子类, 调节性T细胞, 乌司他丁, 小鼠

Objective

To investigate the effect of ulinastatin on the apoptosis of regulatory T cells (Tregs) and cytokine secretion in septic mice.

Methods

A total of 60 male mice were randomized into the sham group, model group and ulinastatin group, 20 mice in each group. The model of sepsis was reproduced by cecal ligation and perforation, the mice in the sham group received celiotomy without ligation and puncture, and the mice in the ulinastatin group were given 100 000 U/kg ulinastatin by tail intravenous injection at 30 min after operation. The apoptosis rates of regulatory T cells were detected by flow cytometry, and the level of interleukin-2 (IL-2), IL-4 and interferon γ were determined by enzyme-linked immunosorbant assay.

Results

The apoptosis rates of Tregs in the sham group, model group and ulinastatin group showed significant differences [(20.1 ± 1.0)%, (10.9 ± 0.8)%, (13.7 ± 0.8)%, F = 10.236, P = 0.030], and the apoptosis rates of Tregs in the ulinastatin group were much lower than those in the sham group, and much higher than those in the model group (all P < 0.05). Meanwhile, the levels of IL-2 [(352 ± 76), (172 ± 59), (249 ± 64) ng/L] and interferon γ [(177 ± 25), (56 ± 14),(109 ± 18) ng/L] in the model group and ulinastatin group decreased obviously as compared with the sham group, and it decreased most in the model group (all P < 0.05). The levels of IL-4 in the model group and ulinastatin group were much higher than those in the sham group, and were highest in the model group [(46 ± 15), (328 ± 81), (242 ± 62) ng/L, all P < 0.05].

Conclusion

Ulinastatin may induce the apoptosis of Tregs, improve the levels of IL-2 and interferon γ, and reduce the levels of IL-4.

Key words: Sepsis, Apoptosis, Cytokines, Regulatory T cells, Ulinastatin, Mice

表1 各组小鼠CD4⁺CD25⁺Treg细胞的凋亡率（ ± s）

组别	细胞数	凋亡率（%）
假手术组	1.58 × 10⁵	20.1 ± 1.0
模型组	1.82 × 10⁵	10.9 ± 0.8^a
乌司他丁组	1.82 × 10⁵	13.7 ± 0.8^ab
F值	?	10.236
P值	?	0.030

表1 各组小鼠CD4⁺CD25⁺Treg细胞的凋亡率（ ± s）

组别	细胞数	凋亡率（%）
假手术组	1.58 × 10⁵	20.1 ± 1.0
模型组	1.82 × 10⁵	10.9 ± 0.8^a
乌司他丁组	1.82 × 10⁵	13.7 ± 0.8^ab
F值	?	10.236
P值	?	0.030

表2 各组小鼠细胞因子IL-2、IL-4及IFN-γ水平变化（ng/L， ± s）

组别	细胞数	IL-2	IL-4	干扰素γ
假手术组	2 × 10⁶	352 ± 76	46 ± 15	177 ± 25
模型组	2 × 10⁶	172 ± 59^a	328 ± 81^a	56 ± 14^a
乌司他丁组	2 × 10⁶	249 ± 64^ab	242 ± 62^ab	109 ± 18^ab
F值	?	8.579	9.679	12.546
P值	?	0.005	0.004	0.003

表2 各组小鼠细胞因子IL-2、IL-4及IFN-γ水平变化（ng/L， ± s）

组别	细胞数	IL-2	IL-4	干扰素γ
假手术组	2 × 10⁶	352 ± 76	46 ± 15	177 ± 25
模型组	2 × 10⁶	172 ± 59^a	328 ± 81^a	56 ± 14^a
乌司他丁组	2 × 10⁶	249 ± 64^ab	242 ± 62^ab	109 ± 18^ab
F值	?	8.579	9.679	12.546
P值	?	0.005	0.004	0.003

