異基因造血干細(xì)胞移植后cGVHD患者調(diào)節(jié)性B細(xì)胞數(shù)量和功能變化及機(jī)制研究.pdf

1、一、人CD19+CD24highCD38high及CD19+CD24highCD27+調(diào)節(jié)性B細(xì)胞特性的研究
　　目的：探討人外周血CD19+CD24highCD38high及CD19+CD24highCD27+調(diào)節(jié)性B細(xì)胞的特性。
　　方法：流式細(xì)胞儀檢測健康志愿者外周血單個核細(xì)胞中 CD19+CD24highCD38high及CD19+CD24highCD27+調(diào)節(jié)性B細(xì)胞（regulatory B cell, Breg

2、）的比例及其凋亡、增殖、B細(xì)胞活化因子受體（B-cell activating factor receptor, BAFF-R）、跨膜蛋白活化因子鈣離子信號調(diào)節(jié)親環(huán)素配體(transmembrane activator calcium modulatorand cyclophilin ligand interactor, TACI)、B細(xì)胞成熟抗原(B cell maturation Ag, BCMA)、結(jié)合的B細(xì)胞活化因子(B-cel

3、l activating factor belonging to the TNF family, BAFF)及一種增殖誘導(dǎo)配體（a proliferation-inducing ligand, APRIL）水平、T細(xì)胞免疫球蛋白黏蛋白結(jié)構(gòu)域相關(guān)分子-3（T cell Ig domain and mucin domain3, Tim-3）表達(dá)、CD20表達(dá)、CD80表達(dá)及 CD86表達(dá)。分別經(jīng) CD40L及 CpG ODN2006刺激后檢

4、測CD19+CD24highCD38high、CD19+CD24highCD27+Breg細(xì)胞分泌的IL-10、TGF-β水平。將CD19+CD24highCD38high與CD19+CD24highCD27+Breg細(xì)胞、CD19+CD24highCD38high與非Breg、CD19+CD24highCD27+與非Breg之間進(jìn)行上述指標(biāo)的比較。
　　結(jié)果：在正常人外周血單個核細(xì)胞中CD19+CD24highCD38high

5、Breg比例為5.88％±3.18%，CD19+CD24highCD27+Breg的比例為14.10%±9.47%，CD19+CD24highCD27+Breg的比例高于CD19+CD24highCD38high Breg（P=0.000）。CD19+CD24highCD38high Breg與CD19+CD24highCD27+Breg相比凋亡水平無差異(28.87%±14.82% vs.28.84%±18.33%, P=0.994)

6、，CD19+CD24highCD38high Breg與非CD19+CD24highCD38high細(xì)胞相比凋亡水平亦無差異(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+細(xì)胞(28.84%±18.33% vs.14.93%±7.83%, P=0.002)。 CD19+CD24highCD38high

7、Breg與CD19+CD24highCD27+ Breg相比增殖水平差異無統(tǒng)計學(xué)意義(21.46%±11.88% vs.17.28%±13.18%, P=0.442)，CD19+CD24highCD38high Breg增殖水平高于非CD19+CD24highCD38high細(xì)胞(21.46%±11.88% vs.6.22%±10.07%, P=0.009)，CD19+CD24highCD27+Breg增殖水平與非CD19+CD24hi

8、ghCD27+細(xì)胞相比差異無統(tǒng)計學(xué)意義(17.28%±13.18%vs.12.96%±11.93%, P=0.570)。CD19+CD24highCD38high Breg細(xì)胞與CD19+CD24highCD27+Breg細(xì)胞均表達(dá)BAFF-R、TACI、BCMA，并且兩群細(xì)胞間BAFF-R、TACI、BCMA的表達(dá)差異無統(tǒng)計學(xué)意義(BAFF-R：97.17%±6.17%vs.96.46%±8.64%, P=0.743; TACI:65

9、.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細(xì)胞BAFF-R、TACI的表達(dá)高于非CD19+CD24highCD38high細(xì)胞(97.17%±6.17%vs.89.56%±7.43%、65.84%±28.60%vs.39.07%±23.92%, P=0.000、0.

