【作者】 高利臣；

【导师】 魏泓；

【作者基本信息】 西南师范大学 ， 动物学， 2004， 硕士

【摘要】 本课题以Mr10 000的羊胎盘免疫调节因子GPIF(goat plancenta immunoregulatory factor，GPIF)为免疫生物活性评价样品材料，对GPIF免疫生物活性进行了评价方法的比较筛选、免疫生物活性评价方法的优化，并在此基础上对GPIF进行了免疫生物活性评价，确立了GPIF免疫生物活性剂量效应曲线，探讨了不同工艺方法及处理因素对GPIF免疫生物活性的影响，与同类药品免疫生物活性进行了对比。 1、GPIF免疫生物活性评价实验样品材料的准备 通过对GPIF免疫生物活性评价实验样品材料的一般理化指标鉴定，光谱、指纹图谱检测和安全性评价，显示由该工艺制备的实验样品材料理化性质稳定，活性成分含量充足，安全可靠，可以用来作为GPIF免疫生物活性的评价。 2、GPIF免疫生物活性评价方法的比较筛选 初步比较筛选了两种形态学免疫生物活性评价方法，E玫瑰花环实验和淋巴细胞转化实验及两种光学比色免疫生物活性评价方法，淋巴细胞增殖实验MTT法和淋巴细胞增殖实验BrdU-ELISA法。并对初步筛选的E玫瑰花环实验和淋巴细胞增殖BrdU-ELISA实验进行了进一步筛选。最终确定GPIF免疫生物活性评价方法为：T细胞来源于胸腺的E玫瑰花环实验和PHA诱导的淋巴细胞增殖BrdU-ELISA实验。 3、GPIF免疫生物活性评价方法的优化 对GPIF E玫瑰花环实验在不影响SRBC活性的基础上用神经氨酸酶处理，经此法处理后E玫瑰花环形成率明显提高；对传统的ConA诱导的人外周血淋巴细胞增殖BrdU-ELISA实验，进行了方法学改进。采用PHA诱导的人外周血淋巴细胞增殖BrdU-ELISA实验效果远远优于ConA诱导增殖效果。 4、GPIF免疫生物活性的评价理学硕d匕论文:羊胎丈免痰调节因子免痰生物翎亏性公卜价的初步研究 无论是E玫瑰花环实验，还是细胞增殖BrdU一ELISA实验，GPIF免疫生物活性都呈现了稳定的剂量效应关系。表现为低浓度(0 .125mg/m1)免疫生物活性较低，随着GPIF浓度升高而活性逐渐增强，到0.smg/m1浓度附近时免疫生物活性达到最高，之后逐渐减弱。GPIF免疫生物活性剂量效应曲线呈正态分布。GPIF是一种细胞免疫增强剂。 5、GPIF免疫生物活性影响因素的探讨 分别对GPIF病毒灭活、冻干工艺及赋形剂的选择、分子截留和制备工艺方法等影响因素进行了对比分析。研究结果显示，病毒灭活、冻干工艺及赋形剂的选择和分子截留对GPIF免疫生物活性没有任何影响，但因制备工艺方法的不同，免疫生物活性也会有改变。免疫生物活性实验证实超滤法提取的GPIF免疫生物活性优于透析法。 6、GPIF与同类药品免疫生物活性比较研究 我们在对比GPIF与所有重庆市制药企业生产的以及在重庆市销售的同类药品胸腺肤注射液、胸腺肤肠溶片(胶囊)和转移因子注射液免疫生物活性时发现，采用此标准制备的GP工F免疫生物活性高于同类产品免疫生物活性。 综上所述，通过对GPIF免疫生物活性评价实验样品材料的准备，显示由该工艺制备的实验样品材料理化性质稳定，活性成分含量充足，安全可靠，可以用来作为GPIF免疫生物活性的评价;通过对GPIF免疫生物活性评价方法的比较筛选及评价方法的优化，为GPIF质量标准建立了较优免疫生物活性评价手段;同时GPIF免疫生物活性剂量效应的发现，为该产品的免疫生物活性深入研究奠定了基础，也为GPIF药效学研究提供了理论资料;对GPIF免疫生物活性影响因素的探讨及与同类药品免疫生物活性的对比，证实GPIF是一种免疫生物活性稳定、且具有高免疫生物活性的细胞免疫增强剂;同时，本研究也为同类产品免疫生物活性评价提供了方法学参考。更多还原

