摘要
目的:通过比较再生障碍性贫血(aplastic anemia,AA)患者和骨髓增生异常综合征(myelodysplastic syndrome,MDS)患者外周血CD3+、CD4+、CD8+T淋巴细胞含量和比值、T-be(tT box expressed in T cells,T-bet)基因的表达及血清中干扰素γ(IFN-γ)水平差异,探讨上述指标对二者发病机制、鉴别诊断及临床治疗的指导意义。
方法:1研究对象:14例AA患者及20例MDS患者及30名健康者作为正常对照(normal control NC)组。其中AA患者14例,男8例、女6例,年龄范围14~66岁,中位年龄26岁,其中慢性再生障碍性贫血(CAA)9例、重型再生障碍性贫血(SAA)5例。MDS患者20例,男9例、女11例,年龄范围27~75岁,中位年龄58岁,按FAB分型,其中难治性贫血(RA)8例、原始细胞过多难治性贫血(RAEB) 12例。正常对照30例,均取自常规体检正常的人群,男女各15例,年龄范围20-75岁,中位年龄55岁。
2研究方法:分离AA、MDS患者及正常对照组周血单个核细胞,采用免疫荧光标记和三色流式细胞术检测周血CD3+,CD4+,CD8+ T细胞占T细胞比例;提取周血单个核细胞RNA,采用实时荧光定量PCR技术检测T-bet基因的表达量,用ELISA法测定患者血清IFN-γ的表达。并对14例AA患者追踪随访6个月,进行治疗前后的对比(均予以环孢素A5mg/kg.d为主的治疗),收集患者基本临床资料进行分析。
结果:1 T淋巴细胞免疫表型CD3+,CD4+,CD8+,CD4+/CD8+表达1.1 AA组CD3十T细胞百分率高于正常对照组(75.338±5.620 VS67.441±12.817,P<0.05);CD4+T细胞低于正常对照组,但相比无统计学差异(31.852±6.425VS33.424±8.415, P>0.05);CD8+T细胞明显高与正常对照组(34.956±6.206 VS27.032±9.046P<0.05); CD4+/CD8+相比对照组显著下降(1.177±0.436 VS1.484±0.378 P<0.05)。1.2 MDS组CD3十T细胞百分率低于正常对照组(62.241±9.102 VS67.441±12.817, P<0.05);低危组CD3十T细胞百分率低于正常对照组,但没有统计学意义(65.803±13.859 VS67.441±12.817, P>0.05);高危组CD3十T细胞百分率低于正常对照组(52.417±12.585 VS67.441±12.817 P<0.05)。MDS组,高危组CD4十T细胞百分率均高于正常对照组(39.283±9.913 VS33.424±8.415 P<0.05, 42.536±10.711 VS33.424±8.415 P<0.05);低危组CD4+T细胞的百分率与正常对照组相比无统计学意义(33.322±9.603 VS33.424±8.415 P>0.05);MDS患者、低危组的CD8十T细胞百分率高于正常对照组,但无统计学意义( 31.213±12.342 VS27.032±9.046,32.101±13.004 VS27.032±9.046 P>0.05);高危组CD8十T细胞百分率低于正常对照组,也没有统计学意义(26.634±3.568 VS 27.032±9.046 P>0.05)。MDS组,高危组CD4+/CD8+高于正常对照组( 1.630±1.063 VS1.484±0.378 P<0.05 , 1.801±1.089 VS1.484±0.378 P<0.05 );低危组的CD4+/CD8+相比正常组没有明显差异(1.3313±0.424VS1.484±0.378 P>0.05)。
1.3 AA与MDS相比:AA组的CD3+T百分率(75.338±5.620)均高于MDS组( 62.241±9.102 )、低危组( 65.803±13.859 )及高危组(52.417±12.585),均有统计学意义(P<0.05)。AA组的CD4+T细胞百分率(31.852±6.425)均明显低于MDS组(39.283±9.913)及高危组(42.536±10.711),均有统计学意义(P<0.05)。AA组与低危组的CD4+T细胞百分率相比无明显差异(31.852±6.425 VS33.322±9.603 P>0.05)。AA组CD8+T细胞的百分率(34.956±6.206)均高于MDS组(31.213±2.342)、低危组(32.101±13.004)、高危组(26.634±3.568),但与低危组比较无明显差异。AA组的CD4+ /CD8+比值(1.177±0.436)均低于MDS组(1.630±1.063)、低危组(1.3313±0.424)、高危组(1.801±0.893),但与低危组比较无明显差异。
