B7-H4介导小鼠间充质干细胞株C3 H10 T1/2对EAE小鼠T细胞亚群的作用

B7-H4 mediated immunomodulatory effects of mouse mesenchymal stem cells C3H10T1/2 on T cell polarization

目的：探讨负性共刺激分子B7-H4在小鼠间充质干细胞株C3H10T1/2（以下简称C3H10）对实验性自身免疫性脑脊髓炎（EAE）小鼠免疫调节的作用及其机制。方法体外试验：构建小鼠B7-H4靶向shRNA稳定转染的C3H10细胞（C3H10-B7-H4细胞），将转染前后的细胞分别与植物血凝素（ PHA）激发的小鼠脾脏淋巴细胞共培养，用ELISA法检测培养上清细胞因子的分泌。体内试验：制备小鼠EAE模型，将50只小鼠分为5组：正常对照组、EAE组、C3H10组（移植 C3H10细胞）、C3H10-NC组（移植shRNA对照质粒转染的C3H10细胞）、C3H10-B7-H4组（移植C3H10-B7-H4细胞），并在免疫后第6天，各组细胞以1×106个/只小鼠尾静脉输注，每日对各组小鼠进行神经功能缺损评分；用ELISA法检测小鼠外周血IL-2、IL-17、IFN-γ、IL-4的表达变化。结果体外试验：B7-H4靶向shRNA转染C3H10可抑制B7-H4的表达；下调表达B7-H4的C3H10细胞对脾细胞分泌IL-2、IL-17、IFN-γ的抑制作用减弱，而对IL-4无明显影响；体内试验：下调表达B7-H4的C3H10细胞对EAE小鼠的治疗作用减弱，表现在神经功能缺损评分增高，发病时间及发病高峰时间均较C3H10组提前。下调表达B7-H4的C3H10细胞治疗EAE小鼠后对血浆中IL-2、IL-17、IFN-γ的表达抑制作用减弱，但对IL-4的表达无影响。结论负性协同刺激分子B7-H4介导了C3H10移植治疗EAE的免疫调节，这一过程可能是调节Th1、Th17等炎性效应性T细胞间接发挥作用。

Objective To investigate the role and mechanism of B7-H4, a negative costimulatory molecule, in mediating the immunomodulatory effects of mesenchymal stem cells C3H10T1/2 （C3H10） on T cell polarization. Methods The lentiviral vectors that carried the shRNA targeting mouse B7-H4 were transfected into mouse mesenchymal stem cells （C3H10-B7-H4）. The cells were co-cultured with PHA-acti-vated mice spleen lymphocytes before and after the transfection. ELISA was performed to detect the concen-trations of cytokines in supernatants of cell culture in order to elucidate the effects of B7-H4 expressed by C3H10 on T cell polarization. A mouse model of experimental allergic encephalitis （EAE） was established. Fifty C57BL/6 mice were divided into five groups including control group, EAE group, C3H10 group （injec-ting EAE mice with C3H10 cells）, C3H10-NC group （ injecting EAE mice with C3H10-NC cells） and＆amp;nbsp;C3H10-B7-H4 group （injecting EAE mice with C3H10-B7-H4 cells）. ELISA was performed to detect the soluble form of IL-2, IL-17, IFN-γ and IL-4 in plasma samples. Results Knocking down the B7-H4 gene with shRNA significantly decreased the expression of B7-H4 on C3H10 cells, which weakened the inhibitory effects of C3H10 cells on the secretion of IL-2, IL-17 and IFN-γ by spleen lymphocytes. The therapeutic effects of C3H10-B7-H4 cells on mice with EAE were weakened after silencing the B7-H4 gene expression, which was manifested as higher nerve function score and earlier onset and bring forwarded peak time of EAE than those of the C3H10 group. Treating EAE mice with C3H10-B7-H4 cells was less efficient in inhibiting the expression of IL-2, IL-17 and IFN-γin plasma. However, knocking down the B7-H4 gene had no signif-icant effect on the expression of IL-4 in terms of treating EAE with C3H10 cells. Conclusion The co-inhib-itor molecule B7-H4 expressed on C3H10 cells mediated the treatment of EAE with C3H10 cells by regula-ting Th1 and Th17 effector T cells.