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

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

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