IgE高亲和力受体结合NPC2对DNP-BSA诱导的肥大细胞脱颗粒和组胺释放的机制探讨

doi:10.11958/20252449

摘要/Abstract

摘要：

目的探讨尼曼-匹克病C2型（NPC2）蛋白通过与免疫球蛋白E（IgE）高亲和力受体（FcεRⅠ）结合对二硝基苯酚-牛血清白蛋白（DNP-BSA）诱导的肥大细胞脱颗粒和组胺释放的调控机制。方法收集2023年6月—12月乌鲁木齐市友谊医院呼吸科确诊的5例过敏性哮喘患者（哮喘组）及5例健康对照者（对照组）。取4~6周龄野生型C57BL/6雄性小鼠（5只）和NPC2基因敲除小鼠（3只）。体外实验随机取2只野生型小鼠分离骨髓来源肥大细胞，经NPC2 siRNA转染建立NPC2敲低组（siNPC2组），并设阴性对照组（NC组）及野生型组（WT组）。体内实验采用卵清蛋白（OVA）建立过敏性哮喘小鼠模型（分组同前）。实时荧光定量逆转录聚合酶链反应（qRT-PCR）检测哮喘组和对照组的外周血中NPC2和FcεRⅠ信使核糖核酸（FcεRⅠ mRNA）的表达；流式细胞术检测肥大细胞分化簇117（CD117）、FcεRⅠ以及溶酶体相关膜蛋白1（CD107a）的表达；酶联免疫吸附试验（ELISA）检测肥大细胞炎性因子、趋化因子、集落刺激因子水平，并检测组胺释放水平。免疫共沉淀（Co-IP）实验验证肥大细胞中NPC2与FcεRⅠ的相互作用；苏木精-伊红（HE）染色法检测小鼠耳片和肺组织病理学改变。结果哮喘组患者外周血中NPC2和FcεRⅠ mRNA表达较对照组升高（P<0.05）。体外实验流式细胞术检测显示，体外诱导培养的小鼠骨髓来源肥大细胞纯度达93.4%。与WT组相比，siNPC2组显著抑制DNP-BSA诱导的炎性因子白细胞介素（IL）-4、IL-5、IL-9、IL-13、肿瘤坏死因子α（TNF-α）、趋化因子CCL2和粒细胞-巨噬细胞集落刺激因子（GM-CSF）释放（P<0.05），并降低组胺释放率和CD107a表达水平。Co-IP实验证实NPC2与FcεRⅠ在肥大细胞中存在直接相互作用。体内实验表明，siNPC2组小鼠肺部炎症损伤较WT组明显减轻。结论 NPC2通过与FcεRⅠ结合促进肥大细胞活化和脱颗粒，敲除NPC2可显著抑制过敏反应。

关键词: 哮喘, 肥大细胞, 细胞脱颗粒, 受体, IgE, 尼曼-皮克病, C型, 组胺

Abstract:

Objective To detect the expression of the high-affinity IgE receptor (FcεRⅠ) and Niemann-Pick disease type C2 protein (NPC2) in mast cells treated with 2,4-dinitrophenyl-conjugated bovine serum albumin (DNP-BSA), and to investigate the regulatory mechanism, by which NPC2 protein binding to FcεRⅠ modulating DNP-BSA-induced mast cell degranulation and histamine release. Methods From June to December 2023, five patients with allergic asthma diagnosed in the Department of Respiratory Medicine at Urumqi Friendship Hospital (asthma group) and five healthy controls (control group) were enrolled. Four- to six-week-old male wild-type C57BL/6 mice (n=5) and NPC2 gene knockout mice (n=3) were selected in this study. For in vitro experiments, bone marrow-derived mast cells (BMMCs) were isolated from two randomly selected wild-type mice. NPC2 knockdown was established via siRNA transfection (siNPC2 group), with corresponding negative control group (NC group) and wild-type (WT group) group. For in vivo experiments, an ovalbumin (OVA)-induced allergic asthma mouse model was established (grouping as above). Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to detect NPC2 and FcεRⅠ messenger RNA (mRNA) expression in peripheral blood from the asthma and control groups. Flow cytometry was used to detect the expression of cluster of differentiation 117 (CD117), FcεRⅠ and lysosome-associated membrane protein 1 (CD107a). Enzyme-linked immunosorbent assay (ELISA) was used to measure levels of inflammatory cytokines, chemokines, colony-stimulating factors and histamine release. Co-immunoprecipitation (Co-IP) was performed to verify the interaction between NPC2 and FcεRⅠ in mast cells. Hematoxylin and eosin (HE) staining was used to examine pathological changes in mouse ear tissue and lung tissue. Results The expression of NPC2 and FcεRⅠ mRNA in peripheral blood was significantly higher in the asthma group compared to the control group (P < 0.05). In vitro flow cytometry confirmed that the purity of the cultured BMMCs reached 93.4%. Compared to the WT group, NPC2 knockdown (siNPC2 group) significantly inhibited DNP-BSA-induced release of inflammatory cytokines [interleukin (IL)-4, IL-5, IL-9, IL-13, tumor necrosis factor-α (TNF-α)], chemokine CCL2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05), and reduced the histamine release rate and CD107a expression level. Co-IP experiments confirmed a direct interaction between NPC2 and FcεRⅠ in mast cells. In vivo experiments showed that NPC2 knockdown/knockout (siNPC2 group) mice exhibited significantly alleviated lung inflammatory injury compared to wild-type (WT group) mice. Conclusion NPC2 promotes mast cell activation and degranulation by binding to FcεRⅠ, and knockout of NPC2 significantly suppresses allergic response.

Key words: asthma, mast cells, cell degranulation, receptors, IgE, Niemann-Pick disease, type C, histamine

中图分类号:

R562.25，R341

图/表 7

参考文献 27

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基因名称	引物序列（5'→3'）	产物大小/bp
NPC2	上游：CCAGCTGAGCAAAGGACAGT	380
	下游：TCTGCTACAGAGCACCTCCT	380
FcεRⅠ	上游：TAAGGTCTCCTTGAACCCTCCA	315
	下游：CAACCATGGCACCTGAGGAA	315
GAPDH	上游：AGGTCGGTGTGAACGGATTTG	123
	下游：TGTAGACCATGTAGTTGAGGTCA	123

基因名称	引物序列（5'→3'）	产物大小/bp
NPC2	上游：CCAGCTGAGCAAAGGACAGT	380
	下游：TCTGCTACAGAGCACCTCCT	380
FcεRⅠ	上游：TAAGGTCTCCTTGAACCCTCCA	315
	下游：CAACCATGGCACCTGAGGAA	315
GAPDH	上游：AGGTCGGTGTGAACGGATTTG	123
	下游：TGTAGACCATGTAGTTGAGGTCA	123

质量浓度	CCL2/（ng/L）								GM-CSF/（ng/L）								IL-4/（ng/L）
质量浓度	WT组			siNPC2组			t		WT组			siNPC2组			t		WT组			siNPC2组			t
0 μg/L	15.23±1.25			14.98±1.18			0.342		8.45±0.72			8.23±0.68			0.384		12.58±1.08			12.31±1.02			0.314
3 μg/L	45.67±3.82^a			28.43±2.65^a			6.432^＊＊		32.18±2.85^a			18.76±1.92^a			6.764^＊＊		38.75±3.24^a			22.46±2.18^a			7.225^＊＊
5 μg/L	78.92±6.54^ab			42.15±3.87^ab			8.381^＊＊		56.34±4.73^ab			29.82±2.74^ab			8.403^＊＊		65.82±5.47^ab			34.27±3.15^ab			8.657^＊＊
F	264.318^＊＊			147.925^＊＊					238.647^＊＊			175.384^＊＊					220.735^＊＊			152.418^＊＊
DNP-BSA含量		IL-5/（ng/L）								IL-9/（ng/L）								IL-13/（ng/L）
DNP-BSA含量		WT组			siNPC2组			t		WT组			siNPC2组			t		WT组			siNPC2组			t
0 μg/L		10.87±0.94			10.65±0.89			0.294		9.63±0.82			9.42±0.78			0.321		11.24±0.97			10.98±0.91			0.338
3 μg/L		35.42±2.96^a			20.18±1.87^a			7.539^＊＊		28.54±2.38^a			16.87±1.65^a			7.033^＊＊		33.67±2.81^a			19.52±1.83^a			7.308^＊＊
5 μg/L		58.76±4.82^ab			30.45±2.79^ab			8.804^＊＊		47.85±3.92^ab			25.63±2.34^ab			8.430^＊＊		62.38±5.16^ab			32.74±3.02^ab			8.586^＊＊
F		249.382^＊＊			198.647^＊＊					196.528^＊＊			158.739^＊＊					260.417^＊＊			215.683^＊＊
DNP-BSA含量			TNF-α/（ng/L）											组胺/%
DNP-BSA含量			WT组			siNPC2组					t			WT组					siNPC2组			t
0 μg/L			13.76±1.15			13.48±1.12					0.302			5.32±0.43					5.18±0.41			0.408
3 μg/L			42.35±3.58^a			24.87±2.32					7.097^＊＊			28.65±2.54^a					15.73±1.48^a			7.612^＊＊
5 μg/L			71.29±5.94^ab			37.56±3.45^ab					8.504^＊＊			58.50±4.32^ab					21.41±2.43^ab			12.961^＊＊
F			232.816^＊＊			173.952^＊＊								378.425^＊＊					149.287^＊＊

