Practical Journal of Organ Transplantation(Electronic Version) ›› 2017, Vol. 5 ›› Issue (2): 116-120.

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Antibody-mediated rejection and membranous nephropathy in renal allograft recipients

  

  • Online:2017-03-20 Published:2021-06-24

肾移植术后抗体介导排斥反应合并膜性肾病

李雪,张明超,陈劲松,徐峰,倪雪峰,程东瑞,季曙明,文吉秋   

Abstract:

Objective Prokaryotic system was used to induce expression and purification of human leukocyto antigen-G1(HLA-G1) protein and to investigate the effect of HLA-G1 on expression of interleukin-10 (IL-10)in hunman B lymphocytes, to study the B cell-mediated immune tolerance mechanism of HLA-G1 in kidney transplantation recipients. Methods We used the prokaryotic expression system to express the HLA-G1 protein and purified it. Human peripheral blood mononuclear cells(PBMCs) were co-cultured with HLA-G1 after Western Blot. Then we detected the expression of IL-10 in B lymphocytes by flow cytometry. Results Results of sodium dodecyl sulfate-polyacrylamide gel(SDS-PAGE) and Western Blot showed that the molecular weight of the purified protein was about 38.2 kDa which was in accord with that of HLA-G1 protein. The expression of IL-10 in B lymphocytes after co-culture with HLA-G1 significantly increased, and the rate of IL-10 on B lymphocytes were 0.1%,0.36%,0.57% and 0.73%, respectively, dependent on the concentration of HLA-G1(concentration gradient: 0,50,100 and 200 ng/ml). Conclusion We used the prokaryotic expression system to express HLA-G1 protein and purified it. The purity was above 95%. HLA-G1 protein induced the expression of IL-10 in B lymphocytes in a concentration dependent manner after co-culture with human PBMC, which suggested that HLA-G1 might play an important role in the establishment of immune tolerance in kidney transplantation.

摘要:

目的 原核体系诱导表达并纯化人类白细胞抗原 -G1(human leukocyte antigen-G, HLA-G1) 蛋白,研究 HLA-G1 分子对人 B 淋巴细胞内白细胞介素 -10(interleukin-10, IL-10)表达水平的影响,进 而探究 HLA-G1 在肾移植受者中 B 细胞介导的免疫耐受机制。方法 通过原核表达体系,构建表达并纯 化 HLA-G1 蛋白,经过蛋白免疫印迹试验(Western Blot)鉴定后,与人外周血单核细胞(peripheral blood mononuclear cell, PBMC)共培养,利用流式细胞仪检测 B 淋巴细胞内 IL-10 的表达情况。结果 十二烷 基硫酸钠 - 聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electropheresis, SDS-PAGE)、 Western Blot 结果表明,纯化后的蛋白分子量大小约为 38.2 kDa,符合 HLA-G1 蛋白的分子量 ;人 PBMC 与 HLA-G1 蛋白共培养后,随着 HLA-G1 蛋白浓度的升高(浓度梯度 :0、50、100 和 200 ng/ml),B 细胞内 IL-10 的表达水平明显增加,分别为 0.1%、0.36%、0.57%、0.73%。结论 通过原核表达系统获得 HLA-G1 蛋白,纯度达 95% 以上,HLA-G1 蛋白与人 PBMC 共培养后,可诱导 B 淋巴细胞表达 IL-10,并具有浓度 依赖性,提示其对肾移植免疫耐受的建立具有重要作用。