GM-CSF与TGF-β联合处理小鼠供体骨髓细胞诱导特异性免疫耐受

The study of the inducing donor specific immunological tolerance by GM-CSF and TGF-βtreated donor’s marrow cell

目的:观察用GM-CSF和TGF-β在体外处理供体骨髓细胞(BM)后,输给受体鼠,观察能否诱导受体鼠对供体鼠淋巴细胞的特异性耐受。方法:体外用GM-CSF与TGF-β联合处理供体BM细胞诱导非成熟树突状细胞(iDC),并检测其成熟度。将BALB/c受体鼠随机分为3组,进行不同的预处理:(1)BM预免疫组:经尾静脉输入体外诱导的iDC,1×105细胞/只;(2)脾细胞预免疫组:经尾静脉输入C57BL/6供体鼠脾细胞,1×105/只;(3)阴性对照组:经尾静脉输入同体积的PBS。每组小鼠均需经过2次预免疫,每次间隔1周。第2次处理后1周,所有组的小鼠均接受相同剂量的C57BL/6来源的脾细胞腹腔注射,1×105/只。脾细胞攻击后3d,检测各组小鼠对C57BL/6小鼠脾细胞的同种异体反应水平,检测指标包括:采用单向混合淋巴细胞培养方法检测受体鼠淋巴细胞对供体鼠淋巴细胞的增殖指数;采用双抗体夹心ELISA检测受体鼠血清中IFN-γ和IL-10水平、FCM检测脾脏CD4+CD25highTreg细胞含量、及NK细胞抑制性受体KLRG1的表达、采用乳酸脱氢酶释放法检测NK细胞杀伤功能。结果:经GM-CSF和TGF-β体外处理的供体BM细胞能够抵抗脂多糖(LPS)促成熟的作用;与脾细胞预免疫组相比,经此BM细胞预处理的小鼠,再次遭遇供体小鼠淋巴细胞时,血清IL-10的含量降低(P<0.05)、脾脏中CD4+CD25highTreg细胞的比例明显升高、对C57BL/6小鼠淋巴细胞增殖反应减弱(P<0.01);NK细胞杀伤率降低。结论:用GM-CSF与TGF-β联合处理供体小鼠BM细胞在一定程度上能够诱导小鼠对供体小鼠脾细胞的免疫耐受,除Treg外,NK细胞可能也参与了诱导免疫耐受。

AIM：To observe whether the donor’s marrow （BM） cells treated by GM-CSF and TGF-β in vitro were beneficial in inducing specific hypoergia to donor’s lymphocyte in recipient.METHODS：Donor’s marrow cell were cultured with GM-CSF and TGF-β in vitro and its maturities were tested by treating with LPS.BALB/c mice were divided into three groups and received treatment as following：（1） BM pretreatment group：donor’s BM cell mentioned above were injected into caudal vein with 105 cell each mouse;（2） donor’s splenocyte group：C57BL/6 mouse＇s splenocyte were injected through caudal vein with 105 cell each mouse;（3） Negative control group：PBS with the same volume were injected through caudal vein.The treatment were performed twice with one week internal in each group.One week after the second treatment,all of the mice were challenged by splenocyte of C57BL/6 mouse in abdominal cavity with 105 cell each mouse.Three days later,the allo-reactivities were tested：the proliferation of recipient mouse lymphocyte to donor’s lymphocyte were test by single mix lymphocyte reaction,the level of serum IFN-γ and IL-10 were tested with ELISA methods.CD4＋CD25 high Treg cell and expression of killer cell lectin-like receptor G1 （KLRG1） on NK cell were tested with flow cytometric technique and the NK cytotoxicity were measured by LDH release method.RESULTS：The donor’s marrow cell treated by GM-CSF and TGF-β in vitro could resist the maturation promotion by LPS.Compared with donor’s lymphocyte pretreatment,pre-treatment with BM cell induced decreased IL-10,increased CD4＋CD25 high Treg cell in spleen,decreased proliferation to donor’s lymphocyte in vitro.Additionally,NK cell cytotoxicity was also decreased.CONCLUSION：The donor’s marrow cell treated by GM-CSF and TGF-βcan induce the donor specific tolerance in some degree,and modulation of NK cell maybe participate in inducing immunological tolerance.