一、人CD19+CD24highCD38high及CD19+CD24highCD27+调节性B细胞特性的研究目的:探讨人外周血CD19+CD24highCD38high及CD19+CD24highCD27+调节性B细胞的特性。方法:流式细胞仪检测健康志愿者外周血单个核细胞中CD19CD24CD38及CD19+CD24highCD27+调节性B细胞(regulatory B cell,Breg)的比例及其凋亡、增殖、B细胞活化因子受体(B-cell activating factor receptor,BAFF-R)、跨膜蛋白活化因子钙离子信号调节亲环素配体(transmembrane activator calcium modulatorand cyclophilin ligand interactor,TACI)、B细胞成熟抗原(B cell maturation Ag,BCMA)、结合的B细胞活化因子(B-cell activating factor belonging to the TNF family,BAFF)及一种增殖诱导配体(a proliferation-inducing ligand,APRIL)水平、T细胞免疫球蛋白黏蛋白结构域相关分子-3(T cell Ig domain and mucin domain 3,Tim-3)表达、CD20表达、CD80表达及CD86表达。分别经CD40L及Cp G ODN2006刺激后检测CD19+CD24highCD38high、CD19+CD24highCD27+Breg细胞分泌的IL-10、TGF-β水平。将CD19+CD24highCD38high与CD19+CD24highCD27+Breg细胞、CD19+CD24highCD38high与非Breg、CD19+CD24highCD27+与非Breg之间进行上述指标的比较。结果:在正常人外周血单个核细胞中CD19+CD24highCD38high Breg比例为5.88%3.18%,CD19+CD24highCD27+Breg的比例为14.10%±9.47%,CD19+CD24highCD27Breg的比例高于CD19+CD24highCD38high Breg(P=0.000)。CD19+CD24highCD38high Bre与CD19+CD24highCD27+Breg相比凋亡水平无差异(28.87%±14.82%vs.28.84%18.33%,P=0.994),CD19+CD24highCD38high Breg与非CD19+CD24highCD38high细胞相凋亡水平亦无差异(28.87%±14.82%vs.23.86%±11.17%,P=0.994),而CD19+CD24highCD27+Breg凋亡水平高于非CD19+CD24highCD27+细胞(28.84%±18.33%vs.14.93%±7.83%,P=0.002)。CD19+CD24highCD38high Breg与CD19+CD24highCD27+Breg相比增殖水平差异无统计学意义(21.46%±11.88%vs.17.28%±13.18%,P=0.442),CD19+CD24highCD38high Breg增殖水平高于非CD19+CD24highCD38high细胞(21.46%±11.88%vs.6.22%±10.07%,P=0.009),CD19+CD24highCD27+Breg增殖水平与非CD19+CD24highCD27+细胞相比差异无统计学意义(17.28%±13.18%vs.12.96%±11.93%,P=0.570)。CD19+CD24highCD38high Breg细胞与CD19+CD24highCD27+Breg细胞均表达BAFF-R、TACI、BCMA,并且两群细胞间BAFF-R、TACI、BCMA的表达差异无统计学意义(BAFF-R:97.17%±6.17%vs.96.46%±8.64%,P=0.743;TACI:65.84%±28.60%vs.71.43%±36.51%,P=0.558;BCMA:91.15%±9.62%vs.90.28%±13.61%,P=0.799)。CD19+CD24highCD38high Breg细胞BAFF-R、TACI的表达高于非CD19+CD24highCD38high细胞(97.17%±6.17%vs.89.56%±7.43%、65.84%±28.60%vs.39.07%±23.92%,P=0.000、0.002),而BCMA的表达(94.13%±3.15%)无差异(P=0.495)。CD19+CD24highCD27+Breg细胞BAFF-R、TACI、BCMA的表达与非CD19+CD24highCD27+细胞(98.71%±1.10%、56.14%±28.75%、94.50%±4.08%)相比差异均无统计学意义(P=0.142,0.130,0.481)。