不同灸法干预免疫抑制大鼠共刺激分子PD-1表达及对PD-1/PD-L1信号通路免疫活化影响的研究

Acup/mox- immune is a recognition from immune regulation mechanism to modulation of acupuncture and moxibustion. It is very important for moxibustion with heat and herbal medicine that regulate the function of the meridians and Zangfu, to promote Yin and Yang balance, to enhance specific and non-specific immune function, and to improve the body immunity. In this study, we are basis on confirming the positive adjustment of moxibustion and herbal cake-separated moxibustion on immune function at the early stage by our research group. The research would systematically expound regulation mechanism of moxibustion, and take immune suppression rat costimulatory molecule PD-1 as the breakthrough point through moxibustion and herbal cake-separated moxibustion. We would focus on the expression of PD-1 after activation of T lymphocytes of immune suppression rats by moxibustion intervention. Meanwhile we would observe the influence of PD-1 negative regulation during immune cell activation and immune tolerance. Then discuss effect of immune activation of adaptability and controllability on the PD-1 / PD-L1 signaling pathway. In comparing the advantages of two different moxibustion methods effect at the same time , we would study the effect of B cell tolerance and inhibition macrophage function through PD-1 regulating. We also observe the degree and duration in the regulation of the immune response. At last, we would explore the mechanism of PD-1/PD-L1 signaling pathway, which plays an important role in regulating the immune function in maintaining steady state and keeping normal immune effect in the periphery.

针灸—免疫是从免疫调控机制上认识针灸调整作用的规律。艾灸借助热力和药物作用调节经络脏腑功能，促进机体阴阳平衡，在增强机体特异性和非特异性免疫功能、提高机体免疫力方面非常重要。本研究在课题组前期研究充分证实艾灸及隔药饼灸对机体免疫功能良性调整作用的基础上，为系统阐释艾灸调控作用机理，以艾灸、隔药饼灸对免疫抑制大鼠共刺激分子PD-1的影响为切入点，重点研究经艾灸干预的免疫抑制动物T淋巴细胞激活后PD-1的表达，观察其在免疫细胞激活和免疫耐受过程中对免疫细胞功能负性调控作用的影响，及对PD-1/PD-L1信号通路免疫活化适度性和可控性的影响；在对两种不同艾灸方法作用效应进行优势比较的同时，研究其在PD-1调节B细胞耐受以及抑制性巨噬细胞的功能中的作用，观察在免疫应答调节中的程度和持续时间，探讨PD-1/PD-L1信号通路在调节机体免疫功能处于自身稳态、维持外围正常免疫效应中发挥重要作用的机制。

研究了负性协同刺激分子PD-1在不同灸法对免疫抑制动物干预过程中T 淋巴细胞上的表达的变化，研究证实免疫抑制模型组血清中PD-1、PD-L1含量均明显增高；脾脏组织和肝脏组织中PD-1的表达率明显增高。隔药饼灸明显提高免疫抑制兔的免疫功能，通过改善血清中PD-1、PD-L1的含量以及脾脏组织和肝脏组织中PD-1的表达率来得以实现。分析了PD-1/PD-L信号通路变化在机体正负刺激信号保持平衡的机理，并对不同灸法的效应进行了比较。采用实时荧光定量PCR技术观察隔药饼灸、艾灸对免疫抑制兔脾组织中PD-1、PD-L1表达率影响，结果显示，空白组脾组织PD1mRNA和PD-L1mRNA呈低水平表达，模型组呈现高表达，差异非常显著；经艾灸干预后，隔药饼组PD-1mRNA、PD-L1mRNA的表达均降低；艾灸组PD1mRNA的表达与模型组比较无差异，PD-L1mRNA的表达与模型组比较有差异。作为配体的PD-L1mRNA的表达较PD1mRNA的表达显著，可能与T细胞的免疫活化有关。经艾灸和隔药饼灸治疗后，脾脏指数明显下降，隔药饼组脾脏和胸腺组织形态恢复的明显；胸腺结构恢复，皮髓界限清楚，皮质淋巴细胞增多，隔药饼灸的作用明显艾灸组。.研究了不同灸法在PD-1调节B细胞耐受以及抑制性巨噬细胞的功能中的作用，对免疫细胞分子的研究显示，艾灸可使分布于B细胞上的CD19和NK细胞上NKG2D含量明显增强；隔药饼灸可使白细胞共同抗原CD45R显著降低，与艾条灸干预比较，呈现明显的特异性。隔药饼灸与艾条灸对广泛存在于活化的T淋巴细胞、B淋巴细胞和巨噬细胞表面的PD-1及PD-L1有抑制作用，呈现出显著的免疫应答特征。.研究了控制隔药饼灸易壮的温度关键环节，分析了达到隔药饼灸过程中产生最大治疗效应在灸法、穴位、壮数等因素的变化规律；强调了热辐射与温度成正相关，温度峰值与热辐射波谷间的关系。