1	Unsinger J, McGlynn M, Kasten KR, et al. IL-7 promotes T cell viability, trafficking, and functionality and improves survival in sepsis[J]. J Immunol, 2010, 184 (7): 3768-3779.
2	Wan YY. Regulatory T cells: immune suppression and beyond[J]. Cell Mol Immunol, 2010, 7 (3): 204-210.
3	Sakaguchi S, Yamaguchi T, Nomura T, et al. Regulatory T cells and immune tolerance[J]. Cell, 2008, 133 (5): 775-787.
4	Cao C, Ma T, Chai YF, et al. The role of regulatory T cells in immune dysfunction during sepsis[J]. World J Emerg Med, 2015, 6 (1): 5-9.
5	Gadina M, O’Shea JJ. Immune modulation: Turncoat regulatory T cells[J]. Nat Med, 2009, 15 (12): 1365.
6	李国辉，马纪林，周红娟，等. 调节性T细胞及辅助性T细胞17在严重脓毒症患者中的表达及对预后影响[J/CD]. 中华危重症医学杂志（电子版），2014，7(6):377-381.
7	Suh HC, Benoukraf T, Shyamsunder P, et al. LPS independent activation of the pro-inflammatory receptor Trem1 by C/EBPε in granulocytes[J]. Sci Rep, 2017 (7): 46440.
8	Shimaoka M, Park EJ. Advances in understanding sepsis[J]. Eur J Anaesthesiol Suppl, 2008 (42): 146-153.
9	Venet F, Pachot A, Debard AL, et al. Increased percentage of CD4⁺CD25⁺ regulatory T cells during septic shock is due to the decrease of CD4⁺CD25^- lymphocytes[J]. Crit Care Med, 2004, 32 (11): 2329-2331.
10	Sakaguchi S, Ono M, Setoguchi R, et al. Foxp3⁺CD25⁺CD4⁺ natural regulatory T cells in dominant self-tolerance and autoimmune disease[J]. Immunol Rev, 2006 (212): 8-27.
11	Taylor AL, Llewelyn MJ. Superantigeninduced proliferation of human CD4⁺CD25^-T cells is followed by a switch to a functional regulatory phenotype[J]. J Immunol, 2010, 185 (11): 6591-6598.
12	Nascimento DC, Alves-Filho JC, Sonego F, et al. Role of rugulatory T cells in long-term immune dysfunction associated with severe sepsis[J]. Crit Care Med, 2010, 38 (8): 1718-1725.
13	Rittirsch D, Huber-Lang MS, Flierl MA, et al. Immunodesign of experimental sepsis by cecal ligation and puncture[J]. Nat Protoc, 2009, 4 (1): 31-36.
14	吴铁军，张丽娜，亢翠翠. 乌司他丁对严重脓毒症患者炎症免疫失衡的调理作用[J]. 中华危重病急救医学，2013，25(4):219-223.
15	Chen X, Wang Y, Luo H, et al. Ulinastatin reduces urinary sepsis-related inflammation by upregulating IL-10 and downregulating TNF-α levels[J]. Mol Med Rep, 2013, 8 (1): 29-34.
16	Feng M, Shu Y, Yang Y, et al. Ulinastatin attenuates experimental autoimmune encephalomyelitis by enhancing anti-inflammatory responses[J]. Neurochem Int, 2014 (64): 64-72.
17	张莹，姚咏明，于燕，等. 高迁移率族蛋白B1对小鼠调节性T细胞与CD4⁺CD25^-T细胞相互作用的影响[J]. 中华外科杂志，2008，46(3):217-220.
18	Hao X, Han J, Xing Z, et al. Urinary trypsin inhibitor attenuated inflammatory response of patients undergoing cardiopulmonary bypass by inducing activated Treg cells[J]. Inflammation, 2013, 36 (6): 1279-1285.
19	Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes[J]. Science, 2002, 298 (5594): 850-854.
20	Curiel TJ, Coukos G, Zou L, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[J]. Nat Med, 2004, 10 (9): 942-949.

