自然杀伤细胞在屋尘螨致敏哮喘小鼠发病中的变化

doi:10.3877/cma.j.issn.1673-5250.2013.01.012

目的

探讨自然杀伤T(NKT)细胞在屋尘螨提取液(HDM)致敏哮喘小鼠发病机制中的作用。

方法

选择6～8周雌性无特定病原体(specific pathogen free，SPF)级BALB/C小鼠16只为研究对象，并随机分为研究组(n＝8)和对照组(n＝8)。研究组采用HDM致敏和激发以建立哮喘小鼠模型。对照组采用0.1 mol/L磷酸盐缓冲液(PBS)替代HDM进行致敏和激发，建立小鼠模型。小鼠气道反应性测定采用肺阻抗法。支气管肺泡灌洗液(BALF)中白细胞介素(IL)-4、干扰素(IFN)-γ含量及血清总免疫球蛋白(Ig)E测定采用酶联免疫法(ELISA)。肺组织T-bet，GATA-3 mRNA表达水平检测采用逆转录-聚合酶链反应(RT-PCR)法。NKT细胞及其成熟细胞比例检测采用流式细胞学技术。

结果

研究组小鼠气道反应性明显高于对照组，两组比较，差异有统计学意义(P<0.01)。两组小鼠BALF中细胞总数和嗜酸性粒细胞(EOC)计数比较，差异有统计学意义(P<0.01)。研究组小鼠BALF中IL-4和总IgE水平均显著高于对照组，而IFN-γ水平显著低于对照组，两组比较，差异均有统计学意义(P<0.01)。研究组小鼠T-bet mRNA水平、NKT及其成熟细胞比例明显低于对照组，而GATA-3 mRNA水平显著高于对照组，两组比较，差异均有统计学意义(P<0.01)。两组小鼠NKT细胞及其成熟细胞比例与小鼠肺组织T-bet mRNA表达水平呈显著正相关性关系(r＝0.767，P<0.01；r＝0.757，P<0.01)，而与GATA-3 mRNA表达水平则呈显著负相关性关系(r＝－0.8871，P<0.01；r＝－0.727，P<0.01)。

结论

NKT细胞可能在屋尘螨致敏哮喘小鼠发病机制中起重要作用。

关键词: 自然杀伤T细胞, 哮喘, 小鼠

Objective

To explore the effect of natural killer T (NKT) cells on the pathology of allergic asthma sensitized with house dust mite extracts(HDM).

Methods

A total of 16 specific pathogen free (SPF) BALB/c mices were included in the study, and randomly divided into research group (n=8) and control group(n=8). Research group was sensitized by HDM to generate the murine model of asthma.Control group was treated with phosphate buffered saline (PBS) instead of HDM. Airway hyperactivity (AHR) was analyzed by pulmonary impedance method. The level of IL-4 and IFN-γ in bronchoalveolar lavage fluid(BALF) and the level of total IgE were detected by ELISA. The mRNA expression of T-bet and GATA-3 was measured by RT-PCR. The number of NKT and the proportion of mature cells were analyzed by flow cytometry.

Results

Compared to control group, AHR of research group was increased significantly(P<0.01). The total cells and eosinophils (EOS) of BALF in research group were significantly higher than those of control group(P<0.01). There had significant differences in the level of IL-4, IFN-γ and total IgE in BALF between two groups(P<0.01). There also had significant differences in the mRNA expression of T-bet, GATA-3, the number of NKT cells and the proportion of mature cells between two groups(P<0.01). The number of NKT cells and the proportion of mature cells had positive correlation with the expression of T-bet (r=0.767, P<0.01; r=0.757, P<0.01), and had negative correlation with the expression of GATA-3(r=-0.8871, P<0.01; r=-0.727, P<0.01).

Conclusions

The NKT cells might play an important role in the pathology of murine model of asthma challenged with HDM extracts.

