| 马丽,沈佳曼,黄芬,等.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[J].浙江中医药大学学报,2020,44(10):941-948. |
| 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：10-16466/j.issn1005-5509-2020-10-003 |
| 中文关键词: 玉屏风散 胸腺上皮细胞 胸腺细胞 淋巴细胞 皮肤免疫功能 益气固表 |
| 英文关键词: Yupingfeng Powder thymic epithelial cells thymocytes lymphocyte skin immune function tonifying Qi and consolidating exterior |
| 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(LY17H160061、LY19H280010) |
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| 中文摘要: |
| [目的]研究益气固表经典方玉屏风散(Yupingfeng Powder，YPF)对BALB/c小鼠胸腺上皮细胞(thymic epithelial cells，TECs)介导的小鼠皮肤T淋巴细胞功能的影响。[方法](1)离体器官培养法研究YPF对BALB/c小鼠表皮淋巴细胞的影响及TECs在该过程中的作用。分离BALB/c小鼠皮肤组织和原代TECs细胞，设对照组(正常培养皮肤组织)，YFP低、中、高剂量组(皮肤组织予25、50、100μg·mL-1 YPF处理)、TEC共培养组(TECs与皮肤组织共培养)、TEC+YPF低、中、高剂量组(TECs与皮肤组织共培养，同时分别加入25、50、100μg·mL-1 YPF处理)，以上各组培养72h后以免疫组化法检测皮肤组织中CD3+、CD4+、CD8+T细胞的数量。(2)研究YPF对BALB/c小鼠胸腺细胞、脾细胞的影响及TECs在该过程中的作用。分离小鼠脾淋巴细胞和胸腺细胞，以噻唑蓝(thiazolyl blue tetrazolium bromide，MTT)法检测25、50、100μg·mL-1 YPF对脾淋巴细胞和胸腺细胞增殖的影响。再采用TECs分别与脾淋巴细胞或胸腺细胞共培养，以对照培养液或上述浓度YPF处理48h，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+细胞比值的变化，酶联免疫吸附法(enzyme linked immunosorbent assay，ELISA)检测脾淋巴细胞和胸腺细胞中γ-干扰素(interferon-γ，IFN-γ)、白介素-2(interleukin-2，IL-2)和IL-4的表达水平。(3)研究YPF对BALB/c小鼠TEC增殖及功能的影响。25、50、100μg·mL-1 YPF直接干预TECs 48h，MTT法检测YPF对TECs增殖的影响，实时荧光定量PCR(quantitative real-time PCR，qPCR)检测TECs中胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin，TSLP)、信号传导及转录激活因子3(signal transducers and activators of transcription 3，STAT3)、肿瘤坏死因子-α(tumor necrosis factor-α，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](1)与对照组比较，TECs与皮肤组织共培养后，皮肤中CD3+、CD4+和CD8+ T细胞数量增加。YPF处理后增加更明显。(2)不同浓度YPF对脾淋巴细胞和胸腺细胞均有促增殖作用(P＜0-01)；与脾淋巴细胞、胸腺细胞单独培养组比较，脾淋巴细胞、胸腺细胞与TECs共培养，并采用不同剂量YPF处理后CD3+、CD4+、CD8+ T细胞数均显著升高，其中脾淋巴细胞+TECs+高剂量YPF组和胸腺细胞+TECs+各剂量YPF组中CD4+/CD8+比值显著升高(P＜0-05，P＜0-01)；ELISA结果显示，TECs与脾淋巴细胞或胸腺细胞共培养后，采用不同剂量YPF处理，IFN-γ水平均升高，YPF中、高剂量组IL-2水平升高(P＜0-05)。(3)不同浓度YPF处理能显著下调TECs中TSLP、STAT3、TNF-α、IL-12和IL-17mRNA水平，显著上调IL-7 mRNA水平(P＜0-01)，增加TECs分泌IL-7的水平(P＜0-05，P＜0-01)。[结论]YPF可增强TECs介导的皮肤T细胞数量增殖，并刺激IFN-γ等细胞因子分泌。YPF还能够促进脾淋巴细胞和胸腺细胞的增殖，增强其功能，并促进T细胞向Th1细胞分化，其机制可能与YPF能促进TECs分泌IL-7有关。本研究提示，YPF具有潜在的促进胸腺介导的皮肤T淋巴细胞免疫功能的作用。 |
| 英文摘要: |
| [Objective] To study the effect of classic prescription of tonifying Qi and consolidating exterior Yupingfeng Powder(YPF) on skin T lymphocyte function mediated by thymic epithelial cells(TECs) in BALB/c mice. [Methods](1) The effect of YPF on epidermal lymphocytes of BALB/c mice and the role of TECs in this process were studied by organ culture in vitro. BALB/c mice skin tissue and primary TECs cells were isolated and divided into control group(normal culture skin tissue)， YFP low， medium and high dose groups(YPF-L， M and H， skin tissue treated with 25， 50， 100μg·mL-1 YPF)， TECs co-culture group(TECs + skin tissue)， TECs + YPF groups(TECs+skin tissue， treated with 25， 50， 100μg·mL-1rPF， respectively). The number of CD3+， CD4+ and CD8+ T cells in skin tissue was detected by immunohistochemistry after 72 hours of culture.