1.4 AA治疗前后的比较,治疗后的AA相比治疗前,CD3+T细胞百分率(68.187±17.647)下降,CD4+T细胞百分率(33.453±8.362)升高,CD8+T细胞百分率(29.370±8.223)下降,CD4+/CD8+比值(1.221±0.425)升高,接近正常。且CD3+T细胞, CD8+T细胞,CD4+ /CD8+比值的变化均有统计学意义(P<0.05)。
2 T-bet基因的表达量的比较
2.1 AA组和正常对照组相比T-bet基因的表达量是增高的,有统计学意义(0.255±0.188 VS0.092±0.058,P<0.05)。
2.2 MDS组正常对照组相比T-bet基因的表达量是减低的(0.085±0.041 VS0.092±0.058,P<0.05);高危组T-bet基因的表达量低于正常对照(0.061±0.018 VS0.092±0.058,P<0.05),低危组T-bet基因的表达量也低于正常对照组,但与正常对照组之间的差异没有统计学意义(0.120±0.039 VS0.092±0.058,P>0.05)。
2.3 AA组T-bet基因的表达量(0.255±0.188)均高于MDS组(0.085±0.041),低危组(0.120±0.039)及高危组(0.061±0.018),均有统计学意义(P<0.05)。
2.4 AA组治疗后T-bet基因的表达量相比治疗前明显下降有统计学意义(0.153±0.0 78 VS0.255±0.188 P<0.05)。
3血清IFN-γ的水平在各组中的比较
3.1 AA组血清IFN - r含量明显高于正常对照组,两组之间比较差异有统计学意义(9.756±2.537 VS 7.552±0.640 P<0.05)。
3.2 MDS组血清中IFN-r含量明显低于正常对照组,差异有统计学意义(5.365±2.338 VS 7.552±0.640 P<0.05)。低危组血清中IFN-r含量(7.845±1.094)明显高于高危组(3.709±1.102),但与正常对照组没有统计学上的差异(7.845±1.094 VS 7.552±0.640 p>0.05)。
3.3 AA组血清IFN - r含量(9.756±2.537)明显高于MDS组(5.365±2.338),低危组(7.845±1.094)及高危组(3.709±1.102),与各组间差异均有统计学意义(P<0.05)
3.4 AA组治疗后的血清IFN - r含量相比治疗前明显下降有统计学意义(7.6822±2.338 VS9.756±2.537 P<0.05)。4 AA组和MDS组的T-bet基因的表达量和血清IFN - r含量的相关系数r值分别是0.64,0.70,均有统计学意义(P<0.05)。
结论:1 T淋巴细胞的免疫表型CD3+、CD4+、CD8+及CD4+/CD8+ AA组较MDS组CD3+T细胞、CD8+T细胞百分率明显增高,CD4+T细胞百分率下降,CD4+/CD8+比值倒置,且AA组治疗前后相比,T淋巴亚群紊乱得到纠正。说明T淋巴细胞免疫功能异常均参与了二者的发病。提示AA患者存在明显的免疫功能亢进,可能与AA的发生、发展有密切的关系,也提示免疫异常是MDS疾病的发生、发展的一个促发因素。流式细胞术检测AA和MDS的T淋巴细胞免疫表型有助于我们明确机体的免疫状态,能更好的帮助我们鉴别两者,指导我们针对两者的免疫状态合理用药。
2 T-bet基因和IFN– r的表达
T-bet基因在AA中的表达量高于正常对照组,MDS中T-bet基因的表达量低于对照组,且AA9.756±2.537较MDS中表达量明显增高,差异有统计学意义。AA患者血清IFN - r含量明显高于对照组,MDS患者则低于正常对照组。且各组的T-bet基因和血清IFN - r含量有明显的相关性。AA组治疗前后T-bet基因和血清IFN - r含量均明显下降。提示AA中存在Th1型细胞反应的亢进,而MDS中存在Th1型抗肿瘤作用的减弱,使得异常造血克隆得以逃脱免疫监视,逐步进展为急性白血病。3提示T-bet等多指标检测,有助于鉴别AA和MDS,并可指导治疗评价疗效。
Objective: To detect the expression levels ofCD3 +, CD4 +, CD8 +, T-bet gene and IFN-γin aplastic anemia and myelodysplastic syndrome patients, and to study its Clinical differential diagnosis significance.
