儿童重型再生障碍性贫血T细胞内调控因子GAS5和mTOR信号途径分子的表达研究

摘要

目的：通过检测再生障碍性贫血（AA）患儿T细胞内mTOR信号途径分子及T细胞内调控因子GAS5的表达水平，初步探讨该信号途径在儿童AA中的改变。方法（1）对16例初治重型AA（SAA）及8例免疫抑制剂治疗（IST）有效的SAA患儿，用流式细胞术（FCM）方法检测外周血CD3＋T细胞内mTOR信号途径分子磷酸化Akt（p-Akt）、磷酸化TSC2（p-TSC2）、磷酸化mTOR （p-mTOR）、磷酸化4EBP1（p-4EBP1）及磷酸化p70S6K（p-p70S6K）的表达水平。（2）用实时荧光定量聚合酶连反应（QRT-PCR）方法检测23例初治SAA及7例IST有效SAA患儿骨髓CD3＋T细胞内调控因子 GAS5的表达水平。结果（1）初治 SAA 组外周血 CD3＋T 淋巴细胞内 p-Akt、p-TSC2、p-mTOR、p-4EBP1及p-p70S6K表达水平均明显高于正常对照组，而低于阳性对照CEM细胞株组；（2）SAA 治疗有效组外周血 CD3＋T 淋巴细胞内 p-Akt、p-TSC2、p-mTOR、p-4EBP1及p-p70S6K的表达水平均均低于初治SAA组；SAA治疗有效组p-Akt、p-TSC2、p-mTORC1、p70S6K的表达水平与正常对照组无明显差异，但p-4EBP1的表达水平高于正常对照组。（3）初治SAA组骨髓CD3＋T淋巴细胞内GAS5的表达量明显低于正常对照组，而显著高于CEM细胞株组。（4）SAA治疗有效组骨髓CD3＋T淋巴细胞内GAS5的表达水平高于初治SAA组，而与正常对照组无明显差异。结论（1）mTOR信号途径的活化程度与疾病状态相关，初治SAA患儿外周血细胞中p-Akt、p-TSC2、p-mTOR、p-4EBP1、p-p70S6K表达升高，说明mTOR信号在SAA患儿中呈活化状态，可能参与AA的T细胞的免疫异常。（2）初治SAA患儿骨髓T细胞内GAS5表达水平明显低于正常儿童而显著高于 CEM细胞，SAA 治疗有效后 GAS5表达水平明显升高，GAS5的表达水平可能与mTOR信号途径的活化存在一定负相关。GAS5可能为负性调控因子。%Objective ToinvestigatethechangesofmTORsignalingpathwayinchildhoodsevere aplastic anemia by detecting the expression levels of the molecules of mTOR signaling pathway and GAS5 in T cells.Methods (1 )The expressions of p-Akt,p-TSC2,p-mTOR,p-4EBP1 ,p-p70S6K in CD3 +T cells in peripheral blood were detected by flow cytometry (FCM).Peripheral blood samples were collected from 1 6 newly diagnosed severe aplastic anemia(SAA)patients and 8 patients with effective immunosuppressive therapy (IST).(2 )The expression of GAS5 in CD3 +T cells in bone marrow mononuclear cells were detected by real time quantitative polymerase chain reaction (RT-PCR).And bone marrow samples were collected from 23 newly diagnosed SAA patients and 7 patients with effective IST.Results (1 )The expressions of p-Akt,p-TSC2,p-mTOR,p-4EBP1 ,p-p70S6K of the newly diagnosed SAA group were significantly higher than the normal control group.(2 )The expressions of p-Akt,p-TSC2,p-mTOR,p-4EBP1 and p-p70S6K of the effective treatment groups were much lower than the newly diagnosed SAA group;the expressions of p-Akt,p-TSC2,p-mTOR,p-p70S6K were similar to the normal control group (P>0.05 ),but the expression of p-4EBP1 was higher.(3 )The expression of GAS5 of the newly diagnosed SAA group were lower than the normal control group.(4) The expression of GAS5 of the effective treatment group was higher than the newly diagnosed SAA group, but was similar to the normal control group.Conclusions (1 )The expressions of p-Akt,p-TSC2, p-mTOR,p-4EBP1 ,p-p70S6K were increased in the newly diagnosed SAA patients,and were much higher than the effective treatment groups,which suggested that the mTOR signaling pathway was activated in SAA patients.And the degree of activation of mTOR signaling pathway was positively associated with the disease states.The signaling pathways may be involved in the T cell-mediated immune abnormalities in the pathogenesis of AA.(2 )The expression of GAS5 in the newly diagnosed SAA patients was decreased but increased in the effective treatment of children with SAA.The expression level of GAS5 may be negatively correlated with the activation of mTOR signaling pathway.