CD19+CD24highCD38high Breg细胞与CD19+CD24highCD27+Breg细胞结合BAFF、APRIL的水平差异均无统计学意义(55.24%±17.46%vs.54.42%±20.36%,P=0.882;61.95%±31.66%vs.63.97%±35.07%,P=0.835)。CD19+CD24highCD38high Breg细胞结合BAFF的水平与非CD19+CD24highCD38high细胞相比差异无统计学意义(55.24%±17.46%vs.58.95%±12.59%,P=0.424),而结合APRIL的水平高于非CD19+CD24highCD38high细胞(55.24%±17.46%vs.35.29%±22.08%,P=0.002)。CD19+CD24highCD27+Breg细胞与非CD19+CD24highCD27+细胞相比结合BAFF、APRIL的水平(59.05%±15.89%,57.76%±33.04%)差异均无统计学意义(P=0.404,0.546)。CD19+CD24highCD38high Breg细胞Tim-3表达水平高于CD19+CD24highCD27+Breg(14.27%±6.97%vs.6.91%±5.88%,P=0.014),CD19+CD24highCD38high Breg细胞Tim-3表达水平高于非CD19+CD24highCD38high细胞(14.27%±6.97%vs.1.44%±1.71%,P=0.000),CD19+CD24highCD27+Breg细胞Tim-3表达水平高于非CD19+CD24highCD27+细胞(6.91%±5.88%vs.1.99%±2.01%,P=0.002)。CD19+CD24highCD38high Breg与CD19+CD24highCD27+Breg相比CD20表达水平差异无统计学意义(96.89%±7.90%vs.97.12%±5.44%,P=0.912),CD19+CD24highCD38high Breg细胞CD20表达水平高于非CD19+CD24highCD38high细胞(88.67%±9.22%,P=0.004),CD19+CD24highCD27+Breg细胞CD20表达水平与非CD19+CD24highCD27+细胞(96.76%±3.38%)相比差异无统计学意义(P=0.798)。经CD40L刺激后分泌IL-10、TGF-β的CD19+CD24highCD38high Breg细胞比例(7.81%±2.67%,7.05%±2.66%)与CD19+CD24highCD27+Breg(7.17%±2.47%,8.14%±2.44%)相比差异均无统计学意义(P=1.000,0.404)。经Cp G ODN2006刺激后分泌IL-10、TGF-β的CD19+CD24highCD38high Breg细胞比例(10.13%±12.19%,7.38%±2.98%)与CD19+CD24highCD27+Breg(9.88%±6.48%,9.03%±3.75%)相比差异均无统计学意义(P=0.421,0.763)。CD19+CD24highCD38high Breg与CD19+CD24highCD27+Breg相比CD80表达水平差异无统计学意义(90.01%±8.78%vs.83.62%±16.51%,P=0.113),CD19+CD24highCD38high Breg细胞CD80表达水平高于非CD19+CD24highCD38high细胞(66.78%±17.32%,P=0.000),CD19+CD24highCD27+Breg细胞CD80表达水平与非CD19+CD24highCD27+细胞相比差异无统计学意义(84.42%±16.89%,P=0.690)。CD19+CD24highCD38high Breg与CD19+CD24highCD27+Breg相比CD86表达水平差异无统计学意义(89.31%±11.00%vs.89.49%±10.87%,P=0.515),CD19+CD24highCD38high Breg细胞CD86表达水平高于非CD19+CD24highCD38high细胞(85.37%±13.64%,P=0.000),CD19+CD24highCD27+Breg细胞CD86表达水平与非CD19+CD24highCD27+细胞相比差异无统计学意义(89.63%±10.74%,P=0.546)。结论:在正常人外周血单个核细胞中CD19+CD24highCD27+Breg的比例高于CD19+CD24highCD38high Breg。CD19+CD24highCD27+Breg凋亡水平高于非CD19+CD24highCD27+细胞。CD19+CD24highCD38high Breg增殖水平高于非CD19+CD24highCD38high细胞。CD19+CD24highCD38high Breg与CD19+CD24highCD27+Breg细胞BAFF-R、TACI、BCMA的表达及结合的BAFF、APRIL均无差异。CD19+CD24highCD38high Breg细胞BAFF-R、TACI的表达高于非CD19+CD24highCD38high细胞。