外周血基因整体表达分析技术实时监测心脏移植免疫排斥状态的初步研究

Real-time monitoring the immune response with nmlti-gene expression in the peripheral blood after heart transplantation？ a preliminary study

目的建立外周血单核细胞检测心脏移植免疫排斥相关基因的研究方法，探讨基因在心脏移植术后免疫排斥监测中的临床价值。方法应用实时荧光定量RT-PCR技术，以SYBR Green Ⅰ作荧光染料，GAPDH作内对照，观察心脏移植术后不同时期病人外周血单个核细胞中与免疫排斥相关基因的mRNA表达水平，并与病人术前基因表达水平对照。结果使用Primer Premier 5．0和Primer Express 3．0软件成功进行引物设计，并建立稳定检测的RT-PCR方法。心脏移植术前外周血单个核细胞16个与免疫排斥相关候选基因的mRNA的相对表达量与正常对照组比较差异无统计学意义（P〉0．05）。1TGA4、FKB、IL1R2、PF4、ITGAM、TGFB1、RHOU7种基因可能与机体免疫排斥状态有更大相关性。结论成功建立了荧光定量RT-PCR方法，检测外周血单个核细胞基因mRNA表达水平简便快速、特异、重复性好、可信度高具有良好的检测心脏移植早期排斥反应的应用前景。

Objective Graft rejective reaction is a major factor affecting heart function and lnng-term survival after heart transplantation. Early identification is critically important for the treatment. We try to develop a micro-invasively monitoring method for the identification of immunologic rejection, by means of gene expression determination in the mononuclear cells of the peripheral blood, and evaluate its clinical usefulness for rejection prediction. Methods The SYBR Green Ⅰ was used as fluorescent dye and the GAPDH as house keeping gene control in the quantitative RT-PCR technique for measuring genes expression of 16 candidate genes involving in various molecular pathways of immunologic rejection after heart transplantation. Results were also compared with that of normal people. Results The expression of 16 immunologic rejection associated genes was not different between normal people and the transplantation recipients before surgery （ P 〉 0.05）. After heart transplantation, the expression of rITGA4, FKB, IL1R 2、 PF4, ITGAM,TGFB1 ,RHOU was associated with immune response. ACt（gene-GAPDH）of 1TGA4,FKB,IL1R 2 among 16 genes reduced significantly 1 month after operation （ P 〈 0.05） then elevated gradually 2 to 3 month post-operatively, ACt（gene - GAPDH）of PF4, ITGAM,TGFB1 ,RHOU increased significantly 1 month after operation（ P 〈 0.01）then dropped rapidly 2 to 3 month post-operatively. No significant difference between pre-operative and post-operative expressions was observed for gene WDR40A. Conclusion A steady real-time quantitative RT-PCR was developed successfully, which has potential in identifying immune associated gene expression and may be valuable for clinical implication.