玉米醇溶蛋白动物体内植入后的免疫学分析方法初探

目的：为了考察玉米醇溶蛋白作为生物医用材料的应用前景，需要检测玉米醇溶蛋白植入后的免疫反应。本实验中我们将玉米醇溶蛋白管植入SD（Sprague Dawley）大鼠皮下，流式细胞仪检测植入后大鼠外周血中CD4^＋和CD8^＋T淋巴细胞含量的变化；对不同部位植入材料的组织学切片方法进行改进，用于后续机体免疫反应分析。方法：雄性SD大鼠八只，随机分成两组：空白对照组、材料植入组。分别于植入后一、二和四周对实验大鼠进行眼眶取血，并用流式细胞仪检测外周血中CD4^＋和CD8^＋T淋巴细胞的含量。皮下或肌袋植入材料四周后，将玉米醇溶蛋白管或三维多孔支架连同周围组织一起取出，用改进的石蜡切片方法制备组织切片，HE染色后观察。结果：玉米醇溶蛋白管植入后一周，蛋白管植入组CD4^＋（T—test，P〈0．01）和CD8^＋（T-test，P〈0．05）T淋巴细胞的含量下降。植入后两周和四周，蛋白管植入组CD4^＋和CD8^＋T淋巴细胞的含量（T—test，P〉0．05）均无明显变化。植入后一、二和四周，与空白对照组相比，蛋白管植入组CD4^＋／CD8^＋的比值（T—Test，P〉0．05）均无显著差异。改进后的切片方法可以制备出完整的、并且耐HE（hematoxylin—eosin）和免疫组化染色处理的组织学切片。结论：利用流式细胞仪检测出了大鼠皮下植入玉米蛋白管后外周血中CD4^＋、CD8^＋T淋巴细胞的含量变化。并且，克服了切片制备过程中材料的脆性问题和贴片不牢固问题，可以进行HE和免疫组化染色处理，为进一步深入地分析玉米醇溶蛋白植入机体后的免疫反应打下了基础。

Immunological Analysis after Implantation of Zein into Rats

Objective： In order to investigate the prospects for that application of zein as a biomedical material, the immune response after implanting should be detected. In this study, we explored the method of detecting CD4 ^＋ and CD8 ^＋ T lymphocytes by flow cytometry and the method of preparing integrated histological sections for further analysis of immune response, after implantation of zein into SD rats. Methods： Eight male SD rats were randomly divided into two groups： normal group without implantation and zein tube implanted group. The changes of CD4^＋ and CD8 ^＋ T lymphocytes in the peripheral blood were detected by flow cytomery at the first, second and fourth week after subcutaneous implantation. Four weeks after implantation, the implanted zein tubes or 3-D zein scaffolds and the surrounding tissue were dissected for the histological observation. Results： One week after subcutaneous implantation, the levels of CD4^＋ （T-test, P〈0.01） and CD8 ^＋ （T-test, P〈0.05） T lymphocytes decreased. Two and four weeks post implantation, no significant difference was shown in the levels of CD4^＋ and CD8 ^＋ （T-test, P〉0.05） T lymphocytes. One, two and four weeks after implantation, the ratio of CD4^＋/CD8 ^＋ had no significant change in the zein tube group compared with the control group （P〉0.05）. Four weeks after implantation, an improved method of preparing integrated paraffin sections of the implanted material was used for fitrther histological observation. Conclusion： The change of CD4^＋ and CD8 ^＋ T lymphocytes in the peripheral blood was successfully detected by flow cytometry. Furthermore, we prepared zein tube and 3-D scaffold sections for histological observation. And these established the foundation for further analysis of immune response in vivo after the implantation of zein.