10、002),而BCMA的表達(dá)(94.13%±3.15%)無差異(P=0.495)。CD19+CD24highCD27+ Breg細(xì)胞BAFF-R、TACI、BCMA的表達(dá)與非 CD19+CD24highCD27+細(xì)胞(98.71%±1.10%、56.14%±28.75%、94.50%±4.08%)相比差異均無統(tǒng)計學(xué)意義(P=0.142,0.130,0.481)。CD19+CD24highCD38high Breg細(xì)胞與 CD19+CD24

11、highCD27+ Breg細(xì)胞結(jié)合BAFF、APRIL的水平差異均無統(tǒng)計學(xué)意義(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細(xì)胞結(jié)合BAFF的水平與非CD19+CD24highCD38high細(xì)胞相比差異無統(tǒng)計學(xué)意義(55.24%±17.46% vs.58.95%±1

12、2.59%，P=0.424),而結(jié)合 APRIL的水平高于非CD19+CD24highCD38high細(xì)胞(55.24%±17.46% vs.35.29%±22.08%, P=0.002)。CD19+CD24highCD27+Breg細(xì)胞與非CD19+CD24highCD27+細(xì)胞相比結(jié)合BAFF、APRIL的水平(59.05%±15.89%，57.76%±33.04%)差異均無統(tǒng)計學(xué)意義(P=0.404,0.546)。CD19+CD2

13、4highCD38high Breg細(xì)胞 Tim-3表達(dá)水平高于 CD19+CD24highCD27+ Breg(14.27%±6.97%vs.6.91%±5.88%, P=0.014)，CD19+CD24highCD38high Breg細(xì)胞Tim-3表達(dá)水平高于非 CD19+CD24highCD38high細(xì)胞(14.27%±6.97% vs.1.44%±1.71%, P=0.000)，CD19+CD24highCD27+Breg細(xì)

14、胞Tim-3表達(dá)水平高于非CD19+CD24highCD27+細(xì)胞(6.91%±5.88% vs.1.99%±2.01%, P=0.002)。CD19+CD24highCD38high Breg與CD19+CD24highCD27+Breg相比CD20表達(dá)水平差異無統(tǒng)計學(xué)意義(96.89%±7.90%vs.97.12%±5.44%, P=0.912)，CD19+CD24highCD38high Breg細(xì)胞CD20表達(dá)水平高于非 CD1

15、9+CD24highCD38high細(xì)胞(88.67%±9.22%, P=0.004)，CD19+CD24highCD27+Breg細(xì)胞CD20表達(dá)水平與非CD19+CD24highCD27+細(xì)胞(96.76%±3.38%)相比差異無統(tǒng)計學(xué)意義(P=0.798)。經(jīng) CD40L刺激后分泌 IL-10、 TGF-β的CD19+CD24highCD38high Breg細(xì)胞比例(7.81%±2.67%,7.05%±2.66%)與CD19+C

16、D24highCD27+Breg(7.17%±2.47%,8.14%±2.44%)相比差異均無統(tǒng)計學(xué)意義(P=1.000,0.404)。經(jīng) CpG ODN2006刺激后分泌 IL-10、 TGF-β的CD19+CD24highCD38high Breg細(xì)胞比例(10.13%±12.19%,7.38%±2.98%)與CD19+CD24highCD27+Breg(9.88%±6.48%,9.03%±3.75%)相比差異均無統(tǒng)計學(xué)意義(P=0

17、.421,0.763)。CD19+CD24highCD38high Breg與CD19+CD24highCD27+Breg相比CD80表達(dá)水平差異無統(tǒng)計學(xué)意義(90.01%±8.78% vs.83.62%±16.51%, P=0.113)，CD19+CD24highCD38high Breg細(xì)胞CD80表達(dá)水平高于非CD19+CD24highCD38high細(xì)胞(66.78%±17.32%, P=0.000)，CD19+CD24high

18、CD27+Breg細(xì)胞 CD80表達(dá)水平與非CD19+CD24highCD27+細(xì)胞相比差異無統(tǒng)計學(xué)意義(84.42%±16.89%, P=0.690)。CD19+CD24highCD38high Breg與CD19+CD24highCD27+Breg相比CD86表達(dá)水平差異無統(tǒng)計學(xué)意義(89.31%±11.00%vs.89.49%±10.87%, P=0.515)，CD19+CD24highCD38high Breg細(xì)胞 CD86表達(dá)

