免疫抑制小鼠灌服蛴螬多肽的疗效分析.docx

免疫抑制小鼠灌服蛴螬多肽的疗效分析 　　 　　关键词：　蛴螬多肽; 小鼠; 免疫功能; 　　Abstract：　This study was performed to investigate the effects of polypeptide extracts from grub on immune function of immunosuppressive mice. The grub polypeptides (GP) were extracted by protease method, and the size of the molecule was determined by electrophoresis. Then the mice were given orally different doses of grub peptides for20 days. All mice except the mice in control group were intraperitoneally injected with cyclophosphamide (CTX) for 3 days before the end of the experiment. The mice were prepared by weighing, collecting blood and aseptic operation for spleen cells. The cell differentiation was induced by lipopolysaccharide (LPS) and concanavalin A (ConA) , and cell proliferation and NO level were detected by MTT and Griess method. The serum haemolysin and macrophage phagocytosis were evaluated by spectrophotometry and flow cytometry, while the immunogenicity was analyzed by delayed hypersensitivity. Data showed that the concentration of polypeptide extracted by enzymolysis was 31.63% and the extraction rate was 37.01 mg/ml, with molecular weight of8 to 9 KDa. The spleen and liver thymus indexes of mice in model group were significantly lower than those of the control and the grub groups. The survival rate of splenetic and myeloid T and B lymphocytes and the secretion of NO in model mice were significantly lower than those of the control and the grub groups. The survival rate of B lymphocyte from bone marrow was significantly higher than that of T lymphocyte, but the secretion of NO in the spleen and bone marrow T lymphocyte was higher than that of B lymphocyte. The absorbance of haemolysin in control group and grub polypeptide group were significantly higher than that in model group. With the increasing of grub polypeptide dose, the content of hemolysin increased gradually, but there was no dose effect relationship. The number of white blood cells in grub groups was higher than that in model group. The ear swelling of the mice in medium and high dose grub polypeptide groups were higher than that of model group. With the increasing of grub dose, the ear swelling thickness increased significantly, and there was a dose effect relationship. The absorbance values of the macrophages from abdominal cavity and bone marrow as well as spleen of grub group were higher than those of model group, and there was a dose effect relationship. The fluorescence intensity of macrophage phagocytosis in grub group was stronger than that of model group, and the number of positive cells of macrophages phagocyting red cell was not significantly higher than that of model group, which suggested that the macrophage phagocytosis was strong. Taken together, polypeptides from grub can significantly enhance immune function in immunosuppressed mice. 　　Keyword：　Grub polypeptide; Mice; Immune function; 　　蛴螬为金龟子科昆虫朝鲜黑金龟子及同属近缘昆虫的干燥幼虫, 在江苏、安徽、山东和东北等地均有分布[1], 神农本草经记载其具有破瘀、止痛和解毒的作用。

文献报道, 蛴螬主要含有脂肪酸类、蛋白质、糖类、生物碱和甾体化合物等[2,3]金哲等[4]发现蛴螬联用羟基喜树碱对人MGC-803胃癌细胞株有显着性抗增殖作用, 陈梅等[5]发现蛴螬提取物能抑制家兔脉络膜新生血管中的血管内皮生长因子和碱性成纤维细胞生长因子表达, 抑制脉络膜新生血管的形成 　　多肽是20种氨基酸以不同的的组成和排列方式构成的从二肽到复杂的线性或环形结构的具有不同生物活性肽类的总称[6], 由于多肽结构样式繁多, 其在人体生理和代谢上表现出多种药理活性, 主要有提高免疫力、降血糖和抗氧化等功能严铭铭等[7]分离得到了单一鹿茸多肽CNT14, 其能够明显促进小鼠海马HT22细胞增殖 　　本研究拟从蛴螬中提取蛴螬多肽, 通过体内试验分析其免疫调节作用, 为蛴螬的开发应用提供试验依据 　　1、 材料与方法 　　1.1 、实验材料 　　环磷酰胺 (CTX) 购于江苏恒瑞医药股份有限公司, DMEM高糖培养基和胎牛血清 (FBS) 均购自美国Gibco公司, RPMI1640培养基购自美国HyClone公司, 刀豆蛋白 (ConA) 和脂多糖 (LPS) 购自美国Sigma公司, 噻唑蓝 (MTT) 购自美国AMRESCO公司, 羟基荧光素二醋酸盐琥珀酰亚胺脂 (CFSE) 购自北京化工厂, 蛴螬多肽 (GP) 由本实验室制备。