质量浓度	CCL2/（ng/L）								GM-CSF/（ng/L）								IL-4/（ng/L）
质量浓度	WT组			siNPC2组			t		WT组			siNPC2组			t		WT组			siNPC2组			t
0 μg/L	15.23±1.25			14.98±1.18			0.342		8.45±0.72			8.23±0.68			0.384		12.58±1.08			12.31±1.02			0.314
3 μg/L	45.67±3.82^a			28.43±2.65^a			6.432^＊＊		32.18±2.85^a			18.76±1.92^a			6.764^＊＊		38.75±3.24^a			22.46±2.18^a			7.225^＊＊
5 μg/L	78.92±6.54^ab			42.15±3.87^ab			8.381^＊＊		56.34±4.73^ab			29.82±2.74^ab			8.403^＊＊		65.82±5.47^ab			34.27±3.15^ab			8.657^＊＊
F	264.318^＊＊			147.925^＊＊					238.647^＊＊			175.384^＊＊					220.735^＊＊			152.418^＊＊
DNP-BSA含量		IL-5/（ng/L）								IL-9/（ng/L）								IL-13/（ng/L）
DNP-BSA含量		WT组			siNPC2组			t		WT组			siNPC2组			t		WT组			siNPC2组			t
0 μg/L		10.87±0.94			10.65±0.89			0.294		9.63±0.82			9.42±0.78			0.321		11.24±0.97			10.98±0.91			0.338
3 μg/L		35.42±2.96^a			20.18±1.87^a			7.539^＊＊		28.54±2.38^a			16.87±1.65^a			7.033^＊＊		33.67±2.81^a			19.52±1.83^a			7.308^＊＊
5 μg/L		58.76±4.82^ab			30.45±2.79^ab			8.804^＊＊		47.85±3.92^ab			25.63±2.34^ab			8.430^＊＊		62.38±5.16^ab			32.74±3.02^ab			8.586^＊＊
F		249.382^＊＊			198.647^＊＊					196.528^＊＊			158.739^＊＊					260.417^＊＊			215.683^＊＊
DNP-BSA含量			TNF-α/（ng/L）											组胺/%
DNP-BSA含量			WT组			siNPC2组					t			WT组					siNPC2组			t
0 μg/L			13.76±1.15			13.48±1.12					0.302			5.32±0.43					5.18±0.41			0.408
3 μg/L			42.35±3.58^a			24.87±2.32					7.097^＊＊			28.65±2.54^a					15.73±1.48^a			7.612^＊＊
5 μg/L			71.29±5.94^ab			37.56±3.45^ab					8.504^＊＊			58.50±4.32^ab					21.41±2.43^ab			12.961^＊＊
F			232.816^＊＊			173.952^＊＊								378.425^＊＊					149.287^＊＊

质量浓度	WT组	siNPC2组	t
0 μg/L	5.32±0.43	5.18±0.43	0.408
3 μg/L	28.65±2.54^a	15.73±1.48^a	7.612^＊＊
5 μg/L	58.50±4.32^ab	21.41±2.43^ab	12.961^＊＊
F	378.425^＊＊	149.287^＊＊