CD19+CD24highCD38high Breg结合APRIL的水平高于非CD19+CD24highCD38high细胞。CD19+CD24highCD38high Breg细胞Tim-3表达水平高于CD19+CD24highCD27+Breg,并且二者均高于非Breg细胞。CD19+CD24highCD38high Breg与CD19CD24CD27Breg分泌IL-10、TGF-β的水平无差异。CD19+CD24highCD38high Breg与CD19+CD24highCD27+Breg均表达CD20。CD19+CD24highCD38high Breg细胞CD80、CD86表达水平高于非CD19+CD24highCD38high细胞。二、c GVHD患者CD19+CD24highCD38high及CD19+CD24highCD27+调节性B细胞数量变化及机制研究目的:探讨异基因造血干细胞移植后c GVHD患者CD19+CD24highCD38high、CD19+CD24highCD27+调节性B细胞数量变化及相关机制。方法:流式细胞仪检测轻-中度cGVHD患者、重度cGVHD患者、无cGVHD患者、免疫耐受者及健康志愿者外周血单个核细胞中CD19+CD24highCD38high、CD19+CD24highCD27+Breg细胞的比例及其凋亡、增殖、BAFF-R、TACI、BCMA、结合的BAFF及APRIL水平。酶联免疫吸附试验(ELISA)测定各组患者血浆中BAFF、APRIL水平。依据血常规淋巴细胞计数及流式细胞仪检测CD19+CD24highCD38high及CD19+CD24highCD27+Breg细胞的比例计数其绝对数。进行各组间比较。结果:轻-中度c GVHD组B细胞绝对数[(3.83±2.20)×107/L]、CD19+CD24highCD38high Breg细胞绝对数[(13.31±13.53)×102/ml]、CD19+CD24highCD27+Breg细胞绝对数[(27.68±32.88)×102/ml]、CD19+CD24highCD38high Breg细胞比例(3.45%±1.75%)、CD19+CD24highCD27+Breg细胞比例(2.43%±1.42%)均低于无c GVHD组的(10.85±10.40)×107/L、(87.36±106.40)×102/ml、(57.59±61.85)×102/ml、6.26%±3.23%、5.06%±3.48%(P<0.05)。轻-中度c GVHD组CD19+CD24highCD38high Breg细胞凋亡(48.40%±19.15%)、血浆BAFF水平[(2877.90±248.76)ng/L]、CD19+CD24highCD38high Breg细胞结合的BAFF水平(67.84%±19.12%)均高于无c GVHD组的22.23%±12.05%、(2495.60±386.78)ng/L、60.76%±18.90%(P<0.05)。重度c GVHD组CD19+CD24highCD38high Breg细胞凋亡(72.34%±19.20%)、血浆BAFF水平[(3171.60±328.50)ng/L]、血浆BAFF/CD19+CD24highCD38high Breg细胞数的比值(15756.00±25144.61)、血浆BAFF/CD19+CD24highCD27+Breg细胞数的比值(8103.10±12623.27)、血浆APRIL/CD19+CD24highCD38high Breg细胞数的比值(16.35±32.03)、血浆APRIL/CD19+CD24highCD27+Breg细胞数的比值(8.53±12.05)、CD19+CD24highCD38high Breg细胞TACI表达水平(93.36%±9.76%)、CD19+CD24highCD38high Breg细胞结合的BAFF水平(79.12%±19.62%)、CD19+CD24highCD27+Breg细胞结合的BAFF水平(79.27%±18.57%)、CD19+CD24highCD38high Breg细胞上结合的APRIL水平(87.64%±23.69%)均高于无c GVHD组(P<0.05)。重度c GVHD组B细胞绝对数[(2.58±3.75)×107/L]、CD19+CD24highCD38high Breg细胞绝对数[(2.97±5.89)×102/ml]、CD19+CD24highCD27+Breg细胞绝对数[(7.24±11.27)×102/ml]、CD19+CD24highCD38high Breg细胞比例(0.70%±0.40%)、CD19+CD24highCD27+Breg细胞比例(2.22%±0.87%)、CD19+CD24highCD38high Breg细胞增殖水平(2.02%±1.68%)、CD19+CD24highCD27+Breg细胞增殖(4.09%±2.44%)均低于无c GVHD组(P<0.05)。