(2)To study the effect of YPF on spleen lymphocytes and thymocytes of BALB/c mice and the role of TECs in this process. Spleen lymphocytes and thymocytes were isolated from mice. The effects of YPF(25， 50， 100μg·mL-1) on proliferation of spleen lymphocytes and thymocytes were detected by thiazolyl blue tetrazolium bromide(MTT) method. Then， TECs were co-cultured with spleen lymphocytes or thymocytes respectively， and treated with control medium or with YPF at the cocentration above for 48h. The number of CD3+， CD4+ and CD8+ T cells and the ratio of CD4+/CD8+ cells in spleen lymphocytes and thymocytes were detected by flow cytometry. The expression levels of interferon-γ(IFN-γ)， interleukin-2(IL-2) and IL-4 in spleen lymphocytes and thymocytes were detected by enzyme linked immunosorbent assay(ELISA). (3) To study the effect of YPF on the proliferation and function of TECs in BALB/c mice. The effects of YPF on the proliferation of TECs were detected by MTT assay. The mRNA levels of thymic stromal lymphopoietin(TSLP)， signal transducers and activators of transcription 3(STAT3)， tumor necrosis factor-α(TNF-α)，IL-7， IL-12 and IL-17 in TECs were detected by quantitative real-time PCR(qPCR). The level of IL-7 secreted by TECs was detected by ELISA. [Results](1)Compared with control group， the number of CD3+， CD4+ and CD8+ T cells in skin was increased in TECs co-culture group. The increase was more obvious after YPF treatment.(2)The results showed that different doses of YPF could promote the proliferation of spleen lymphocytes and thymocytes(P＜0-01). Compared with spleen lymphocytes and thymocytes alone， the number of CD3+， CD4+， CD8+ T cells in the co-culture groups(spleen lymphocytes or thymocytes + TECs + different doses of YPF) significantly increased. Among which，the ratio of CD4+/ CD8+ in spleen lymphocyte + TECs + YPF-H group and thymocyte + TECs + each dose of YPF groups were significantly increased(P＜0-05，P＜0-01). ELISA results showed that IFN-γ levels increased after spleen lymphocytes or thymocytes were co-cultured with TECs， and IL-2 level in YPF-M and YPF-H groups increased(P＜0-05). (3)The levels of TSLP，STAT3， TNF-α， IL-12 and IL-17 in TECs+different doses of YPF were significantly down-regulated， and the level of IL-7 mRNA was significantly up-regulated(P＜0-01). The level of IL-7 secreted by TECs also increase(P＜0-05，P＜0-01). [Conclusion] YPF can enhance the proliferation of skin T cells mediated by TECs and stimulate the secretion of IFN-γ and other cytokines. YPF can also promote the proliferation and function of spleen lymphocytes and thymocytes， and promote the differentiation of T cells into Th1 cells. The mechanism may be related to YPF promoting TECs to secrete IL-7. This study suggests that YPF has potential to promote thymus mediated immune function of skin T lymphocytes. |
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