【作者】 刘英姿；

【导师】 邓旭明； 孙永祥；

【作者基本信息】 吉林大学 ， 兽医， 2010， 博士

【摘要】 碎米花杜鹃（Spiciferum）为杜鹃花科（Ericaceae）杜鹃花属（Rhododendron）植物,为小灌木,主要产于我国云南中部至东南部以及贵州西部等地。生于海拔800-1200米的山坡灌丛、松林或次生林园中。碎米花杜鹃的乙酸乙酯层提取物Proanthocyanidin A-1（PAA-1）是一种原花青素类二聚体化合物。原花青素是植物中广泛存在的一大类多酚化合物的总称。近年来研究发现,原花青素具有抗氧化、保护心血管、抗癌、抗血小板聚集、免疫调节、保护肝肾功能等多种药理活性。原花青素作为天然药物提取的有效成分,其多种药理作用决定了原花青素类制剂的开发在临床方面具有广阔的前景。国内外主要针对原花青素的抗氧化活性以及防治一些如癌症、心血管疾病等一些慢性病的研究,而对于其免疫调节活性的报道却极少。一种物质研究其是促进还是抑制免疫细胞的增殖,对其免疫调节活性的研究和发现新的药物都是非常重要的。体外研究表明,PAA-1具有很好的免疫调节作用,通过各种浓度（5、25、50、100 mg/L）体外刺激能单独或协同LPS或者Con A、anti-CD3mAb刺激Balb/c小鼠脾淋巴细胞增殖。Con A和LPS作为淋巴细胞的有丝分裂原,能刺激淋巴细胞有丝分裂,所以常被用来刺激T、B淋巴细胞,作为评估机体细胞和体液免疫功能的指标。Con A、anti-CD3mAb直接作用于T淋巴细胞,而LPS直接刺激B淋巴细胞。T淋巴细胞参与细胞免疫,而B淋巴细胞主要参与体液免疫反应。PAA-1可以协同LPS或Con A、anti-CD3mAb体外刺激小鼠脾淋巴细胞增殖,证明PAA-1可提高机体特异性的细胞免疫和体液免疫应答。自然杀伤细胞（NK细胞）在机体防御系统中发挥重要作用,能通过抗肿瘤和抗病毒而发挥重要作用。NK细胞主要能识别病毒感染细胞,以及肿瘤细胞并对其起到杀伤作用,从而保护机体免受损伤。试验发现,PAA-1通过体外各种浓度刺激能激活NK细胞对YAC-1细胞的杀伤活性,说明PAA-1对于机体的非特异性免疫应答也存在一定的促进作用。为进一步研究PAA-1体外促进淋巴细胞增殖的机制,运用流式细胞仪检测PAA-1对小鼠脾淋巴细胞表面标志CD4和CD8 T淋巴细胞亚群比例的影响,从分子水平上对其机理进行研究。CD4细胞识别MHC II类分子递呈的抗原,并通过辅助性T细胞Th1介导细胞免疫反应,通过Th2细胞介导体液免疫。CD8细胞识别MHC I类分子递呈的抗原,通过细胞毒性T细胞（Ts）调节细胞免疫反应。实验证明, PAA-1体外刺激不仅增加了CD4⁺CD8⁺双阳性细胞数量,而且诱导了CD4⁺CD8⁺向CD4⁺以及CD8⁺单阳性细胞方向分化增殖,显示PAA-1能刺激T淋巴细胞的分化成熟,从而发挥辅助性或杀伤性的作用。PAA-1体外能显著刺激T淋巴细胞分化成熟,表明PAA-1是通过增加成熟T淋巴细胞的比率而发挥免疫增强作用。此外,为了进一步了解这种机制,采用双抗夹心ELISA法对Th细胞分泌的多种细胞因子进行了测定。细胞因子是一种蛋白质多肽,是细胞信号转导的分子。它们由多种类型的细胞分泌,并相互之间紧密联系,共同发挥作用。细胞因子在免疫系统中发挥重要作用,对抗各种免疫、炎症以及感染性疾病。当机体受到各种感染侵袭时,各种免疫细胞（如T细胞和巨噬细胞）分泌细胞因子对抗感染。此外,细胞因子还可以激活免疫细胞,刺激它们产生更多的细胞因子。细胞因子分泌量,对机体对抗各种感染的能力密切相关。试验发现,PAA-1各浓度在体外能协同Con A增加Th1细胞分泌的细胞因子（IL-2、IL-12、IFN-γ、TNF-a）,却抑制Th2细胞分泌的细胞因子（IL-4、IL-10）。这些结果表明,Th1可能为PAA-1的靶细胞,PAA-1使Th1/Th2的平衡向Th1方向移动,这对许多的Th2占优势的免疫紊乱性疾病的治疗具有重要的意义。巨噬细胞是参与免疫应答的一种重要细胞,具有抗原加工、提呈和免疫调节等作用,在机体特异性免疫应答中发挥重要作用。巨噬细胞可吞噬消化异物、杀灭多种病原微生物,并吞噬处理机体自身衰老死亡的组织细胞,是机体非特异性免疫的重要因素。PAA-1各种浓度体外能显著提高腹腔巨噬细胞能量代谢水平以及吞噬中性红的能力,说明PAA-1可促进巨噬细胞增殖并增强巨噬细胞的吞噬能力。只有活化的巨噬细胞的才能发挥吞噬作用,对机体的免疫反应才具有意义。巨噬细胞在免疫活化或受刺激时,能释放大量的一氧化氮自由基和活性氧等细胞效应分子,以杀伤入侵病原体和肿瘤细胞。一氧化氮（NO）是一种重要的介质分子,参与机体的多种生理与病理过程,包括神经传递和炎症;在免疫功能的调节中也起着十分广泛而复杂的作用。既是天然免疫中的一个重要免疫分子,能够发挥非特异性杀菌和杀伤肿瘤细胞的作用,又是一种重要的信使分子,可以调节获得性免疫。通过Griess法测定证实PAA-1各种浓度体外能增加小鼠腹腔巨噬细胞NO的分泌量。具有强大的杀灭微生物活性的活性氧杀灭外来入侵物的过程中,氧消耗的突然增长,生成超氧阴离子（O₂^-）,从O₂^-开始,在细胞膜上进行的一系列反应导致了过氧化氢（H₂O₂）、羟自由基（·OH）、单线态氧（¹O₂）以及其他活性物质的生成,这一现象即呼吸爆发。呼吸爆发伴随着吞噬作用而产生,是吞噬细胞杀灭被吞噬微生物的另一重要机制。由于超氧阴离子（O₂^-）是呼吸爆发活动中的关键产物,因而O₂^-生成量,成为检测和评价吞噬细胞杀菌功能的重要指标。NBT还原实验证明,通过一定浓度的PAA-1体外刺激,可以增强小鼠腹腔巨噬细胞分泌的活性氧还原NBT的能力。巨噬细胞富含溶酶体酶,如酸性磷酸酶、非特异性酯酶、溶菌酶等,测定这些酶的活性也是衡量巨噬细胞功能的指标之一。经过PAA-1的体外刺激,细胞溶酶体酶的活性也提高了,说明PAA-1能可活化巨噬细胞,增强巨噬细胞的吞噬能力。体外试验证明了PAA-1的免疫增强作用,要确定其对动物体的免疫调节作用,还应进行临床的体内试验。选用1日龄肉鸡,7日龄时进行新城疫免疫,11日龄时分别添加不同剂量PAA-1（0、5、10、15 mg/kg）10天,在14和21日龄分别采血测新城疫HI抗体效价,21日龄屠宰后测免疫器官胸腺、法氏囊、脾脏指数,结果为PAA-1能提高新城疫HI抗体效价水平,增强免疫器官指数,进一步证明了PAA-1在动物体内的增强机体细胞免疫和体液免疫的作用,为PAA-1开发成为新型的免疫增强剂提供一定的科学与实验技术依据。更多还原