重度c GVHD组CD19+CD24highCD38high Breg细胞比例、CD19+CD24highCD38high Breg细胞增殖水平均低于轻-中度c GVHD组(P<0.05)。重度c GVHD组CD19+CD24highCD38high Breg细胞凋亡、血浆BAFF水平、血浆BAFF/CD19+CD24highCD38high Breg细胞数的比值、血浆BAFF/CD19+CD24highCD27+Breg细胞数的比值、血浆APRIL/CD19+CD24highCD38high Breg细胞数的比值、血浆APRIL/CD19+CD24highCD27+Breg细胞数的比值、CD19+CD24highCD38high Breg细胞上结合的BAFF水平、CD19+CD24highCD27+Breg细胞结合的BAFF水平均高于轻-中度c GVHD组(P<0.05)。免疫耐受组B细胞绝对数[(13.30±15.84)×107/L]、CD19+CD24highCD38high Breg细胞绝对数[(168.05±220.05)×102/ml]、CD19+CD24highCD38high Breg细胞比例(11.40%±7.94%)、CD19+CD24highCD27+Breg细胞凋亡(47.91%±20.39%)、CD19+CD24highCD38high Breg细胞增殖(33.75%±13.47%)、血浆BAFF水平[(3152.80±752.48)ng/L]、血浆APRIL水平[(87.68±19.32)ng/L]、CD19+CD24highCD38high Breg细胞上结合的BAFF水平(68.16%±17.46%)、CD19+CD24highCD38high Breg细胞上结合的APRIL水平(81.89%±21.98%)均高于正常对照组的(4.68±2.96)×107/L、(27.48±24.99)×102/ml、5.88%±3.18%、28.84%±18.33%、21.46%±11.88%、(2218.80±229.32)ng/L、(69.83±10.27)ng/L、55.24%±17.46%、61.95%±31.66%(P<0.05)。免疫耐受组CD19+CD24highCD27+Breg细胞绝对数[(42.11±33.25)×102/ml]、CD19+CD24highCD27+Breg细胞比例(4.14%±2.21%)、CD19+CD24highCD38high Breg细胞凋亡(15.18%±14.48%)、CD19+CD24highCD27+Breg细胞增殖(7.01%±5.62%)均低于正常对照组的(90.48±57.31)×102/ml、14.10%±9.47%、28.87%±14.82%、17.26%±13.18%(P<0.05)。无c GVHD组B细胞绝对数、CD19+CD24highCD27+Breg细胞凋亡(54.49%±21.29%)、血浆APRIL水平、CD19+CD24highCD38high Breg细胞上结合的BAFF水平、CD19+CD24highCD38high Breg细胞上结合的APRIL水平、CD19+CD24highCD27+Breg细胞上结合的APRIL水平均高于正常对照组(P<0.05)。无c GVHD组CD19+CD24highCD27+Breg细胞绝对数、CD19+CD24highCD27+Breg细胞比例、CD19+CD24highCD38high Breg细胞凋亡低于正常对照组(P<0.05)。免疫耐受组CD19+CD24highCD38high Breg细胞绝对数、CD19+CD24highCD38high Breg细胞比例、血浆BAFF水平均高于无c GVHD组(P<0.05)。免疫耐受组CD19+CD24highCD27+Breg细胞TACI表达水平(58.50%±28.08%)低于无c GVHD组的78.18%±28.75%(P<0.05)。各组间CD19+CD24highCD38high Breg、CD19+CD24highCD27+Breg细胞BAFF-R、BCMA表达水平无差异(P<0.05)。结论:异基因造血干细胞移植后c GVHD患者同无c GVHD者相比CD19+CD24highCD38high Breg、CD19+CD24highCD27+Breg细胞数量减低,且在重度c GVHD患者中CD19+CD24highCD38high Breg数量低于轻-中度c GVHD。在免疫耐受者中CD19+CD24highCD38high Breg数量较正常对照升高,但CD19+CD24highCD27+Breg细胞数量较正常对照减低。Breg细胞数量的变化与BAFF、APRIL及其受体变化所致凋亡和增殖水平的变化有关。三、c GVHD患者CD19+CD24highCD38high及CD19+CD24highCD27+调节性B细胞功能变化及机制研究目的:探讨异基因造血干细胞移植后c GVHD患者CD19+CD24highCD38high、CD19+CD24highCD27+调节性B细胞功能变化及相关机制。