Methods: The study included 14 cases of aplastic anemia(AA)、20 cases of myelodysplastic Syndrome (MDS) and 30 cases normal control(NC).There were 14 cases of aplastic anemia (8 males and 6 females,aging form 14 to 66 with a median age of 26),including chronic aplastic anemia(CAA)9cases and 5cases of acute aplastic anemia (SAA). There were 20 cases of myelodysplastic Syndrome (9males and 11 females, aging form 27to 75 with a median age of 58), including refractory anemia(RA) 8cases,refractory anemia with excess blasts (RAEB)12cases.Take 30 normal cases as controls. (15males and 15 females, aging form 20 to 75 with a median age of 55) . the expression levels of CD3 +, CD4 +,CD8+ was detected by the immunofluorescence and flow cytometry. The expression levels of T-bet were measured by the real-time quantitative reverse transcription polymerase chain reaction. and The expression levels of IFN-γwere measured by the ELISE. We studied the expression of these makers in patients who,s post-treatment in 6 months .Finally, collect the clinical date to parallel.
Results: 1 1The expression level of CD3 +, CD4 +, CD8 + ,CD4+/ CD8+
1.1 The levels of CD3(+)T cells in AA were higher than those in control group(75.338±5.620 VS67.441±12.817,P<0.05),No differences of the levels of CD4 (+)T cells were between the both AA and control group(31.852±6.425VS33.424±8.415, P>0.05), The levels of CD8(+)T cells in AA were higher than those in control group(34.956±6.206 VS27.032±9.046P <0.05), The ratios of CD4+/CD8+ were lower than those of control group (1.177±0.436 VS1.484±0.378 P<0.05)
1.2 The levels of CD3(+)T cells in MDS were lower than those in control group(62.241±9.102 VS67.441±12.817, P<0.05),No differences of the levels of CD3 (+)T cells were between the both low-risk group and control group (65.803±13.859 VS67.441±12.817, P>0.05), The levels of CD3(+)T cells in high-risk group were lower than those in control group(52.417±12.585 VS67.441±12.817 P<0.05)。The levels of CD4(+)T cells in MDS and high-risk group were lower than those in control group( 39.283±9.913 VS33.424±8.415, 42.536±10.711 VS33.424±8.415 P <0.05),No differences of the levels of CD4 (+)T cells were between the both low-risk group and control group(33.322±9.603 VS33.424±8.415 P>0.05), The levels of CD8(+)T cells in high-risk group were lower than those in control group(26.634±3.568 VS 27.032±9.046 P>0.05); there no difference between the other groups. and control group, The ratios of CD4+/CD8+ in MDS and high-risk group were higher than those in control group(1.630±1.063 VS1.484±0.378,1.801±1.089 VS1.484±0.378 P<0.05), No differences of the ratios of CD4+/CD8+ were between in low-risk group and control group(1.3313±0.424.MDS VS1.484±0.378 P>0.05);
1.3 The levels of CD3(+)T cells in AA group were higher than those in MDS group, low-risk group and high-risk group. The levels of CD4(+)T cells in AA group were lower than those in MDS group and high-risk group, No differences of the levels of CD4(+)T cells were between in AA group and low-risk group. The levels of CD8(+)T cells in AA group were higher than those in MDS group, low-risk group and high-risk group, but there were no differences between AA group and low-risk group. The ratios of CD4+/CD8+ in AA group were lower than those in MDS group and high-risk group, but there were no differences between AA group and low-risk group. 1.4 The patients with AA were serially analyzed before and after treatment, the levels of CD3 + were decreased, The levels of CD4 + were increase, The levels of CD8 + were decreased, The ratios of CD4 + / CD8 +were decreased to close normal. And the changes of CD3 +, CD8 +, CD4 + / CD8 + were statistically significant.