【Abstract】 Spiciferum are shrubs of Ericaceae and Rhododendron, the main production area is westen part of GUIZHOU, central and south east region of YUNNAN in China. Proanthocyanidin A-1 （PAA-1） extracted from Rhododendron spiciferum in ethylacetate is a Dimer compound of Proanthocyanidins. Proanthocyanidins （PAs） are general of Polyphenols. Recently researchs have found that PAs have many medical benefits including antioxidant, cardiovascular protection, anti-cancer, anti-platelet aggregation, immunomodulation and protection of liver and kidney function. So development of PAs drugs has great clinical prospects. Researchers have focused on potent antioxidant capacity, there have few reports related to immunomodulatory effects of PAs.In the present studies, the immunomodulatory effects of PAA-1 were investigated in vitro. Different concentrations of 5, 25, 50 or 100 mg/L PAA-1 alone or PAA-1 plus Con A, or anti-CD3 mAb, or PAA-1 plus LPS can stimulate cell proliferation of plenocytes. A number of studys reported that potentiation of Con A, anti-CD3 mAb or LPS-induced proliferation of immunocyte by a certain compound suggested whose important effect in immune function. The evaluation of substances that either promote or inhibit immunocyte proliferation is crucial to the study of immunomodulatory and drug discovery. Con A and anti-CD3 mAb act directly on T cells, while LPS acts on B cells. T cells are involved in cell-mediated immunity whereas B cells are primarily responsible for humoral immunity. Therefore, PAA-1 could enhance both cellular immunity and humoral immunity.NK cells are a part of innate immune system and play a major role in defending the host from both tumors and virally infected cells. Different concentrations of PAA-1 significantly enhanced NK function to kill YAC-1 target cells. PAA-1 also can enhance the nonspecific immunity.To research the mechanism of stimulating cell proliferation of plenocytes of PAA-1 from cell level, effects of PAA-1 to ratio of two main lymphocyte T subsets （CD4+ cells or T helpers, and CD8+ cells or T cytotoxic/suppressors） in mice were detected by flow cytometer. CD4 T cells recognize antigens presented by major histocompatibility