19、水平高于非 CD19+CD24highCD38high細(xì)胞(85.37%±13.64%, P=0.000)，CD19+CD24highCD27+Breg細(xì)胞CD86表達(dá)水平與非CD19+CD24highCD27+細(xì)胞相比差異無統(tǒng)計學(xué)意義(89.63%±10.74%, P=0.546)。
　　結(jié)論：在正常人外周血單個核細(xì)胞中 CD19+CD24highCD27+ Breg的比例高于CD19+CD24highCD38high Breg

20、。 CD19+CD24highCD27+ Breg凋亡水平高于非CD19+CD24highCD27+細(xì)胞。 CD19+CD24highCD38high Breg增殖水平高于非CD19+CD24highCD38high細(xì)胞。CD19+CD24highCD38high Breg與 CD19+CD24highCD27+Breg細(xì)胞 BAFF-R、TACI、BCMA的表達(dá)及結(jié)合的BAFF、APRIL均無差異。CD19+CD24highCD38h

21、igh Breg細(xì)胞 BAFF-R、 TACI的表達(dá)高于非CD19+CD24highCD38high細(xì)胞。CD19+CD24highCD38high Breg結(jié)合APRIL的水平高于非CD19+CD24highCD38high細(xì)胞。CD19+CD24highCD38high Breg細(xì)胞Tim-3表達(dá)水平高于CD19+CD24highCD27+Breg，并且二者均高于非Breg細(xì)胞。CD19+CD24highCD38high Breg與

22、 CD19+CD24highCD27+ Breg分泌 IL-10、 TGF-β的水平無差異。CD19+CD24highCD38high Breg與 CD19+CD24highCD27+ Breg均表達(dá) CD20。CD19+CD24highCD38high Breg細(xì)胞 CD80、 CD86表達(dá)水平高于非CD19+CD24highCD38high細(xì)胞。
　　二、cGVHD患者 CD19+CD24highCD38high及 CD19+

23、CD24highCD27+調(diào)節(jié)性B細(xì)胞數(shù)量變化及機(jī)制研究
　　目的：探討異基因造血干細(xì)胞移植后 cGVHD患者 CD19+CD24highCD38high、CD19+CD24highCD27+調(diào)節(jié)性B細(xì)胞數(shù)量變化及相關(guān)機(jī)制。
　　方法：流式細(xì)胞儀檢測輕-中度cGVHD患者、重度cGVHD患者、無cGVHD患者、免疫耐受者及健康志愿者外周血單個核細(xì)胞中 CD19+CD24highCD38high、CD19+CD24highCD

24、27+Breg細(xì)胞的比例及其凋亡、增殖、BAFF-R、TACI、BCMA、結(jié)合的BAFF及APRIL水平。酶聯(lián)免疫吸附試驗(ELISA)測定各組患者血漿中BAFF、APRIL水平。依據(jù)血常規(guī)淋巴細(xì)胞計數(shù)及流式細(xì)胞儀檢測CD19+CD24highCD38high及CD19+CD24highCD27+Breg細(xì)胞的比例計數(shù)其絕對數(shù)。進(jìn)行各組間比較。
　　結(jié)果：輕-中度 cGVHD組 B細(xì)胞絕對數(shù)[(3.83±2.20)×107/L]、

25、CD19+CD24highCD38high Breg細(xì)胞絕對數(shù)[(13.31±13.53)×102/ml]、CD19+CD24highCD27+ Breg細(xì)胞絕對數(shù)[(27.68±32.88)×102/ml]、CD19+CD24highCD38high Breg細(xì)胞比例(3.45%±1.75%)、CD19+CD24highCD27+Breg細(xì)胞比例(2.43%±1.42%)均低于無 cGVHD組的(10.85±10.40)×107/L、

26、(87.36±106.40)×102/ml、(57.59±61.85)×102/ml、6.26%±3.23%、5.06%±3.48%(P<0.05)。輕-中度cGVHD組CD19+CD24highCD38high Breg細(xì)胞凋亡(48.40%±19.15%)、血漿BAFF水平[(2877.90±248.76) ng/L]、CD19+CD24highCD38high Breg細(xì)胞結(jié)合的BAFF水平(67.84%±19.12%)均高于無c

