人来源脂肪干细胞对特发性血小板减少性紫癜小鼠疗效分析

Efficacy analysis of human-derived adipose stem cells on idiopathic thrombocytopenic purpura mice

目的通过注射人来源脂肪干细胞(hADSC),了解hADSC能否改善特发性血小板减少性紫癜(ITP)小鼠血小板水平和调控辅助性T细胞(Th)细胞免疫功能紊乱。方法选择无特定病原体级健康白变种实验室(BALB/c)雌性小鼠30只,体质量为15~25 g,鼠龄6~8周。选择年龄21~36岁女性健康志愿者6例,通过外科医生无菌条件下获取的不再临床应用的人体脂肪组织。应用贴壁细胞培养法及梯度离心法,将分离后脂肪干细胞鉴定、培养和增殖;将30只BALB/c小鼠随机分为正常组、对照组和治疗组,除了正常组,对照组和治疗组小鼠按免疫法建立小鼠ITP模型。建模后,治疗组输注hADSC,而对照组则注射等量的磷酸盐缓冲溶液(PBS)。再用血细胞分析仪检测各组外周血血小板,用酶联免疫吸附分析(ELISA)法检测外周血血小板相关免疫球蛋白(PAIgG)和Th1/Th2/Th3/Th17细胞因子水平,并用逆转录-聚合酶链式反应(RT-PCR)检测脾脏组织中相关因子mRNA表达。结果与正常组干扰素γ(IFN-γ)(128.44 pg/mL±10.29 pg/mL)、白细胞介素(IL)2(20.14 pg/mL±2.65 pg/mL)、IL-17(22.42 pg/mL±5.70 pg/mL)、IL-4(53.67 pg/mL±8.27 pg/mL)、IL-10(68.28 pg/mL±5.53 pg/mL)及转化生长因子β_(1)(TGF-β_(1))(117.21 pg/mL±15.15 pg/mL)相比,ITP小鼠外周血IFN-γ(164.16 pg/mL±11.76 pg/mL)、IL-2(31.48 pg/mL±4.61 pg/mL)、IL-17(29.92 pg/mL±3.26 pg/mL)水平明显升高,IL-4(31.17 pg/mL±5.35 pg/mL)、IL-10(41.40 pg/mL±5.73 pg/mL)、TGF-β_(1)(63.96 pg/mL±7.47 pg/mL)水平明显降低,差异均有统计学意义(P<0.05)。治疗组输注hADSC后,其外周血血小板[(580±89)×10^(9)/L]明显上升,PAIgG(18.26 pg/mL±0.84 pg/mL)水平明显下降(均P<0.01),同时Th1/Th2/Th3/Th17细胞因子IFN-γ(130.59 pg/mL±14.16 pg/mL)、IL-2(22.09 pg/mL±3.78 pg/mL)、IL-17(24.19 pg/mL±6.79 pg/mL)水平下降,而IL-4(48.81 pg/mL±6.02 pg/mL)、IL-10(62.35 pg/mL±7.09 pg/mL)、TGF-β_(1)(104.47 pg/mL±16.30 pg/mL)的水平均升高(均P<0.05)。通过对小鼠脾脏组织检测相应的Th细胞因子mRNA表达,其表达变化与外周血细胞因子结果基本一致。结论hADSC可能通过调控Th1/Th2/Th3/Th17细胞及其因子水平,进而改善ITP机体细胞免疫功能紊乱,改善ITP小鼠血小板水平。

Objective To investigate the effect of human adipose-derived stem cells(hADSC) on improving platelet levels and regulating helper T cell (Th) immune dysfunction in idiopathic thrombocytopenic purpura (ITP) mice via hADSC injection.Methods Thirty female specific pathogen free and healthy white mutant laboratory(BALB/c) mice weighted 15-25 g and aged 6-8 weeks were sacrificed.Six healthy female volunteers aged 21-36 years old were enrolled,and the non-clinical adipose tissue was obtained under aseptic conditions by surgeons.The isolated adipose stem cells were identified,cultured and proliferated by adherent cell culture and gradient centrifugation.The BALB/c mice were randomly divided into normal group,control group and treatment group.The normal group was given no treatment,and the mice in control group and treatment group were immunized to establish ITP model.After modeling,the treatment group was treated with hADSC,while control group was treated with the same amount of phosphate buffered solution(PBS).Peripheral blood platelets in each group were detected by blood cell analyzer,the levels of platelet-associated immunoglobulin(PAIgG) and Th1/Th2/Th3/Th17 cytokines in peripheral blood were detected by enzyme-linked immunosorbent assay(ELISA),and mRNA expression of related factors in spleen tissue was detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Compared with normal group of interferon-γ(IFN-γ)(128.44 pg/mL±10.29 pg/mL),interleukin(IL)-2(20.14 pg/mL±2.65 pg/mL),IL-17(22.42 pg/mL±5.70 pg/mL),IL-4(53.67 pg/mL±8.27 pg/mL),IL-10(68.28 pg/mL±5.53 pg/mL) and transforming growth factor β_(1)(TGF-β_(1))(117.21 pg/mL±15.15 pg/mL),the peripheral blood IFN-γ(164.16 pg/mL±11.76 pg/mL),IL-2(31.48 pg/mL±4.61 pg/mL),IL-17(29.92 pg/mL±3.26 pg/mL) level of ITP mice were significantly increased,and IL-4(31.17 pg/mL±5.35 pg/mL),IL-10(41.40 pg/mL±5.73 pg/mL),TGF-β_(1)(63.96 pg/mL±7.47 pg/mL) were significantly decreased,and the differences were statistically significant(P<0.05).After infusion of hADSC,the peripheral blood platelets[(580±89)×10^(9)/L]in treatment group was increased significantly,and level of PAIgG(18.26 pg/mL±0.84 pg/mL) decreased significantly(P<0.01).Meanwhile,the levels of Th1/Th2/Th3/Th17 cytokines IFN-γ(130.59 pg/mL±14.16 pg/mL),IL-2(22.09 pg/mL±3.78 pg/mL),IL-17(24.19 pg/mL±6.79 pg/mL) were decreased,and IL-4(48.81 pg/mL±6.02 pg/mL),IL-10(62.35 pg/mL±7.09 pg/mL),TGF-β_(1)(104.47 pg/mL±16.30 pg/mL) were increased(P<0.05).The detecting results of mRNA expression of corresponding factors of Th1/Th2/Th3/Th17 in spleen tissue of mice showed that the expression changes were basically consistent with the cytokine results.Conclusion It is demonstrated that hADSC could improve cellular immune dysfunction of ITP and improve platelet level of ITP mice via regulating Th1/Th2/Th3/Th17 cells and their factors.