蛴螬多肽对免疫抑制小鼠免疫功能的影响

Effects of polypeptide extracts from grub on immune function of immunosuppressive mice

目的观察蛴螬多肽提取物对免疫抑制小鼠的免疫功能的影响。方法采取蛋白酶水解提取蛴螬多肽,电泳确定相对分子质量大小,然后每天给小鼠灌服不同剂量多肽连续20 d,除对照组外其余小鼠在实验结束前连续3 d腹腔注射环磷酰胺,试验结束小鼠称体质量、采血、无菌操作制备脾脏细胞,体外培养添加脂多糖和刀豆蛋白A诱导细胞分化,采用MTT和Griess法检测细胞增殖率和NO分泌量;分光光度法和流式技术检测血清溶血素及巨噬细胞吞噬功能,迟发型超敏反应分析免疫原性。结果酶解提取蛴螬多肽质量浓度为37.01 mg/ml,提取率为31.63%,Mr在8 000～9 000。模型组小鼠脾脏肝脏胸腺指数显著低于对照组和蛴螬组,小鼠脾脏和骨髓T、B淋巴细胞存活率及NO分泌量明显低于对照组和蛴螬组,其中骨髓B淋巴细胞存活率显著高于T淋巴细胞,但脾脏和骨髓T淋巴细胞NO分泌量高于B淋巴细胞;对照组和蛴螬多肽组小鼠溶血素吸光度值显著高于模型组,随着蛴螬多肽剂量增加溶血素含量逐渐提高,但不存在剂量效应关系;蛴螬组小鼠血液白细胞数均高于模型组,随着剂量增加白细胞数提高;中高剂量蛴螬多肽组小鼠耳肿胀度高于模型组,随着蛴螬剂量增加耳肿胀度显著增加,存在剂量效应关系;蛴螬组小鼠腹腔骨髓脾脏巨噬吸光度值高于模型组,随着蛴螬多肽剂量增加吸光度值提高,存在剂量效应关系;流式细胞技术检测结果蛴螬组巨噬细胞吞噬鸡红细胞荧光强度强于模型组,巨噬细胞吞噬鸡红细胞阳性细胞数不明显高于模型组,结果提示巨噬细胞吞噬能力强。结论蛴螬多肽具有明显增强免疫抑制小鼠免疫功能的作用。

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.