【作者】 韦艳霞；

【导师】 程卫东；

【作者基本信息】 兰州大学 ， 中西医结合临床， 2012， 硕士

【摘要】 目的：1.验证红芪多糖3(HPS-3)对正常小鼠胸腺细胞是否具有免疫调节作用。2.建立和优化适合HPS-3作用后小鼠胸腺组织蛋白质样品的双向电泳技术,获得HPS-3作用后小鼠胸腺组织蛋白二维凝胶电泳图谱,为后续PD Quest软件分析和差异蛋白质点的鉴别、分析奠定基础。3.寻找HPS-3作用后小鼠胸腺组织差异表达的蛋白质点,探讨HPS-3调节免疫功能的分子生物学水平机制,为寻找HPS-3促进正常小鼠胸腺细胞增殖的靶标蛋白提供理论依据。方法：1.正常小鼠分别给予不同剂量HPS-3(50mg/(kg·d),100mg/(kg-d))14天,测定胸腺、脾脏指数及T淋巴细胞增殖能力,并用透射电镜观察小鼠胸腺细胞的超微结构。2.运用标准裂解法提取100mg/(kg-d)HPS-3组小鼠的胸腺组织总蛋白,然后分别采用三氯乙酸(TCA)-丙酮沉淀法和磷酸三丁酯-丙酮-甲醇蛋白纯化法纯化所得蛋白,以固相pH梯度胶条进行双向凝胶电泳(2-DE),并对等电聚焦程序进行改良和优化；同时将磷酸三丁酯-丙酮-甲醇蛋白纯化法运用于A549细胞蛋白的纯化并进行双向电泳,银染显色后比较所得凝胶图像。3.采用2所建立和优化的2-DE技术对小鼠胸腺组织蛋白进行分离,所得凝胶银染显色、Image Scanner扫描获取图谱后,通过PD Quest软件对其进行分析、寻找差异蛋白质点。结果：1.低剂量HPS-3(50mg/(kg·d))对小鼠胸腺指数、脾脏指数及T淋巴细胞增殖能力无显著影响,而0mg/(kg·d) HPS-3能显著增加小鼠胸腺、脾脏指数,增强T淋巴细胞增殖反应。电镜观察显示：100mg/(kg·d) HPS-3可以促进胸腺细胞分裂、增殖,50mmg/(kg.d)HPS-3无显著影响。2.磷酸三丁酯-丙酮-甲醇蛋白纯化法能减少蛋白降解、增加蛋白的溶解性,所得蛋白的2-DE图谱蛋白点可达到1165士12,而且对酸性蛋白及低丰度蛋白的表达也优于TCA-丙酮沉淀法；聚焦条件的优化,使横向和纵向拖尾有明显改善,成功建立了背景清晰,蛋白分离良好、分辨率较高的胸腺组织蛋白的双向电泳体系。磷酸三丁酯-丙酮-甲醇蛋白纯化法也适用于A549细胞蛋白提取纯化。3.实验所得2-DE图谱经PDQuest软件分析得出以下结果：空白组分离得到1106±37个蛋白点,HPS-3组得到1165±12个蛋白质点,两组共有的蛋白点中表达量差异两倍以上的有192个。结论：1.HPS-3能促进小鼠胸腺细胞增殖,提高小鼠免疫力。2.建立了HPS-3作用后小鼠胸腺组织蛋白质提取纯化的方法,采用磷酸三丁酯-丙酮-甲醇蛋白纯化法可以更有效地提取胸腺组织蛋白；利用优化后的2-DE技术获得了HPS-3作用后小鼠胸腺组织蛋白2-DE图谱。3.HPS-3可以显著影响正常小鼠胸腺组织蛋白质表达。更多还原

【Abstract】 Objective:1.To verify whether the third polysaccharides from Hedysarum polybotys saccharide (HPS-3) can regulate the immunity of mouse thymus organizations.2. To choose the best method for preparing the protein of the mouse thymus tissue treated with HPS-3through comparison the two-dimensional gel electrophoresis (2-DE) maps and give others some reference. It is also a strong basis for the identification of differential protein spots and the possible target-related.3. To find out the differential protein spots expression in mouse thymus organizations treated with HPS-3, and discuss the molecular biology mechanism of enhancing the immunity with HPS-3.Methods:l.The mice were treated with different doses of HPS-3(50mg/kg/day,100mg/kg/day) for fourteen days, the thymus gland index, spleen index and proliferation index of T lymphocyte were detected and the ultrastructure of mouse thymocyte through transmission electron microscope (TEM) was observed at the same time.2. Thymus totally proteins from the mice, which have given HPS-3in100mg/kg/d for fourteen days, were extracted using standard tissue lysis method, then trichloroacetic acid (TCA)-acetone precipitation method and tri-n-butylphosphate:acetone:methanol protein purification method were used respectively to purify the thymus protein. In the meantime, the tri-n-butylphosphate:acetone:methanol protein purification method was utilized to precipitate the protein from A549cell. The proteins were separated by means of immobilized pH gradient based on two-dimensional gel electrophoresis, and the condition of isoelectric focusing (IEF) was also adjusted and optimized, then the gels were stained by Beyotime Fast Sliver Stain kit, digitized images of2D gels that were generated using Image Scanner were analyzed by PD Quest software.3. The protein of mouse thymus organizations were separated utilizing2-DE which have been chose from the second method, analyzed the digitized images of2D gels with PD Quest software and found out the protein spots which differentially expressed between the two groups.Results:1.Compared with control group, thymus gland index and spleen index were raised obviously, T lymphocyte proliferation was promoted in HPS-3dosed with100mg/kg/day, but which treated with50mg/kg/day didn’t cause observable change. The ultrastructure characteristics of mice thymocyte showed that the mice which were given HPS-3with100mg/kg/day could promote the thymocyte disintegrating and proliferating.2. Tri-n-butylphosphate:acetone: methanol protein purification method not only decreased protein decomposition, but also increased protein solubility. There were1165±12spots expressed in the2-DE mapping which the protein has been precipitated through the tri-n-butylphosphate:acetone:methanol protein purification method, and better expression in acidic protein and low abundance than the TCA-acetone precipitation method. Besides, the focus voltage for gel electrophoresis was optimized on the traditional focus conditions, and a two-dimensional electrophoresis system with a clear background and good protein separation was successfully established for thymus tissue. Horizontal and vertical tail was improved significantly by the optimized focusing condition, and a high-resolution two-dimensional electrophoresis map with clearer protein spots and more complete separation was obtained easily, the tri-n-butylphosphate:acetone:methanol protein purification method was also suitable for the protein from A549cells.3. Analytic results of the2-DE mapping with PD Quest software is that:there were1106±37spots expressed in control group, and1165±12spots displayed in HPS-3group, there were192protein spots which were uifferentially expressed with more than twofold increased or decreased volume between control and HPS-3group.Conclusion:1. HPS-3can promote the thymocyte proliferating and elevate the immune responses of the mouse.2. The method of extracting thymus tissue protein was established. The protein spots in2-DE map were significantly increased using tri-n-butylphosphate:acetone: methanol protein purification method; in addition, by using the optimized method described above, satisfactory2-DE maps of thymus tissue has been obtained, which lays a foundation for the further study of the proteome of thymus tissue treated with HPS-3.3. HPS-3significant impact the expression of the mouse thymus organizations proteins.更多还原