生血合剂影响慢性再障患者外周血单个核细胞T-bet/GATA-3及相关信号分子、细胞因子表达的体外研究

In Vitro Study of Shengxue Mixture Interfering the Expressions of T-bet GATA-3 Relevant Signal Transduction MolecuLar and Cytokine of Peripheral Blood Mononuclear Cell from Patients of Chronic Aplastic Anemia

目的:转录因子T-bet、GATA-3及相关信号通路在Th1、Th2分化平衡及免疫反应调节中发挥重要的作用,本研究拟从体外研究健脾补肾活血中药复方生血合剂对慢性再生障碍性贫血(CAA)患者外周血单个核细胞(PBMNC)T-bet、GATA-3及相关信号分子、细胞因子表达的影响。方法:纳入CAA患者10例,男5例,女5例,年龄42～73岁,采集外周血前3天内未服用中药及环孢素。生血合剂制成无菌针剂(每毫升含2 g生药),环孢菌素A(CsA)用无菌RPMI1640配制成浓度为1 mg/mL的溶液。分离培养患者外周血单个核细胞(PBMNC),加或不加药物培养72 h,分为空白对照组、大剂量生血合剂组(200 mg/mL)、小剂量生血合剂组(100 mg/mL)和环孢素组(400ng/mL),采用实时荧光定量PCR(Realtime FQ-PCR)检测培养后PBMNC转录因子T-bet、GATA-3及信号分子STAT4、STAT6 mRNA表达情况,采用酶联免疫吸附法(ELISA)检测PBMNC培养上清IFN-γ、IL-12、IL-4水平。结果:CAA患者PBMNC体外培养后,空白对照组T-bet、STAT4、IFN-γ、IL-12表达均明显高于小剂量生血合剂组、大剂量生血合剂组和环孢素组(P<0.05),小剂量生血合剂组T-bet、STAT4、IFN-γ、IL-12显著高于大剂量生血合剂组、环孢素组(P<0.05),大剂量生血合剂组T-bet、STAT4、IFN-γ、IL-12与环孢素组之间没有显著性差异(P>0.05);各组GATA-3、STAT6、IL-4之间比较无明显差异(P>0.05)。结论:生血合剂能在体外下调CAA患者PBMNC T-bet、STAT4、IFN-γ、IL-12表达,并且呈现一定的剂量依赖性。

Objective：Transcription factors T-bet（T-box expressed in T cell）,GATA-3（GATA binding protein 3）and relevant signal transduction pathways play important roles in the balance,differentiation of Th1/Th2 cells and regulation of immunological responses.This study aims to explore the effects of Shengxue mixture（SXM）on the expressions of T-bet,GATA-3,relevant signal transduction Molecular and Cytokine of peripheral blood mononuclear cell（PBMNC）from patients of chronic aplastic anemia（CAA）in vitro.Methods：10 patients with CAA（5 male,5 female,age range from 42 to 73）were included.They all didn＇t take Chinese materia medica or cyclosporin A（CsA）within 3 days before drawing peripheral blood.SXM was made into aseptic parenteral solution containing 2 grams of crude drug per milliliter.CsA was diluted into solution（1 mg/mL of concentration）with aseptic cell cμLture fluid（RPMI1640）.PBMNC from CAA patients was isolated and cultured in 72 h with or without drugs.They were divided into four groups,i.e.group of aplastic anemia PBMNC without adding drugs（group Blank）,group of aplastic anemia PBMNC with adding high-dose Shengxue Mixture（group HDSXM）,group of aplastic anemia PBMNC with adding low-dose Shengxue mixture（group LDSXM）and group of aplastic anemia PBMNC with adding CsA（group CsA）.Finally,mRNA expressions of PBMNC transcriptional factors and signaling molecule were determined with methods of realtime fluorescent quantitation polymerase chain reaction（Realtime FQ-PCR）,and levels of cytokine in the supernatant were detected by applying enzyme linked immunosorbent assay（ELISA）.Results：After culturing in vitro,expressions of T-bet,STAT4,IFN-γ and IL-12 in blank group were significantly higher than those in LDSXM group,HDSXM group,and CsA group（P0.05）,while expressions of T-bet,STAT4,IFN-γ and IL-12 in LDSXM group were higher than those in HDSXM group and CsA group（P0.05）.There was no significant difference between HDSXM group and CsA group in terms of above indexes（P0.05）.In the expressions of GATA-3,STAT6 and IL-4,no significant difference existed between these groups（P0.05）.Conclusion：Shengxue Mixture can lower the expressions of T-bet,STAT4,IFN-γ and IL-12 from PBMNC of CAA patients in vitro,and is dose dependent in a certain extent.