细胞培养板和酶标板购自美国Corning公司, 二氧化碳培养箱为日本SANYO产品, XSZ-D2倒置生物显微镜为重庆光学仪器厂产品, 粉碎机 (小型) 购于日本日立公司6～8周龄健康昆明小鼠, 体质量20～22 g, 购于中国军事医学科学院实验动物中心 　　1.2、 蛴螬多肽的提取纯化及鉴定 　　1.2.1 、提取 　　取5 g蛴螬粉于含100 ml蒸馏水的烧杯中并调pH至8.0, 向其加入木瓜蛋白酶搅拌, 置于55℃水浴锅水浴2.5 h, 然后置于100℃沸水10 min终止酶解反应, 10 000 r/min离心15 min, 收集上清为蛴螬多肽粗提取物;粗提取物与10%三氯乙酸1.25 ml摇匀, 静置10 min, 4 000 r/min离心15 min, 取3 ml上清液加入2 ml双缩脲试剂混匀, 静置10 min后2 000 r/min离心10 min, 取上清于540 nm处测吸光度值, 同时制作谷胱甘肽标准曲线, 计算蛴螬多肽质量浓度及提取率 　　1.2.2、 纯化 　　采用Mr 10 000膜超滤粗提物得>Mr10 000和 　　1.2.3 、鉴定 　　采用Tricine-SDS-PAGE法鉴定蛴螬多肽相对分子质量, 首先按4∶1取蛴螬多肽和5×loading Buffer混合, 沸水浴10 min, 冷至室温备用;然后按照凝胶配方制胶;其次灌胶:取制胶架、1.5 mm制胶板、制胶夹和电泳槽一套, 组装并检查是否漏胶, 用加样枪缓缓注胶于装置内, 加样后用去离子水压平界面, 静置等待分层, 同理加入浓缩胶, 插入10孔梳子, 准备电泳;最后点样:装备好电泳设备后槽中加入电泳液, 依次加点样蛋白Marker 5?l和样品30?l。

　　1.3 、动物分组与给药处理 　　取50只健康昆明小鼠随机分对照组、模型组 (环磷酰胺组) 、蛴螬多肽组;蛴螬组小鼠每天1次灌服不同剂量蛴螬多肽 (50、100、200 mg/kg) , 对照组和模型组灌服等量生理盐水, 连续灌服20 d, 实验结束前3 d, 除对照组外其余小鼠每天腹腔注射环磷酰胺 (CY) 50 mg/kg, 连续3 d 　　1.4 、小鼠白细胞数与脏器指数的测定 　　实验结束小鼠称体质量, 取全血于抗凝管中, 采用全血细胞分析仪检测白细胞数;颈椎脱臼处死小鼠, 摘取脾脏、肝脏、肾脏, 生理盐水清洗, 滤纸吸干后称质量, 按照公式计算脏器指数=脏器质量 (mg) /体质量 (g) 　　1.5、 小鼠迟发型变态反应 (DTH) 检测 　　末次给药后小鼠腹部剃毛, 均匀涂抹50?l二甲苯致敏, 4 d后在小鼠右耳均匀涂抹20?l二甲苯, 24 h后将小鼠颈椎脱臼处死, 用直径为8 mm打孔器在左右耳相同位置取下圆耳片称质量, 记录致敏前后质量之差即为其耳胀程度 　　1.6、 小鼠淋巴细胞和巨噬细胞制备 　　处死小鼠无菌操作取脾于200目细胞筛研碎, 收集细胞悬液, 3 000 r/min离心4 min, 弃上清PBS悬浮细胞, 加红细胞裂解液1～2 min, 3 000 r/min离心4 min,。