27、GVHD組的22.23%±12.05%、(2495.60±386.78) ng/L、60.76%±18.90%(P<0.05)。重度cGVHD組CD19+CD24highCD38high Breg細(xì)胞凋亡(72.34%±19.20%)、血漿 BAFF水平[(3171.60±328.50) ng/L]、血漿BAFF/CD19+CD24highCD38high Breg細(xì)胞數(shù)的比值(15756.00±25144.61)、血漿BAFF/CD1

28、9+CD24highCD27+ Breg細(xì)胞數(shù)的比值(8103.10±12623.27)、血漿APRIL/CD19+CD24highCD38high Breg細(xì)胞數(shù)的比值(16.35±32.03)、血漿APRIL/CD19+CD24highCD27+ Breg細(xì)胞數(shù)的比值(8.53±12.05)、CD19+CD24highCD38high Breg細(xì)胞 TACI表達(dá)水平(93.36%±9.76%)、CD19+CD24highCD38hi

29、gh Breg細(xì)胞結(jié)合的BAFF水平(79.12%±19.62%)、CD19+CD24highCD27+ Breg細(xì)胞結(jié)合的BAFF水平(79.27%±18.57%)、CD19+CD24highCD38high Breg細(xì)胞上結(jié)合的APRIL水平(87.64%±23.69%)均高于無cGVHD組(P<0.05)。重度 cGVHD組 B細(xì)胞絕對數(shù)[(2.58±3.75)×107/L]、CD19+CD24highCD38high Breg細(xì)

30、胞絕對數(shù)[(2.97±5.89)×102/ml]、CD19+CD24highCD27+Breg細(xì)胞絕對數(shù)[(7.24±11.27)×102/ml]、CD19+CD24highCD38high Breg細(xì)胞比例(0.70%±0.40%)、 CD19+CD24highCD27+ Breg細(xì)胞比例(2.22%±0.87%)、CD19+CD24highCD38high Breg細(xì)胞增殖水平(2.02%±1.68%)、CD19+CD24highC

31、D27+Breg細(xì)胞增殖(4.09%±2.44%)均低于無 cGVHD組(P<0.05)。重度 cGVHD組CD19+CD24highCD38high Breg細(xì)胞比例、CD19+CD24highCD38high Breg細(xì)胞增殖水平均低于輕-中度cGVHD組(P<0.05)。重度cGVHD組CD19+CD24highCD38high Breg細(xì)胞凋亡、血漿BAFF水平、血漿BAFF/CD19+CD24highCD38high Breg

32、細(xì)胞數(shù)的比值、血漿 BAFF/CD19+CD24highCD27+ Breg細(xì)胞數(shù)的比值、血漿APRIL/CD19+CD24highCD38high Breg細(xì)胞數(shù)的比值、血漿APRIL/CD19+CD24highCD27+Breg細(xì)胞數(shù)的比值、CD19+CD24highCD38high Breg細(xì)胞上結(jié)合的BAFF水平、CD19+CD24highCD27+ Breg細(xì)胞結(jié)合的BAFF水平均高于輕-中度 cGVHD組(P<0.05)。免

33、疫耐受組 B細(xì)胞絕對數(shù)[(13.30±15.84)×107/L]、CD19+CD24highCD38high Breg細(xì)胞絕對數(shù)[(168.05±220.05)×102/ml]、CD19+CD24highCD38high Breg細(xì)胞比例(11.40%±7.94%)、CD19+CD24highCD27+ Breg細(xì)胞凋亡(47.91%±20.39%)、CD19+CD24highCD38high Breg細(xì)胞增殖(33.75%±13.47

34、%)、血漿BAFF水平[(3152.80±752.48) ng/L]、血漿APRIL水平[(87.68±19.32) ng/L]、CD19+CD24highCD38high Breg細(xì)胞上結(jié)合的BAFF水平(68.16%±17.46%)、CD19+CD24highCD38high Breg細(xì)胞上結(jié)合的APRIL水平(81.89%±21.98%)均高于正常對照組的(4.68±2.96)×107/L、(27.48±24.99)×102/ml

35、、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)。免疫耐受組 CD19+CD24highCD27+ Breg細(xì)胞絕對數(shù)[(42.11±33.25)×102/ml]、CD19+CD24highCD27+Breg細(xì)胞比例(4.14%±2.21%)、CD19+

