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| 玉屏风散对小鼠胸腺上皮细胞介导的皮肤T淋巴细胞功能的影响 |
| Effect of Yupingfeng Powder on the function of skin T lymphocyte mediated by thymic epithelial cells in mice |
| DOI： |
| 中文关键词: 玉屏风散 胸腺上皮细胞 胸腺细胞 淋巴细胞 免疫功能 |
| 英文关键词: Yupingfeng Powder Thymic epithelial cells thymocytes Lymphocyte immune function |
| 基金项目:国家自然科学基金面上项目(81473575)；浙江省自然科学基金一般项目(LY19H280010) |
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| 中文摘要: |
| [目的]研究玉屏风散对BALB/c小鼠胸腺上皮细胞（TECs）及TECs介导的小鼠皮肤T淋巴细胞功能的影响。[方法]分离BALB/c小鼠皮肤组织和TECs细胞，给予50μg·mL-1玉屏风散和5ng·mL-1胸腺基质淋巴细胞生成素（TSLP）培养皮肤组织、50μg·mL-1玉屏风散作用于TECs与皮肤组织共培养体系72 h。免疫组化法检测皮肤组织中CD3、CD4、CD8阳性细胞数量的变化。分离小鼠脾淋巴细胞和胸腺细胞，MTT法检测25、50、100μg·mL-1玉屏风散对脾淋巴细胞和胸腺细胞增殖的影响；TECs分别与脾淋巴细胞和胸腺细胞共培养,25、50、100μg·mL-1玉屏风散作用于共培养体系48h,实验分为对照组、共培养（TECs）组、共培养低、中、高剂量（TECs＋YPF-L、M、H）组，流式细胞术检测脾淋巴细胞和胸腺细胞中CD3＋、CD4＋和CD8＋T细胞数量及CD4/CD8比值的变化，ELISA检测脾淋巴细胞和胸腺细胞分泌IFN（干扰素）-γ、白介素（IL）-2和IL-4的水平。25、50、100μg·mL-1玉屏风散干预TECs 48h，MTT法检测玉屏风散对TECs增殖的影响，qPCR检测TECs中TSLP、信号传导及转录激活因子（STAT）3、肿瘤坏死因子（TNF）-α、IL-7、IL-12和IL-17 mRNA水平变化，ELISA检测TECs分泌IL-7的水平。[结果]与对照组比较，TECs介导后，共培养组皮肤组织内CD3、CD4和CD8阳性细胞数量增加,且CD3和CD4阳性细胞数量增加较明显。不同浓度玉屏风散对脾淋巴细胞和胸腺细胞均有促增殖作用（P<0.01），与对照组比较，共培养各剂量组脾淋巴细胞和胸腺细胞中CD3+、CD4+、CD8+T细胞数量显著升高、共培养高剂量组脾淋巴细胞中和共培养各剂量组胸腺细胞中CD4/CD8比值显著升高，ELISA结果显示共培养各剂量组脾淋巴细胞和胸腺细胞分泌的IFN-γ水平升高、共培养中高剂量组IL-2水平升高（P<0.05，P<0.01）。qPCR结果显示不同浓度玉屏风散均显著下调TECs中TSLP、STAT3、TNF-α、IL-12和IL-17水平，显著上调IL-7水平，增加TECs分泌IL-7的水平（P<0.05，P<0.01）。[结论] 在TECs的介导下，玉屏风散可提高皮肤组织中T淋巴细胞的数量和功能；玉屏风散直接处理对体外脾淋巴细胞和胸腺细胞具有促增殖作用，TECs介导后，玉屏风散能够增强这些细胞的功能，并促进T细胞向Th1细胞分化，其机制可能与其增强TECs分泌的IL-7水平有关。 |
| 英文摘要: |
| [Objective] To study the effect of Yupingfeng Powder on thymic epithelial cells (TECs) and skin T lymphocyte function mediated by TECs in BALB/c mice. [Methods] The skin tissue of BALB/c mice and TECs were isolated，cultured which joined with 50μg·mL-1 Yupingfeng Powder and 5ng·mL-1 thymic stromal lymphopoietin (TSLP)，50μg·mL-1Yupingfeng Powder were joined the co culture of TECs and skin tissue system for 72h. The changes of CD3, CD4, CD8 positive cells in the skin were detected by immunohistochemistry. Mice spleen lymphocytes and thymocytes were isolated, the effects of 25, 50, 100μg·mL-1 Yupingfeng Powder on the proliferation of spleen lymphocyte and thymocyte were detected by MTT; TECs co cultured with spleen lymphocyte and thymocyte respectively, 25, 50, 100μg·mL-1 Yupingfeng Powder were joined in co culture system for 48h, the experiment was divided into control group, co cultured (TECs) group, co cultured low, medium and high dose (TECs + YPF-L, M, H) group, the changes of CD3＋, CD4＋ and CD8＋T cells and CD4/CD8 ratio in spleen lymphocyte and thymocyte flow were detected by flow cytometry, and ELISA was used to detect the levels of interferon (IFN)-γ, Interleukin(IL)-2 and IL-4. 25, 50, 100μg·mL-1 Yupingfeng Powder intervened in TECs for 48h, MTT was used to detect the effect of Yupingfeng Powder on the proliferation of TECs, TSLP, signal transduction and activator of transcription (STAT)3, tumor necrosis factor (TNF)-α, IL-7, IL-12, and IL-17 mRNA in TECs were detected by qPCR, IL-7 secreted by TECs was detected by ELISA. [Results] Compared with the control group, the number of CD3, CD4 and CD8 positive cells in the skin of co cultured group increased, and the increase of CD3 and CD4 positive cells was more obvious. Yupingfeng Powder could promote the proliferation of spleen lymphocytes and thymocytes after 48h (P<0.01); Compared with the control group, the number of CD3＋, CD4＋ and CD8＋T cells of the co cultured each dose groups in spleen lymphocyte and thymocytes significantly increased, CD4/CD8 ratio of the co cultured high dose groups in spleen lymphocyte and co cultured each dose groups in thymocytes was significantly increased, the results of ELISA shown that IFN-γ level of the co cultured each dose groups in spleen lymphocytes and thymocytes increased, the IL-2 level of the co cultured medium and high-dose groups increased(P<0.05, P<0.01). The results of qPCR shown that the levels of TSLP, STAT3, TNF-α, IL-12 and IL-17 were significantly decreased, IL-7 mRNA level was significantly increased of Yupingfeng Powder each dose groups in TECs, IL-7 secretion increased of Yupingfeng Powder each dose groups in TECs (P<0.05, P<0.01). Conclusion: Under the mediation of TECs, Yupingfeng powder can improve the quantity and function of T lymphocyte in skin tissue. The direct treatment of Yupingfeng powder can promote the proliferation of spleen lymphocytes and thymocytes in vitro, after the mediation of TECs, Yupingfeng powder can enhance the function of these cells and promote the differentiation of T cells into Th1 cells, its mechanism may be related to the enhancement of IL-7 level secreted by TECs. |
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