环孢菌素A对慢性再生障碍性贫血患者T-bet、GATA-3、相关信号分子、细胞因子及Th1/Th2的影响

Impact of Cyclosporine A on the Expression of T-bet,GATA-3,Relevant Signal Transduction Molecules,Cytokine and Th1/Th2 Balance in Patients with Chronic Aplastic Anemia

本研究探讨转录因子T-bet、GATA-3及相关信号通路在慢性再生障碍性贫血(CAA)免疫发病中的作用,从Th细胞失衡、转录因子及相关信号通路水平研究环孢菌素(CsA)治疗慢性AA的免疫调节机理。采用实时荧光定量PCR(real-time FQ-PCR)检测慢性AA患者治疗前和CsA治疗6月后外周血单个核细胞(PBMNC)T-bet、GATA-3及STAT4、STAT6mRNA表达;采用流式细胞术、酶联免疫吸附法(ELISA)检测慢性AA患者治疗前和CsA治疗6月后外周血Th1、Th2比例及PBMNC培养上清IFN-γ、IL-12、IL-4水平,并与正常人进行比较。结果表明:慢性AA患者PBMNC T-bet、STAT4mRNA表达、T-bet/GATA-3比值、Th1比例、Th1/Th2比值、PBMNC培养上清IFN-γ、IL-12表达均明显高于正常组(p<0.01),经CsA治疗6月后,患者T-bet、STAT4表达、T-bet/GATA-3比值、Th1比例、IFN-γ、IL-12表达均有所下降,但T-bet、STAT4、T-bet/GATA-3比值、Th1比例、IFN-γ表达仍未达到正常水平,GATA-3、STAT6mRNA表达、Th2比例、IL-4表达在治疗前后与正常人比较均无明显差异(p>0.05)。结论:IFN-γ/T-bet、IL-12/STAT4通路的异常活化及Th平衡向Th1型偏移在AA免疫异常的发病过程中起到关键的作用;CsA能通过下调IFN-γ/T-bet、IL-12/STAT4通路的异常活化及纠正Th1过度极化而减轻AA异常亢进的细胞免疫,解除造血抑制。

The study was aimed to explore the effects of T-bet（T-box expressed in T cell）,GATA-3（GATA binding protein 3） and relevant signal transduction pathways on the immune-related pathogenesis of chronic aplastic anemia（CAA）,and to investigate the immunological regulation mechanism in the treatment of CAA by using cyclosporine A（CsA） at the level of Th cell imbalance,transcriptional factors,and relevant signal pathways.The real-time fluorescent quantitative polymerase chain reaction（real-time FQ-PCR） was used to determine the mRNA expression of T-bet,GATA-3,signal transducers and activators of transcription 4（STAT4） and signal transducers and activators of transcription 6（STAT6） in peripheral blood mononuclear cell（PBMNC） of CAA patients before and after treatment with CsA;the flow cytometry（FCM） and enzyme linked immunosorbent assay（ELISA） were used to determine the Th1/Th2 proportion in peripheral blood,and levels of IFN-γ,IL-12 and IL-4 in PBMNC-cultured supernatant.Healthy people were included to test the above indexes.The results showed that the mRNA expression of PBMNC T-bet,STAT4,T-bet/GATA-3 ratio,Th1 proportion,Th1/Th2 ratio and levels of IFN-γ and IL-12 in PBMNC-cultured supernatant of CAA patients were significantly higher than those of healthy people（p0.01）.After treating with CsA for 6 months of CsA treatment,expression of T-bet,STAT4,T-bet/GATA-3 ratio,Th1 proportion,IFN-γ and IL-12 levels were lower than before,however,the expression of T-bet,STAT4,T-bet/GATA-3 ratio,Th1 proportion and IFN-γ had not been reduced to normal state.Compared to healthy people,no significant difference existed in the mRNA expression of GATA-3,STAT6,Th2 proportion,as well as level of IL-4 before and after treatment（p0.05）.It is concluded that the abnormal activation of IFN-γ/T-bet and IL-12/STAT4 pathways,as well as Th balance deviating to Th1 excursion play vital roles in the immunological pathogenesis of AA.CsA lowers the abnormal activation of IFN-γ/T-bet and IL-12/ STAT4 pathways to correct Th1 hyperpolarization,which may reduce the abnormally activated cell-mediated immunity and relax hematopoietic depression of AA patients.