HLA-G参与羊膜间充质干细胞抑制淋巴细胞增殖的机制研究

Mechanism of HLA-G Participation in Inhibiting Lymphocyte Proliferation by Amniotic Mesenchymal Stem Cells

本研究旨在探讨羊膜间充质干细胞(human amniotic mesenchymal stem cells,HAMSC)抑制淋巴细胞增殖的作用机制,证明人类白细胞抗原G(HLA-G)参与了HAMC的免疫抑制功能。从胎膜上分离、培养和扩增HAMSC,用流式细胞术进行表型鉴定,同时检测膜表面和胞质中HLA-G的含量;用ELISA法检测培养上清中HLA-G的含量;MTT法检测混合培养后HAMSC对淋巴细胞增殖的影响。结果表明,HAMSC膜表面和胞质均表达HLA-G,膜表面和胞质HLA-G的含量分别是(16.75±3.871)%和(39.14±4.274)%,在细胞培养上清中的含量是5.2 ng/ml。HAMSC及其上清加入淋巴细胞混合培养后,淋巴细胞抑制率增高,当HLA-G特异性抗体加入后这种抑制效应减低。结论:HAMSC表面和胞质都表达HLA-G,同时也分泌HLA-G到上清液中。HLA-G是HAMSC的免疫抑制功能的关键因素之一,这为HAMSC在移植后抑制排斥反应的临床应用提供了理论依据。

This study was aimed to investigate the inhibitory machanism of human amniotic mesenchymal stem cells （HAMSC） on lymphocyte proliferation and to validate the participation of the nonclassic human leukocyte antigen （HLA） class Ⅰ molecule （HLA-G） in immunosuppressive action of HAMSC.HAMSC were isolated from fetal membranes of human placentas,and were cultured and expanted.The phenotypes of HAMSC were identified by flow cytometry,at same time the HLA-G levels on membrane surface and in cytoplasm were detected by flow cytometry.The soluble HLA-G （sHLA-G） level in HAMSC supernatants was determined by ELISA,MTT assay was used to examine the effect of mixed cultured HAMSC on proliferation of lymphocytes.The results showed that both surface and cytoplasm of HAMSC expressed HLA-G,the average rates of HLA-G expression on surface and in cytoplasm were （16.75 ±3.871） ％ and （39.14 ±4.274） ％,respectivaly.The sHLA-G level in cell culture supematant was 5.2 ng/ml.After HAMSC and culture supematants were added in the MLR,the inhibitory rate on lymphocyte proliferation increased obvionsly,meanwhile the inhibitory rate on lymphocyle proliferation decreased when the HLA-G antibody was added in MLR.It is concluded that the surface and cytoplasm of HAMSC express HAL-G,at same time HAMSC secrete the HLA-G to supernatants of culture.The HLA-G is one of critical factors inhibiting immunofunction of HAMSC.This study contributes to improve the clinical therapeutic trails for using the HAMSC to prevent rejection.