方法:流式细胞仪检测轻-中度c GVHD患者、重度c GVHD患者、无c GVHD患者、免疫耐受者及健康志愿者外周血单个核细胞中CD19+CD24highCD38high及CD19+CD24highCD27+Breg细胞分别经CD40L及Cp G ODN2006刺激后分泌IL-10、TGF-β水平,同时检测STAT3磷酸化(p STAT3)、My D88、CD40、TLR9、CD80、CD86及Tim-3水平,并进行各组间比较。结果:经CD40L刺激后轻-中度cGVHD、重度c GVHD患者CD19CD24CD38Breg细胞分泌IL-10的水平(2.32%±0.42%、0.56%±0.54%)均低于无c GVHD者(5.88%±3.11%)(P<0.05),TGF-β水平各组间无差异;重度c GVHD患者CD19+CD24highCD27+Breg细胞分泌IL-10的水平(2.02%±1.90%)低于无c GVHD者(4.94%±3.81%)(P<0.05),TGF-β水平各组间无差异。经Cp G ODN2006刺激后CD19+CD24highCD38high Breg细胞分泌IL-10、TGF-β的水平均无差异;轻-中度c GVHD、重度c GVHD患者CD19+CD24highCD27+Breg细胞分泌IL-10的水平(4.02%±1.91%、1.69%±1.25%)均低于无c GVHD者(8.80%±5.98%)(P<0.05),TGF-β水平各组间无差异。轻-中度c GVHD、重度c GVHD患者CD19+CD24highCD38high Breg细胞p STAT3水平(6.48%±10.20%、3.06%±4.80%)均低于无c GVHD者(14.79%±7.05%)(P<0.05);My D88水平各组间无差异。重度c GVHD患者CD19+CD24highCD27+Breg细胞p STAT3、My D88水平(3.31%±2.09%,41.31%±29.52%)均低于无c GVHD者(27.98±12.65,69.41%±24.09%)(P<0.05)。重度c GVHD患者CD19+CD24highCD38high、CD19+CD24highCD27+Breg细胞Tim-3水平(2.07%±0.58%、2.59%±2.44%)均低于无c GVHD者(11.35%±9.72%,16.34%±22.52%)(P<0.05)。免疫耐受者CD19+CD24highCD27+Breg细胞Tim-3水平(23.49%±23.19%)高于正常对照(6.91%±5.88%)(P<0.05)。CD19+CD24highCD38high、CD19+CD24highCD27+Breg细胞CD40、TLR9、CD80、CD86水平各组间均无差异。结论:异基因造血干细胞移植后cGVHD患者与无cGVHD者相比CD19+CD24highCD38high、CD19+CD24highCD27+Breg细胞分泌IL-10的功能减弱,分泌TGF-β的功能无异常。CD19+CD24highCD38high Breg功能的减弱与CD40介导的STAT3信号转导通路上p STAT3水平减低有关。CD19+CD24highCD27+Breg分泌IL-10的功能的减弱与CD40介导的STAT3信号转导通路上p STAT3水平减低及TLR9介导的My D88信号转导通路上My D88水平减低均有关。c GVHD患者与无c GVHD者相比CD19+CD24highCD38high、CD19+CD24highCD27+Breg细胞Tim-3表达减低,而在免疫耐受者CD19+CD24highCD27+Breg细胞Tim-3表达较正常对照升高。四、对c GVHD具有生物标记意义的B细胞相关基因筛选目的:筛选对cGVHD具有生物标记意义的B细胞相关基因。方法:利用基因芯片方法分别对轻-中度cGVHD患者、重度cGVHD患者、无c GVHD患者、免疫耐受者及健康志愿者外周血单个核细胞的基因表达谱进行检测,进行组间比较,筛选出与B细胞相关的差异表达基因。应用荧光定量PCR对筛选出的差异表达基因进行验证。结果:基因芯片结果显示cGVHD患者与无c GVHD患者相比共有284条表达差异基因,其中与B细胞相关的有5条,分别为CDKN2A、SOX4、ZBTB32、CD70、IL21R。免疫耐受患者与无c GVHD患者相比共有800条表达差异基因,其中与B细胞相关的有3条,分别为NFAM1、CD27、BST1。应用荧光定量PCR对基因芯片筛选出的表达差异基因进行验证,结果显示c GVHD患者与无c GVHD患者相比CDKN2A基因的表达水平降低(P=0.001)。免疫耐受患者与无c GVHD患者相比CD27基因的表达水平升高(P=0.038),而BST1基因的表达水平减低(P=0.022)结论:c GVHD患者与无c GVHD患者相比CDKN2A基因的表达水平降低。免疫耐受患者与无c GVHD患者相比CD27基因的表达水平升高,而BST1基因的表达水平减低。这些基因的变化有望成为具有潜在生物标记意义的指标用于指导临床工作。