complex class II （MHC II） and mediate both cellular immune response through Th1 cells and humoral immune response through Th2 cells. CD8 recognizes antigens presented by MHC I and mediates cellular immune responses through cytotoxic T cells. In our study, a significant up regulation of the CD4+, CD8+ and CD4+CD8+ double positive cell populations after PAA-1 treatment was found. It means that PAA-1 possess immune-stimulating capacity.We found that PAA-1 has a significant stimulatory effect on inducing the release of Th1 cytokines （IL-2, IL-12, IFN-γ, and TNF-α） and decreasing the release of Th2 cytokines （IL-4, IL-10） with Con A by ELISA kit in vitro. The results showed that PAA-1 can exert the effect of immunological regulation by up-adjusting the Th1/Th2 arms of the immune system. PAA-1 might be useful for the correction of Th2 dominant pathological disorders.Macrophages play an important role in immunity. Different concentration PAA-1 can enhance proliferation of peritoneal macrophages and macrophage phagocytic function on neutral red significantly. Phagocytic activity is accompanied by the production of reactive oxygen (O^2-) and NO as well as the release of lysosomal enzymes such as acid phosphatase （AP） and these products is involved in killing and digesting microbial pathogens. NO plays an important role in immunity through bactericidal and antitumor.In vitro PAA-1 can increase NO secreted by macrophage. The higher reduction in NBT assay represented higher activity of oxidase enzyme, reflecting the stimulation of phagocytes in proportional to intracellular killing. The concentration of 50 mg/L PAA-1 can stimulate macrophage secret more O^2- to reduce NBT dye. For lysosomal enzyme activity, transformation of p-NPP to the colored compound by the acid phosphatase of the stimulated macrophages was correlated to degranulation in phagocytosis. For lysosomal enzyme activity, the 50 mg/L concentration PAA-1 exhibited the maximum potency. These results showed that PAA-1 can active macrophage, and increase Macrophage phagocytosis.In vivo different concentrations of 0, 5, 10, 15 mg/kg PAA-1 were fed to 11-day chickens for ten days, HI antibody titer against NDV in chicken and immune organs index were detected in clinical Trials. The results showed that PAA-1 can enhance antibody level of ND and immune organs index of chickens.All of the above tests provided scientific and experimental foundations for developing new kind immune adjuvant of PAA-1.更多还原