过表达肝细胞生长因子的人牙髓来源间充质干细胞通过调节Th2/Th1、Th17/Treg平衡改善变应性鼻炎的研究

Study of HGF/hDPSCs ameliorating allergic rhinitis through regulating Th1/Th2 and Th17/Treg balance

  • 摘要:
    背景 间充质干细胞治疗变应性鼻炎是研究热点,过表达肝细胞生长因子的间充质干细胞在其中的作用和机制有待进一步探究。
    目的 构建过表达肝细胞生长因子的人牙髓来源间充质干细胞(hepatocyte growth factor-overexpressing human dental pulp-stem cells,HGF/hDPSCs),研究其对变应性鼻炎的作用和可能机制。
    方法 提取人牙髓来源间充质干细胞,利用腺病毒载体Ad5将靶基因HGF转入,以构建HGF/hDPSCs,并评估其HGF分泌能力和鼻黏膜分布。采取卵清蛋白腹腔注射致敏、滴鼻激发的方式构建变应性鼻炎小鼠模型。根据致敏、激发和干预措施的区别,将小鼠分为4组:WT-PBS组(阴性对照组),AR-PBS组(阳性对照组),AR-hDPSCs组(hDPSCs治疗组),AR-HGF/hDPSCs组(HGF/hDPSCs治疗组)。利用症状评分、病理学分析(HE、PAS染色)监测各组小鼠变应性鼻炎症状和鼻黏膜局部炎症情况。采取免疫组织化学染色(CD3、CD19)、ELISA(总IgE、OVA特异IgE)和PCR(IFN-γ、IL-5、IL-13、IL-17A、Foxp3)探究免疫应答机制。
    结果 HGF/hDPSCs可在体外和体内稳定表达HGF,经尾静脉注射后可迁移至小鼠鼻黏膜。症状评分显示,HGF/hDPSCs可改善变应性鼻炎小鼠挠鼻、喷嚏、流涕等过敏症状(P<0.05)。病理分析和免疫组化结果显示,变应性鼻炎小鼠鼻黏膜免疫反应以T淋巴细胞聚集为主要特征。应用HGF/hDPSCs可减少鼻黏膜淋巴细胞浸润和杯状细胞增生等鼻黏膜炎症表现,并降低病理损伤评分(P<0.01)。ELISA结果表明,HGF/hDPSCs可下调血清总IgE和OVA特异性IgE表达水平(P<0.01)。PCR结果表明,HGF/hDPSCs可抑制变应性鼻炎小鼠脾Th1细胞(IFN-γ)、Th2细胞(IL-5、IL-13)应答水平,改变了Th2/Th1平衡;下调Th17细胞(IL-17A)应答水平(P<0.05),不影响Treg细胞(Foxp3)应答水平(P>0.05),改变了Th17/Treg平衡。
    结论 HGF/hDPSCs可通过调节Th2/Th1、Th17/Treg平衡缓解变应性鼻炎小鼠的鼻部过敏症状,减轻鼻黏膜炎症。

     

    Abstract:
    Background Mesenchymal stem cells (MSCs) therapy for allergic rhinitis (AR) is a research hotspot in this field, and the effectiveness and mechanism of hepatocyte growth factor (HGF)-overexpressing MSCs in AR treatment remains to be further explored.
    Objective To evaluate the function of HGF-overexpressing human dental pulp stem cells (HGF/hDPSCs) in AR treatment and discover the underlying mechanisms in a mouse model.
    Methods Firstly, dental pulp stem cells were isolated from human and transfected with the target gene HGF using the Adenovirus 5 (Ad5) vector to create HGF/hDPSCs. The in vivo study was performed to determine the secretion and distribution of HGF in nasal mucosa. An allergic rhinitis mouse model was established through intraperitoneal injection of ovalbumin (OVA) for sensitization and nasal drop for challenge. Based on differences in sensitization, challenge, and intervention, the mice were divided into 4 groups: WT-PBS group (negative control group), AR-PBS group (positive control group), AR-hDPSCs group (hDPSCs treatment group), and AR-HGF/hDPSCs group (HGF/hDPSCs treatment group). Symptom scoring and pathological analysis, including HE and PAS staining, were used to monitor the symptoms of allergic rhinitis and inflammation of the nasal mucosa. Finally, the immune response mechanism was explored using immunohistochemical staining for CD3 and CD19, ELISA for total IgE, and OVA-specific IgE, and qPCR for IFN-γ, IL-5, IL-13, IL-17A, and Foxp3.
    Results HGF/hDPSCs expressed HGF stably in vitro and in vivo and migrated to the nasal mucosa after tail vein injection. Symptom scores indicated that HGF/hDPSCs significantly improved allergic symptoms such as nasal scratching, sneezing, and runny nose in mice with allergic rhinitis (P<0.05). Pathological analysis and immunohistochemistry revealed that the nasal mucosal immune response of allergic rhinitis mice was mainly characterized by T lymphocyte aggregation. The application of HGF/hDPSCs significantly reduced nasal mucosal inflammation, including lymphocyte infiltration and goblet cell hyperplasia, as well as pathological damage scores (P<0.01). ELISA results indicated that HGF/hDPSCs could significantly down-regulate the sera levels of total IgE and OVA-specific IgE (P<0.01). The qPCR results indicated that HGF/hDPSCs could inhibit the response of Th1 cells (IFN-γ) and Th2 cells (IL-5, IL-13) in the spleen of mice with allergic rhinitis, thereby altering Th2/Th1 balance. Additionally, HGF/hDPSCs were found to down-regulate the response of Th17 cells (IL-17A) (P<0.05), without any effect on the response of Treg cells (Foxp3) (P>0.05), thus changing the Th17/Treg balance.
    Conclusion HGF/hDPSCs have the potential to alleviate nasal allergic symptoms and reduce nasal mucosal inflammation in mice with OVA-induced allergic rhinitis by regulating the Th2/Th1 and Th17/Treg balance.

     

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