奥马珠单抗调控Wnt/β-catenin信号通路对哮喘小鼠气道平滑肌细胞增殖和迁移的影响

doi:10.13481/j.1671-587X.20260518

摘要/Abstract

摘要：

目的探讨奥马珠单抗通过调控无翅型MMTV整合位点家族蛋白（Wnt）/β-连环蛋白（β-catenin）信号通路对哮喘小鼠气道平滑肌细胞（ASMCs）增殖和迁移的影响。方法将12只小鼠随机分为正常组和模型组，每组6只，采用卵清蛋白和氢氧化铝混合液致敏及激发的方式构建哮喘小鼠模型。取2组小鼠支气管组织，分离原代ASMCs，采用免疫荧光染色法鉴定ASMCs中α-平滑肌肌动蛋白（α-SMA）阳性表达。ASMCs分为对照组、哮喘组、奥马珠单抗组、iCRT3（Wnt/β-catenin信号通路抑制剂）组和奥马珠单抗+SKL2001（Wnt/β-catenin信号通路激活剂）组。对照组ASMCs为正常组小鼠支气管组织分离的ASMCs；其他组ASMCs均为模型组小鼠支气管组织分离的ASMCs，并进行相应处理。采用5-乙炔基-2'-脱氧尿苷（EdU）染色法和细胞计数试剂盒8（CCK-8）法检测各组ASMCs中EdU阳性细胞百分比及增殖活性，细胞划痕实验法检测各组ASMCs细胞划痕愈合率，酶联免疫吸附试验（ELISA）法检测各组ASMCs上清液中白细胞介素（IL）-6、IL-17、肿瘤坏死因子α（TNF-α）、IL-33和IL-13水平，Western blotting法检测各组ASMCs中增殖细胞核抗原（PCNA）、基质金属蛋白酶9（MMP-9）、Wnt 3a和β-catenin蛋白表达水平。结果在正常组和模型组小鼠支气管组织中分离的细胞中，98%以上的细胞呈α-SMA阳性，表明成功分离ASMCs。与对照组比较，哮喘组ASMCs中EdU阳性细胞百分比，细胞增殖活性，划痕愈合率，上清液中IL-6、IL-17、TNF-α、IL-33和IL-13水平以及细胞中PCNA、MMP-9、Wnt 3a和β-catenin蛋白表达水平均明显升高（P<0.05）。与哮喘组比较，奥马珠单抗组和iCRT3组ASMCs中EdU阳性细胞百分比，细胞增殖活性，划痕愈合率，上清液中IL-6、IL-17、TNF-α、IL-33及IL-13水平以及细胞中PCNA、MMP-9、Wnt 3a和β-catenin蛋白表达水平均明显降低（P<0.05）；与奥马珠单抗组比较，奥马珠单抗+SKL2001组ASMCs中上述指标均明显升高（P<0.05）。结论奥马珠单抗可抑制哮喘小鼠ASMCs增殖、迁移和炎症反应，其作用机制与抑制Wnt/β-catenin信号通路有关。

关键词: 奥马珠单抗, 哮喘, 气道平滑肌细胞, 细胞增殖, 细胞迁移

Abstract:

Objective To investigate the effect of omalizumab on the proliferation and migration of the airway smooth muscle cells （ASMCs） in the asthmatic mice by regulating wingless-type MMTV integration site family（Wnt）/β-catenin signal pathway. Methods Twelve mice were randomly divided into normal group and model group. The mouse model of asthma was constructed by sensitizing and stimulating the mixture of ovalbumin and aluminum hydroxide. The primary ASMCs were isolated from the bronchial tissue of the mice in normal group and model group. Immunofluorescence staining method was used to detect the positive expression of alpha-smooth muscle actin （α-SMA） in ASMCs. The ASMCs were divided into control group， asthma group， omalizumab group， iCRT3 （Wnt/β-catenin signaling pathway inhibitor） group， and omalizumab+SKL2001 （Wnt/β-catenin pathway activator） group. The ASMCs in control group were isolated from the bronchial tissues of mice in normal group. The ASMCs in other groups were isolated from the bronchial tissues of mice in model group and received corresponding treatments. 5-ethynyl-2'-deoxyuridine （EdU） staining method and cell counting kit-8 （CCK-8） method were used to detect the percentages of EdU-positive cells and proliferation activities in ASMCs in various groups. Wound healing assay was used to detect the wound healing rates in ASMCs in various groups. Enzyme-linked immunosorbent assay （ELISA） method was used to detect the levels of interleukin （IL）-6， IL-17， tumor necrosis factor-α （TNF-α）， IL-33， and IL-13 in the supernatants of ASMCs in various groups. Western blotting method was used to detect the expression levels of proliferating cell nuclear antigen （PCNA）， matrix metalloproteinase 9 （MMP-9）， Wnt 3a， and β-catenin proteins in ASMCs in various groups. Results Over 98% of the cells isolated from the bronchial tissues of the mice in normal group and model group exhibited positive α-SMA expression， confirming the successful isolation of ASMCs. Compared with control group， the EdU-positive rate in ASMCs， proliferation activity， wound healing rate， the levels of IL-6， IL-17， TNF-α， IL-33， and IL-13 in the supernatant， and the expression levels of PCNA， MMP-9， Wnt 3a， and β-catenin proteins in ASMCs in asthma group were significantly increased （P<0.05）. Compared with asthma group， the EdU-positive rates in ASMCs， proliferation activities， wound healing rates， the levels of IL-6， IL-17， TNF-α， IL-33， and IL-13 in the supernatants， and the expression levels of PCNA， MMP-9， Wnt 3a， and β-catenin proteins in ASMCs in omalizumab group and iCRT3 group were significantly decreased （P<0.05）. Compared with omalizumab group， the above indices in ASMCs were significantly increased in omalizumab+SKL2001 group （P<0.05）. Conclusion Omalizumab inhibits the proliferation， migration and inflammatory response of ASMCs in the asthmatic mice， and its mechanism is related to inhibiting Wnt/β-catenin signal pathway.

Key words: Omalizumab, Asthma, Airway smooth muscle cells, Cell proliferation, Cell migration

中图分类号:

R562.25

任爽,高晓超,王燕,刘艳梅,孟文霞. 奥马珠单抗调控Wnt/β-catenin信号通路对哮喘小鼠气道平滑肌细胞增殖和迁移的影响[J]. 吉林大学学报(医学版), 2026, 52(5): 1369-1376.

Shuang REN,Xiaochao GAO,Yan WANG,Yanmei LIU,Wenxia MENG. Effects of omalizumab on proliferation and migration of airway smooth muscle cells in asthmatic mice by regulating Wnt/β-catenin signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2026, 52(5): 1369-1376.

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Group	Percentage ofEdU-positive cell(η/%)	Proliferation activity
Control	10.69±0.75	0.46±0.05
Asthma	28.74±1.92^*	1.27±0.16^*
Omalizumab	14.96±0.85^△	0.59±0.05^△
iCRT3	16.22±0.91^△	0.67±0.07^△
Omalizumab+SKL2001	20.89±1.73^#	0.89±0.08^#

Group	IL-6	IL-17	TNF-α	IL-33	IL-13
Control	131.67±7.22	26.69±1.58	54.62±3.25	15.69±1.04	43.68±2.59
Asthma	276.69±15.56^*	87.72±5.16^*	165.54±10.27^*	46.78±2.63^*	89.76±5.05^*
Omalizumab	162.28±10.43^△	39.83±2.25^△	78.86±4.46^△	26.81±1.54^△	56.81±2.96^△
iCRT3	150.67±8.89^△	46.69±2.58^△	83.61±5.29^△	28.67±1.83^△	61.62±4.04^△
Omalizumab+SKL2001	212.36±12.26^#	60.63±4.18^#	123.37±7.75^#	37.44±2.15^#	69.22±4.05^#

Group	PCNA	MMP-9	Wnt 3a	β-catenin
Control	1.06±0.19	0.71±0.09	0.54±0.06	0.15±0.02
Asthma	2.73±0.25^*	2.05±0.22^*	1.38±0.19^*	0.81±0.09^*
Omalizumab	1.56±0.14^△	1.08±0.15^△	0.69±0.07^△	0.29±0.03^△
iCRT3	1.71±0.16^△	1.19±0.18^△	0.80±0.09^△	0.35±0.04^△
Omalizumab+SKL2001	2.08±0.18^#	1.52±0.17^#	1.01±0.13^#	0.57±0.06^#