36、CD24highCD38high Breg細(xì)胞凋亡(15.18%±14.48%)、CD19+CD24highCD27+Breg細(xì)胞增殖(7.01%±5.62%)均低于正常對照組的(90.48±57.31)×102/ml、14.10%±9.47%、28.87%±14.82%、17.26%±13.18%(P<0.05)。無cGVHD組B細(xì)胞絕對數(shù)、CD19+CD24highCD27+Breg細(xì)胞凋亡(54.49%±21.29%)、血漿APR

37、IL水平、CD19+CD24highCD38high Breg細(xì)胞上結(jié)合的BAFF水平、CD19+CD24highCD38high Breg細(xì)胞上結(jié)合的APRIL水平、CD19+CD24highCD27+Breg細(xì)胞上結(jié)合的APRIL水平均高于正常對照組(P<0.05)。無cGVHD組CD19+CD24highCD27+Breg細(xì)胞絕對數(shù)、CD19+CD24highCD27+Breg細(xì)胞比例、CD19+CD24highCD38high

38、Breg細(xì)胞凋亡低于正常對照組(P<0.05)。免疫耐受組CD19+CD24highCD38high Breg細(xì)胞絕對數(shù)、CD19+CD24highCD38high Breg細(xì)胞比例、血漿BAFF水平均高于無cGVHD組(P<0.05)。免疫耐受組CD19+CD24highCD27+ Breg細(xì)胞 TACI表達(dá)水平(58.50%±28.08%)低于無 cGVHD組的78.18%±28.75%(P<0.05)。各組間 CD19+CD24h

39、ighCD38high Breg、CD19+CD24highCD27+ Breg細(xì)胞BAFF-R、BCMA表達(dá)水平無差異(P<0.05)。
　　結(jié)論：異基因造血干細(xì)胞移植后 cGVHD患者同無 cGVHD者相比CD19+CD24highCD38high Breg、CD19+CD24highCD27+ Breg細(xì)胞數(shù)量減低，且在重度cGVHD患者中CD19+CD24highCD38high Breg數(shù)量低于輕-中度cGVHD。在免疫

40、耐受者中CD19+CD24highCD38high Breg數(shù)量較正常對照升高，但CD19+CD24highCD27+ Breg細(xì)胞數(shù)量較正常對照減低。Breg細(xì)胞數(shù)量的變化與BAFF、APRIL及其受體變化所致凋亡和增殖水平的變化有關(guān)。
　　三、cGVHD患者 CD19+CD24highCD38high及 CD19+CD24highCD27+調(diào)節(jié)性B細(xì)胞功能變化及機(jī)制研究
　　目的：探討異基因造血干細(xì)胞移植后 cGVHD患

41、者 CD19+CD24highCD38high、CD19+CD24highCD27+調(diào)節(jié)性B細(xì)胞功能變化及相關(guān)機(jī)制。
　　方法：流式細(xì)胞儀檢測輕-中度cGVHD患者、重度cGVHD患者、無cGVHD患者、免疫耐受者及健康志愿者外周血單個核細(xì)胞中 CD19+CD24highCD38high及CD19+CD24highCD27+Breg細(xì)胞分別經(jīng)CD40L及CpG ODN2006刺激后分泌IL-10、TGF-β水平，同時檢測STAT3

42、磷酸化(pSTAT3)、MyD88、CD40、TLR9、CD80、CD86及Tim-3水平，并進(jìn)行各組間比較。
　　結(jié)果：經(jīng)CD40L刺激后輕-中度cGVHD、重度cGVHD患者CD19+CD24highCD38high Breg細(xì)胞分泌IL-10的水平(2.32%±0.42%、0.56%±0.54%)均低于無cGVHD者(5.88%±3.11%)(P＜0.05)，TGF-β水平各組間無差異；重度 cGVHD患者CD19+CD24

43、highCD27+Breg細(xì)胞分泌IL-10的水平(2.02%±1.90%)低于無cGVHD者(4.94%±3.81%)(P＜0.05)，TGF-β水平各組間無差異。經(jīng) CpG ODN2006刺激后CD19+CD24highCD38high Breg細(xì)胞分泌 IL-10、TGF-β的水平均無差異；輕-中度cGVHD、重度cGVHD患者CD19+CD24highCD27+Breg細(xì)胞分泌IL-10的水平(4.02%±1.91%、1.69%

