Tim-1+CD19+调节性B细胞在肾移植受者外周血的鉴定与功能研究
Identification and functional study of Tim-1 + CD19 + regulatory B cell in kidney transplantation recipients
目的 观察肾移植受者外周血中T细胞免疫球蛋白黏蛋白分子1 (Tim-1)+ CD19+B细胞比例,研究调节性B细胞的功能特性.方法 2009年12月至2010年6月解放军第三○九医院全军器官移植中心所行亲属活体肾移植供者和受者35对,肾移植受者移植术前(术前组,n=35)及术后4周(术后组,n =35)采集外周血,以健康供者作为对照组(n=35),采用流式细胞术分析各组外周血CD19+B细胞数并分选各组Tim-1+ CD19+B细胞.分离供、受者外周血淋巴细胞进行混合培养后,分别加入Tim-1+ CD19+和Tim-1 - CD19+B细胞,并以此分为Tim-1+组和Tim-1 -组,不加B细胞作为空白组,Annexin V-FITC/PI双染法观察各组细胞凋亡情况,流式细胞术分析各组细胞因子含量.结果 术前组、术后组和对照组CD19+B细胞绝对值分别为(170 ±90)、(202 ±99)、(155±71)个/μl,术后组高于对照组且差异有统计学意义(P =0.0300);3组Tim-1+ CD19+B细胞比率分别为(2.20±0.98)%、(35.46±10.66)%、(1.95±0.95)%,术后组高于对照组和术前组(均P<0.01).混合淋巴细胞培养中,Tim-1+组、Tim-1 -组和空白组细胞早期凋亡率分别为(45.31±12.37)%、(10.92±2.14)%、(1.93±0.26)%,Tim-1+组高于Tim-1 -组和空白组(均P <0.05);3组细胞晚期凋亡率分别为(21.32±5.67)%、(2.32±0.31)%、(1.27±0.19)%,Tim-1+组高于Tim-1 -组和空白组(均P <0.05).Tim-1+组培养上清液中白细胞介素10的水平显著高于Tim-1 -组[(5.32±0.37)pg/ml比(2.46±0.25) pg/ml,P=0.0001],干扰素γ水平低于Tim-1 -组[(1.51±0.22) pg/ml比(4.69±0.32)pg/ml,P=0.0015].结论 肾移植受者外周血中存在Tim-1+ CD19+B细胞,具有促进淋巴细胞凋亡的功能.Tim-1+ CD19+B细胞作为一群新的调节性B细胞亚群,在移植免疫反应中发挥重要作用.
更多Objective To observe the ratio of Tim-1 + CD19 + B cell in the peripheral blood of kidney transplantation recipients and elucidate its functions.Methods From December 2009 to June 2010,a total of 35 pairs of kidney transplant recipients were selected and divided into 3 groups:healthy donors as control ( n =35 ),pre-transplantation ( n =35 ) and post-transplantation ( n =35 ).The profiles of Tim-1 + CD19 + B cell in kidney transplantation donors and recipients were analyzed and sorted by flow cytometry (FCM). Mixed lymphocyte culture (MLC) was carried out between kidney transplantation donors and recipients.After the additions of Tim-1 + CD19 + and Tim-1 -CD19 + B cells,there were 3 groups:Tim-1 +,Tim-1 - and blank.Lymphocyte proliferation and inhibition status were evaluated by propidium iodide uptake and Annexin V binding.And the cytokine levels were detected by FCM.Results The absolute values of peripheral CD19 + B cells were ( 170 ± 90),(202 ± 99 ),( 155 ± 71 ) cells/μl in the pre-transplantation,post-transplantation and control groups respectively,post-transplantation group were higher than control group (P=0.0300).The Tim-1+CD19+ cell ratios were (2.20±0.98)%,(35.46±10.66)% and (1.95±0.95 ) % in three groups.And the differences were statistically significant ( both P < 0.01 ).Tim-1 + CD19 +B and Tim-1 - CD19 + B cells were added into MLC respectively.The early apoptotic cells of the Tim-1 + group were higher than those in the Tim-1 - group [ (45.31 ± 12.37 ) % vs ( 10.92 ± 2.14) %,P < 0.05 ] and significantly higher than the blank group [ ( 1.93 ±0.26) %,P <0.01 ].Late apoptotic and dead cells of the Tim-1 + group were higher than those in the Tim-1 - group [ (21.32 ± 5.67 ) % vs (2.32 ± 0.31 ) %,P<0.01] and the blank group [(1.27 ±0.19)%,P <0.05].The interleukin 10 levels in MLC supematant of the Tim-1 + group were significantly higher than those in the Tim-1 - group [ (5.32 ± 0.37) pg/ml vs (2.46 ±0.25) pg/ml,P =0.0001 ].However,the interferon-γ levels were lower than those in the Tim-1- group [(1.51 ±0.22) pg/ml vs (4.69 ±0.32) pg/ml,P=0.0015].Conclusion Present in the peripheral blood of kidney transplantation recipients,Tim-1 + CD19 + B cell has the capacity of promoting lymphocytic apoptosis. As a new regulatory subset of B cells,it plays important roles in the immune responses of transplantation.
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