小鼠心脏移植排斥反应中miR-494与TH17细胞分化的相关性研究

Correlation between miR-494 and TH 17 cell differentiation in murine cardiac transplant rejection

目的研究小鼠心脏移植排斥反应中小分子RNA494（miR-494）与辅助性T淋巴细胞17（TH17细胞）分化的相关性。方法分别建立颈部异位心脏移植模型：对照组，供者和受者均为C57BL／6小鼠；实验组，供者为Balb／c小鼠，受者为C57BL／6小鼠。利用逆转录聚合酶链反应（RT-PCR）检测移植物中miR-494与IL-17AmRNA的表达。分离移植物中CD4^＋T淋巴细胞、CD8^＋T淋巴细胞及CD45^＋细胞，检测各组细胞中miR-494与IL-17AmRNA的表达。体外分离C57BL／6小鼠脾细胞，利用免疫磁珠法进行CD4^＋T淋巴细胞分选，定向分化为TH1、TH2、TH17和调节性T淋巴细胞（Treg细胞）亚群，采用RT-PCR检测各细胞亚群IL-17AmRNA及miR-494的表达。结果小鼠心脏移植术后7d，实验组移植物中IL-17AmRNA表达高于对照组，而miR-494的表达低于对照组（均P〈0．05）。移植物CD4^＋T淋巴细胞IL-17A mRNA表达高于对照组，而miR-494的表达降低（均P〈0．05）。体外培养TH17细胞亚群中，miR-494的表达显著低于Tn1、TH2和Treg细胞。结论miR-494可能与移植排斥反应中TH17细胞分化密切相关，miR-494可能通过调控TH17细胞在移植排斥反应中发挥作用。

Objective To investigate correlation between microRNA （miR-494） and TH 17 cell differentiation in murine cervical heterotopic cardiac transplant model. Method The heterotopic cardiac transplant models of Balb/c→C57BL/6 mice were established as experimental group, and those of C57BL/6→C57BL/6 mice as control group. Real timepolymerase chain reaction（PCR） was used to detect miR-494 and interleukin（IL）-17A mRNA expression in the grafts. CD4^＋ T cells, CD8^＋ T cells and CD45^＋ myeloid cells were isolated from the grafts, and miR-494 and IL-17 mRNA expression was detected. In vitro, lymphocytes in the spleen from C57BL/6 mice were harvested, and CD4^＋ T cells were isolated with MACS and then stimulated to TH 1, TH2, TH 17, Treg subset cells. The expression of IL-17A mRNA and miR-494 in different T subsets was examined by Reverse transcription-polymerase chain reaction （RT-PCR）. Result Two grafts from each study group were harvested on the 7th day post-transplantation. In experimental group, the IL-17A mRNA expression was increased, while the expression of miR-494 was decreased as compared with control group with the difference being significant between two groups. The expression of IL-17A mRNA in CD4^＋ T cells of the grafts was significantly increased, while that expression of miR-494 was decreased. In vitro, the expression of miR-494 in TH 17 cells was significantly lower than that in TH 1, TH2 and Treg cells. Conclusion miR-494 is related closely to TH 17 cells differentiation in the transplant rejection, which may play a role in transplant rejection through regulating T. 17 ceils.