44、±1.25%)均低于無cGVHD者(8.80%±5.98%)(P＜0.05)，TGF-β水平各組間無差異。輕-中度cGVHD、重度cGVHD患者CD19+CD24highCD38high Breg細(xì)胞pSTAT3水平(6.48%±10.20%、3.06%±4.80%)均低于無cGVHD者(14.79%±7.05%)(P＜0.05)； MyD88水平各組間無差異。重度 cGVHD患者CD19+CD24highCD27+Breg細(xì)胞 pST

45、AT3、MyD88水平(3.31%±2.09%，41.31%±29.52%)均低于無 cGVHD者(27.98±12.65,69.41%±24.09%)(P＜0.05)。重度cGVHD患者 CD19+CD24highCD38high、CD19+CD24highCD27+Breg細(xì)胞 Tim-3水平(2.07%±0.58%、2.59%±2.44%)均低于無cGVHD者(11.35%±9.72%，16.34%±22.52%)(P＜0.05)

46、。免疫耐受者CD19+CD24highCD27+Breg細(xì)胞Tim-3水平(23.49%±23.19%)高于正常對照(6.91%±5.88%)(P＜0.05)。CD19+CD24highCD38high、CD19+CD24highCD27+Breg細(xì)胞CD40、TLR9、CD80、CD86水平各組間均無差異。
　　結(jié)論：異基因造血干細(xì)胞移植后 cGVHD患者與無 cGVHD者相比CD19+CD24highCD38high、CD19

47、+CD24highCD27+Breg細(xì)胞分泌IL-10的功能減弱，分泌 TGF-β的功能無異常。CD19+CD24highCD38high Breg功能的減弱與 CD40介導(dǎo)的STAT3信號轉(zhuǎn)導(dǎo)通路上pSTAT3水平減低有關(guān)。CD19+CD24highCD27+Breg分泌IL-10的功能的減弱與CD40介導(dǎo)的STAT3信號轉(zhuǎn)導(dǎo)通路上pSTAT3水平減低及TLR9介導(dǎo)的MyD88信號轉(zhuǎn)導(dǎo)通路上MyD88水平減低均有關(guān)。cGVHD患者與無

48、cGVHD者相比CD19+CD24highCD38high、CD19+CD24highCD27+Breg細(xì)胞Tim-3表達(dá)減低，而在免疫耐受者CD19+CD24highCD27+Breg細(xì)胞Tim-3表達(dá)較正常對照升高。
　　四、對cGVHD具有生物標(biāo)記意義的B細(xì)胞相關(guān)基因篩選
　　目的：篩選對cGVHD具有生物標(biāo)記意義的B細(xì)胞相關(guān)基因。
　　方法：利用基因芯片方法分別對輕-中度 cGVHD患者、重度 cGVHD患者、

49、無cGVHD患者、免疫耐受者及健康志愿者外周血單個核細(xì)胞的基因表達(dá)譜進(jìn)行檢測，進(jìn)行組間比較，篩選出與B細(xì)胞相關(guān)的差異表達(dá)基因。應(yīng)用熒光定量PCR對篩選出的差異表達(dá)基因進(jìn)行驗證。
　　結(jié)果：基因芯片結(jié)果顯示cGVHD患者與無cGVHD患者相比共有284條表達(dá)差異基因，其中與B細(xì)胞相關(guān)的有5條,分別為CDKN2A、SOX4、ZBTB32、CD70、IL21R。免疫耐受患者與無cGVHD患者相比共有800條表達(dá)差異基因，其中與B細(xì)胞相關(guān)

50、的有3條,分別為NFAM1、CD27、BST1。應(yīng)用熒光定量PCR對基因芯片篩選出的表達(dá)差異基因進(jìn)行驗證，結(jié)果顯示 cGVHD患者與無 cGVHD患者相比CDKN2A基因的表達(dá)水平降低(P=0.001)。免疫耐受患者與無cGVHD患者相比CD27基因的表達(dá)水平升高(P=0.038)，而BST1基因的表達(dá)水平減低(P=0.022)
　　結(jié)論：cGVHD患者與無cGVHD患者相比CDKN2A基因的表達(dá)水平降低。免疫耐受患者與無cGVH