荧光定量RT—PCR实时检测心脏移植患者外周血单核细胞16种免疫排斥基因整体表达变化的初步研究

Preliminary research on 16 peripheral blood monocyte immune rejection relative genes expression after heart transplantation with fluorescent quantitation RT-PCR techniques.

目的探讨外周血单核细胞免疫排斥相关基因的检测对心脏移植排斥反应的诊断价值。方法采取心脏移植术患者外周血，应用实时荧光定量RT-PCR技术，观察心脏移植术后不同时期外周血单个核细胞中与免疫排斥相关多个基因系统的mRNA表达水平，并与术前和正常组的基因表达水平对照。结果心脏移植术前外周血单个核细胞16种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）。初步筛选出与机体免疫排斥状态有更大相关性的7种基因，术后3个月内心脏功能稳定组较术前和正常对照组ITGA4、FKB、ILlR-2mRNA表达水平上调，PF4、ITGAM、TGFβ1、RHOU表达水平降低。这与临床观察的移植术后1～3个月免疫排斥概率最大相吻合。结论检测外周血单个核细胞基因mRNA表达水平的荧光定量RT—PCR方法简便快速、特异、重复性好、可信度高，但在检测心脏移植排斥反应方面值得进一步深入研究。

Objective To construct the micro-invasive immune rejection monitoring methods with peripheral blood mononuclear cell gene expression detection and evaluate the clinic rejection estimation value. Methods The SYBR Green I was used as fluorescent dye and the GAPDH as house keeping gene control in the quantitation RT-PCR technique to observe the 16 immune rejection relative genes expression features after heart transplantation. results were also compared with that of the normal people. Results The 16 immune rejection relative genes expression were no different between normal people and the transplantation recipients before surgery （ P 〉 0.05 ）. After heart transplantation the expression of ITGA4, FKB, IL1R-2 up regulated and the level of PF4, ITGAM,TGFβ1, RHOU down regulated. The results were similar with the clinic observation that the immune rejection often occurs in the first 3 months after heart transplantation. It implied that these 7 genes may play an important role in the acute immune rejection after transplantation. Conclusion The real time quantitation RT-PCR methods were constructed successfully to detect the multiple immune relative genes expression and is of clinic applicable.