[1]	庄燕, 戴林峰, 张海东, 陈秋华, 聂清芳. 脓毒症患者早期生存影响因素及Cox 风险预测模型构建[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(05): 372-378.
[2]	杨瑾, 刘雪克, 张媛媛, 金钧, 韦瑶. 肠道微生物来源石胆酸对脓毒症相关肝损伤的保护作用[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(04): 265-274.
[3]	张霞, 张瑞, 郑志波, 张勤. 紫草素调控乳酸化修饰和线粒体功能改善脓毒症心肌病小鼠的预后[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(04): 275-284.
[4]	张婧琦, 江洋, 孙佳璐, 唐兴喆, 赵宇飞, 崔颖, 李信响, 戴景月, 傅琳, 彭新桂. 基于肾周CT特征结合血清肌酐水平探讨脓毒症伴急性肾损伤的早期识别[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(04): 285-292.
[5]	钟雅雯, 王煜, 王海臻, 黄莉萍. 肌苷通过抑制线粒体通透性转换孔开放缓解缺氧/复氧诱导的人绒毛膜滋养层细胞凋亡[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(05): 525-533.
[6]	唐亦骁, 陈峻, 连正星, 胡海涛, 鲁迪, 徐骁, 卫强. 白果内酯对小鼠肝缺血再灌注损伤保护作用研究[J/OL]. 中华移植杂志(电子版), 2024, 18(05): 278-282.
[7]	郑俊, 吴杰英, 谭海波, 郑安全, 李腾成. EGFR-MEK-TZ三联合分子的构建及其对去势抵抗性前列腺癌细胞增殖与凋亡的影响[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 503-508.
[8]	黄程鑫, 陈莉, 刘伊楚, 王水良, 赖晓凤. OPA1 在乳腺癌组织的表达特征及在ER阳性乳腺癌细胞中的生物学功能研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(05): 275-284.
[9]	张英信, 林婷, 张剑文. 构建靶向HLA-A2且表达PD-L1的CAR-Treg细胞及验证其对CD4⁺T细胞抑制作用[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(05): 719-728.
[10]	杜霞, 马梦青, 曹长春. 造影剂诱导的急性肾损伤的发病机制及干预靶点研究进展[J/OL]. 中华肾病研究电子杂志, 2024, 13(05): 279-282.
[11]	成人脓毒症患者β-内酰胺类抗生素延长输注专家共识编写组. 成人脓毒症患者β-内酰胺类抗生素延长输注专家共识[J/OL]. 中华重症医学电子杂志, 2024, 10(04): 313-324.
[12]	陈曦, 吴宗盛, 郑明珠, 邱海波. 胸腺萎缩在脓毒症免疫紊乱中的研究进展[J/OL]. 中华重症医学电子杂志, 2024, 10(04): 379-383.
[13]	杨翔, 郭兰骐, 谢剑锋, 邱海波. 转录组学在脓毒症诊疗中的临床研究进展[J/OL]. 中华重症医学电子杂志, 2024, 10(04): 384-388.
[14]	陈惠英, 邱敏珊, 邵汉权. 脓毒症诱发肠黏膜屏障功能损伤的风险因素模型构建与应用效果[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(05): 448-452.
[15]	傅新露, 李之岳, 卢丹. 妊娠合并结肠癌穿孔致脓毒症休克一例并文献复习[J/OL]. 中华产科急救电子杂志, 2024, 13(04): 227-231.

阅读次数

全文

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	0	0	0	0

摘要

最新录用	在线预览	正式出版

0	0	28

来源	本网站	其他网站

次数	8	21
比例	28%	72%

选择文件类型/文献管理软件名称

选择包含的内容

Effect of ulinastatin on the apoptosis of regulatory T cells and cytokine secretion in septic mice

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Effect of ulinastatin on the apoptosis of regulatory T cells and cytokine secretion in septic mice