Key words: natural killer T cells, asthma, mice

图1 两组小鼠气道反应性比较

Figure 1 Comparison of airway hyperresponsiveness assay between two groups

表1 两组小鼠BALF中细胞总数和EOS计数比较(±s，×10⁵/mL)

Table 1 Comparison of the number of total cells and EOS count in BALF between two groups(±s，×10⁵/mL)

组别	n	细胞总数	EOS计数
研究组	8	7.32±1.25	1.77±0.67
对照组	8	2.12±0.49	0.0
t	?	－10.90	－7.43
P	?	0.00	0.00

表1 两组小鼠BALF中细胞总数和EOS计数比较(±s，×10⁵/mL)

Table 1 Comparison of the number of total cells and EOS count in BALF between two groups(±s，×10⁵/mL)

组别	n	细胞总数	EOS计数
研究组	8	7.32±1.25	1.77±0.67
对照组	8	2.12±0.49	0.0
t	?	－10.90	－7.43
P	?	0.00	0.00

表2 两组小鼠BALF中IL-4，IFN-γ和血清中总IgE含量比较(±s, pg/mL)

Table 2 Comparison of the levels of IL-4, IFN-γ and IgE in BALF between two groups(±s, pg/mL)

组别	n	IL-4	IFN-γ	总IgE
研究组	8	137.52±22.30	314.21±187.83	19.45±3.48
对照组	8	62.23±16.15	733.11±168.09	7.57±1.24
t	?	－7.73	4.70	－9.09
P	?	0.00	0.00	0.00

表2 两组小鼠BALF中IL-4，IFN-γ和血清中总IgE含量比较(±s, pg/mL)

Table 2 Comparison of the levels of IL-4, IFN-γ and IgE in BALF between two groups(±s, pg/mL)

组别	n	IL-4	IFN-γ	总IgE
研究组	8	137.52±22.30	314.21±187.83	19.45±3.48
对照组	8	62.23±16.15	733.11±168.09	7.57±1.24
t	?	－7.73	4.70	－9.09
P	?	0.00	0.00	0.00

表3 两组小鼠肺组织T-bet，GATA-3 mRNA表达水平比较(±s)

Table 3 Comparison of the level of T-bet and GATA-3 mRNA expression of lung between two groups(±s)

组别	n	T-bet mRNA	GATA-3 mRNA
研究组	8	0.70±0.10	1.11±0.11
对照组	8	0.97±0.10	0.79±0.16
t	?	5.42	－4.61
P	?	0.00	0.00

表3 两组小鼠肺组织T-bet，GATA-3 mRNA表达水平比较(±s)

Table 3 Comparison of the level of T-bet and GATA-3 mRNA expression of lung between two groups(±s)

组别	n	T-bet mRNA	GATA-3 mRNA
研究组	8	0.70±0.10	1.11±0.11
对照组	8	0.97±0.10	0.79±0.16
t	?	5.42	－4.61
P	?	0.00	0.00

表4 两组小鼠NKT细胞及其成熟细胞比例比较(±s，%)

Table 4 Comparison of the proportions of NKT cells and mature NKT cells between two groups(±s，%)

组别	n	NKT细胞	成熟NKT细胞
研究组	8	20.61±2.90	62.46±5.25
对照组	8	31.39±3.80	73.91±3.29
t	?	6.38	5.22
P	?	0.00	0.00

表4 两组小鼠NKT细胞及其成熟细胞比例比较(±s，%)

Table 4 Comparison of the proportions of NKT cells and mature NKT cells between two groups(±s，%)

组别	n	NKT细胞	成熟NKT细胞
研究组	8	20.61±2.90	62.46±5.25
对照组	8	31.39±3.80	73.91±3.29
t	?	6.38	5.22
P	?	0.00	0.00

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The Effect of Natural Killer T Cells on the Pathogenesis of Allergic Asthma Challenged With House Dust Mite Extracts

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The Effect of Natural Killer T Cells on the Pathogenesis of Allergic Asthma Challenged With House Dust Mite Extracts