【摘要】 目的探讨3个拷贝人源分子佐剂C3d(hC3d3)与绒毛膜促性腺激素β亚基(hCGβ)的融合蛋白hCGβ-hC3d3作用于人外周血单个核细胞(PBMC)对hCGβ抗原免疫原性的影响。方法用L-亮氨酸甲基酯(L- LME)或B细胞磁珠阴性分离试剂盒对人PBMC进行细胞分离纯化;实验分成B细胞、B+T细胞、PBMC和Raji细胞4组。分别应用1、10及100 nmol／L不同浓度的hCGβ、hCGβ-hC3d3及美洲商陆(PWM)等抗原体外作用于以上细胞10～12 d,采用氚标记的胸腺嘧啶核苷(3H-TdR)掺入法了解不同抗原刺激后各组细胞的增殖情况;用酶联免疫吸附试验(ELISA)检测培养上清液中抗hCGβ抗体的分泌水平;而酶联免疫斑点法(ELISpot)可测定各种抗原刺激后特异抗体分泌的细胞数量。结果与hCGβ相比,融合蛋白hCGβ-hC3d3能使各组细胞增殖明显升高,且呈浓度依赖性。应用100 nmol／L的不同抗原培养12 d后,取培养上清液进行ELISA检测,经hCGβ及PWM刺激后,上清液中均未检测到抗hCGβ特异性抗体;经hCGβ-hC3d3培养后则在B+T细胞及PBMC组中见低水平抗hCGβ抗体。采用ELISpot法分析抗hCGβ特异性抗体生成细胞,经hCGβ刺激的细胞仅有数个散在的阳性细胞;而经hCGβ-hC3d3刺激后,阳性细胞明显增多(P<0．05)。结论hC3d3明显促进人免疫活性细胞对hCGβ抗原的免疫原性。更多还原

【Abstract】 Objective To probe into the fact if molecular adjuvant hC3d3 promotes the immunogenicity of human immuno-competent cells on the human chorionic gonadotropinβ(hCGβ) antigen in case of fused with the antigen. Methods The isolated B cells (by Human B Cell Isolation Kit II ) , the combined B cells and T cells (by L-LME), peripheral blood mononuclear cell (PBMC) and Raji cells were treated in vitro respectively with 1, 10, 100 nmol/L hCGβ, hCGβ-hC3d3 or PWM for 10-12 days. The cell proliferation was determined by incorporation of [3H] thymidine. Anti-hCGβantibody levels in the 10-12 days culture supernatants were measured by indirect ELISA. The Ab-secreting cells in the 10-day cultured lymphocytes were detected by the enzyme-linked immunospot (ELISpot) assay. Results It was found that the proliferation of B cell, the combined B and T cell, PBMC and Raji following exposure to hCGβ-C3d3 fusion protein was significantly higher than that of hCGβalone. The low level of anti-hCGβantibody could be produced in the combined B cells and T cells, and PBMC with 100 nmol/L hCGβ-C3d3, but no anti-hCGβantibody was observed in all co -cultured cells with hCGβalone. The anti-hCGβantibody- secreting cells were significantly increased after the B cell, B and T cell, and PBMC treated with hCGβ-C3d3 fusion protein comparing to the hCGβalone. Conclusion The human molecular adjuvant hC3d3 improves the immunogenicity in human immuno-competent cells on the hCGβantigen if fused with the antigen.更多还原