【Abstract】 In this paper, the preparation of goat plancenta immunoregulatory factor(GPIF) avivity samples, the comparison among several methods for evaluating immunobiologic activity and the choosing of optimum methods have been systematically studied. On the base of these, we have evaluated the immnobiologic acitivity of GPIF, finded the dose-effect activity curve of GPIF; and analyzed influencing factors of different grafts, disposal means on GPIF immunobiologic activity. Further, we compared GPIF immunobiologic activity with the similar drugs.1.The preparation of GPIF avivity evaluation samples: By identifling the physiochemistry items, UV spectrum data, Chromatographic Fingerprints test and safty evaluation, we found GPIF activity evaluation samples were eligible.2.The comparison of methods of immunobiologic activity evaluation: In this paper, we primitivily selected two kinds of morphologic methods to evaluate GPIF immunobiologic activity: E rosettes experiment, Lymphocyte transformation experiment, and two kinds of optic colorimetry immunobiologic activity evaluation methods: cell proliferation MTT assay, cell proliferation BrdU-ELISA assay. And further studied E rosettes experiment and lymphocyte proliferation BrdU-ELISA assay. Finally, we ensure immunobiologic activity evaluation methods of GPIF wre T cells from pigs thymus of E rosettes experiment and PHA inducing lymphocyte proliferation BrdU-ELISA assay.3.The choosing of optimum evaluating methods of immunobiologic activity: If we disposed sheep blood red cells with neuraminic acid enzyme, the E rosettes forming ratio remarkbly increased; We improved the conventional method of BrdU-ELISA assay on lymphocyte proliferation induced by ConA. The results showed that lymphocyte proliferation induced by PHA was better than by ConA.4. Immunobiologic activity evaluation of GPIF: Both E rosettes experiment and lymphocyte proliferation assasy showed that immunobiologic activity of GPIF steadily exited dose-effect relations, namely, the low concentration (0.125mg/ml) activity was lowwer, with the concentration increasing, immunobiologic activity of GPIF gradually increased, when it was at the 0.5mg/ml, immunobiologic activity of GPIF reached summit. After that, the immunobiologic activity gradually weakened. The immunobiologic activity of GPIF curve was normal school.5.Analyse of influencing factors for immunobiologic activity of GPIF: The analyse results of influencing factors of high temperature diposaling virus, freezy-dry craft, choose of excipents, different Mr interception, preparation graft on GPIF indicated that high temperature diposaling virus, freezy-dry craft, choose of excipents, different Mr interception did not exited any influencing factors on GPIF. But, when preparation graft changed, the GPIF immunobiologic activity also changed with it. Activity evaluation assay proved that ultrafilter graft was better than dialysis.6.The comparison of GPIF immunobiologic activity with the similar drugs. When we compared the immunobiologic activity of GPIF to thymopolypeptides solution, thymopolypeptides enteric-coated tablets and tansfer factor solution, the checking results indicated that immunobiologic activity of GPIF was higher than the similar drugs, which were produced and saled in Chongqing.In conclusion, the results of checking of physiochemistry items, UV spectrum data, Chromatographic Fingerprints test and safty evaluation indicated that GPIF avivity evaluation samples were eligible. Through the comparison of methods for evaluating immunobiologic activity and the choosing of optimum methods to evaluating immunobiologic activity, we created assay methods evaluating immunobiologic activity for quality standard of GPIF.The research of dose-effect of GPIF can also supply theoretical materials for pharmacology. That the experiments on the factors which affect the immunobiologic activity of GPIF and the comparison with similar drugs demonstrated that GPIF was a kind of cellar immunoregulating drug with stable and high immunobiologic activity. At更多还原