2 The expression levels of T-bet gene and IFN-γ
2.1 The expression levels of T-bet gene
2.1.1 The level of t-bet gene in AA group was significantly higher than that in control group(0.255±0.188 VS0.092±0.058,P<0.05)。
2.1.2 The level of T-bet gene in MDS group was lower than those in and control group(0.085±0.041 VS0.092±0.058,P<0.05);The levels of t-bet gene in low-risk group and high-risk group were lower than that in control group, but there were no differences between control group and low-risk group.
2.1.3 The level of t-bet gene in AA group was significantly higher than that in MDS group, low-risk group and high-risk group.
2.1.4. The patients with AA were serially analyzed before and after treatment, compared with the untreated group ,the level of T-bet gene were decreased(0.153±0.078 VS0.255±0.188 P<0.05)。
2.2 The Serum levels of IFN - r
2.2.1 The level of IFN-γin AA group was significantly higher than that in control group(9.756±2.537 VS 7.552±0.640 P<0.05)。
2.2.2 The level of IFN-γin MDS group was lower than those in control group. (5.365±2.338 VS 7.552±0.640 P<0.05);The levels of IFN-γin low-risk group were higher than that in high-risk group ,but there were no differences between control group and low-risk group.
2.2.3 The level of IFN-γin AA group was significantly higher than that in MDS group, low-risk group and high-risk group.
2.2.4 The patients with AA were serially analyzed before and after treatment, compared with the untreated group ,the level of IFN-γwere decreased。(9.756±2.537 VS7.6822±2.338 P<0.05)。
2.2.5 The levels of t-bet gene in aplastic anemia group and myelodysplastic syndrome group were positively correlated with those of IFN-γ,Pearson respective:o.64、0.7 0(P﹤0.01)
Conclusions:1 T lymphocyte immune phenotype testing
Although there were no differences of the levels of CD3+, CD4 +, CD8 + CD4/CD8 between the low-risk group and the control group. But when AA group was compared with MDS group, low-risk group and high-risk group, all the indicators are statistically significant. And after treatment all the indicators were changed in patients with AA .These changes reveal that abnormality of immune function plays an important role at the onset of AA. and MDS. Immunologic abnormality to promote the advancement of the myelodysplastic syndrome. by flow cytometry Detecting AA and MDS T-lymphocyte immune phenotype helps us to clear the body's immune status, and can better help us both identify and guide clinical treatment.
2 The expression levels of t-bet gene and IFN-γ.
The expression levels of t-bet gene and IFN-γin AA group was significantly higher than the MDS group and the normal control group, and compared with the untreated group, the levels of t-bet gene and IFN-γin patients with AA were decreased。There exist the closely correlate between the etiopathogenesis of aplastic anemia with the levels of t-bet gene and IFN-γ.the Immunologic abnormality is the main etiological factor to the aplastic anemiathe. Existing the immune state heterogeneity in the MDS group, Immunologic abnormality to promote the advancement of the MDS. But patients in the MDS group have ununiformity in immune state. So identify immunization the factor Precisely effectiveness in the myelodysplastic syndrome, we must the carry out more investigation.
3 The multivariable indicators could be used for to guide clinical identification and diagnosis purposes.
引文
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