有机苯与造血细胞凋亡及机体免疫变化的相关性研究
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摘要
[目的]研究有机苯对不同程度慢性苯中毒患者的细胞免疫、体液免疫功能的影响。进一步探讨有机苯的免疫毒性,以及免疫抑制治疗(Immunosuppressivetherapy,IST)对苯中毒相关性再障(Benzene poisoning-induced aplasticanemia,BPAA)患者免疫功能的改善和骨髓造血功能的恢复。研究凋亡相关基因Caspase-9及Bcl-2分别在BPAA、原发性再障及非原发性造血系统疾病引起的全血细胞减少患者的造血干细胞凋亡过程中的变化及其意义,重点讨论该凋亡相关基因在苯发病机制中的作用。
[方法]选择有机苯中毒患者68例,其中轻度中毒25例、中度中毒15例、重度中毒(即BPAA)28例,及正常对照20例,采用流式细胞仪(flow cytometry,FCM)方法检测其外周血T淋巴细胞亚群;采用免疫散射比浊法检测免疫球蛋白、补体。将28例BPAA患者根据T细胞亚群有无异常分成两组,CD4~+/CD8~+比值正常患者为A组,CD4~+/CD8~+比值降低患者为B组。A组不给予IST治疗,B组给予IST治疗。采用半定量RT-PCR方法检测28例BPAA组患者及20例正常对照骨髓单个核细胞(bone marrow mononuclear cell,BMMNC),对两者Caspase-9和Bcl-2的基因表达的异同进行比较。产物DNA用凝胶分析系统采集图像并分析Caspase-9,Bcl-2的吸光度值数据以均数±标准差((?)±S)的形式表示,有机苯致轻、中、重度中毒组与正常对照组间比较采用单因素方差分析,A与B治疗组比较以及BPAA等全血细胞减少组与正常对照组比较采用组间t检验,数据用SPSS13.0软件包做统计分析,检验水准α=0.05。
[结果]
1.慢性苯中毒患者T淋巴细胞亚群的变化:有机苯致轻度、中度中毒组患者CD8~+T细胞比值与正常对照相比无明显统计学差异(P＞0.05),而CD4~+T细胞和CD4~+/CD8~+比值与正常对照相比,两者均较正常值明显降低,差异有统计学意义(P＜0.05);BPAA组患者CD3~+T细胞比值与正常对照相比无明显统计学差异(P＞0.05),而CD8~+T细胞的增高、CD4~+T细胞和CD4~+/CD8~+比值的降低与正常对照组相比,差异有统计学意义(P＜0.05)。IST治疗后B组患者6个月后随访CD4~+/CD8~+比值恢复正常。
2.慢性苯中毒患者免疫球蛋白及补体的变化:有机苯致轻度与中度中毒组患者免疫球蛋白及补体水平与正常对照组相比,差异无统计学意义(P＞0.05):BPAA组患者IgG、IgA、C3下降及IgM增高与正常对照相比,差异有统计学意义(P＜0.05);a_1、a_2、β球蛋白及白蛋白水平与正常对照组相比,差异无统计学意义(P＞0.05),而γ球蛋白降低与正常对照组相比,差异有统计学意义(P＜0.05)。
3.BPAA患者Caspase-9、Bcl-2的变化:(1)Caspase-9:BPAA组Caspase-9和内参照β-actin吸光度的比值为1.09±0.12,显著高于正常对照组0.83±0.41,差异有统计学意义(P＜0.05);原发性AA的Caspase-9和内参照β-actin吸光度的比值0.89±0.34与正常对照组0.83±0.41相比差异无统计学意义(P＞0.05):非原发性造血系统疾病引起的全血细胞减少组的Caspase-9和内参照β-actin吸光度的比值0.81±0.32与正常对照组0.83±0.41相比差异无统计学意义(P＞0.05)。(2)Bcl-2:BPAA组Bcl-2和内参照β-actin吸光度的比值为0.81±0.38,显著低于正常对照组1.12±0.46,差异有统计学意义(P＜0.05);原发性AA组Bcl-2和内参照β-actin吸光度的比值0.68±0.11,显著低于正常对照组1.12±0.46,差异有统计学意义(P＜0.05);非造血系统疾病引起的全血细胞减少组Bcl-2和内参照β-actin吸光度的比值1.87±0.26,显著高于正常对照组1.12±0.46,差异有统计学意义(P＜0.05)。
[结论]
1.T淋巴细胞亚群、免疫球蛋白和补体的异常提示BPAA患者存在明显的细胞及体液免疫功能异常。T细胞免疫调节机制紊乱、淋巴细胞功能和比例失调在有机苯对造血干细胞损伤中起重要的作用。
2.BPAA的发病机制可能与上调凋亡通路中的Caspase-9以及下调Bcl-2的表达有关。
3.T细胞亚群有无异常可能成为BPAA患者选择应用IST的一项参考指标。
[意义]探讨免疫功能及凋亡相关基因的异常在有机苯中毒发病机制中的作用,进一步加深对BPAA免疫抑制治疗的认识,为BPAA在分子病因学方面开阔了新思路。
Objective: To study the effect of cellular immunity and humoral immunity about mild,moderate,and severe poisoning patients induced by organic benzene;and further understand the benzene' lymphotoxity. Try to know whether IST therapy can improve the immunity dysfunction and recover the bone marrow hematopoietic injury. To study the variation and significance of Caspase-9 and Bcl-2 gene in the BMMNC apoptosis process of BPAA patients,aplastic anemia(AA) patients,and cytopenia patients with non-hematopoietic diseases. Two genes and their effects on BPAA development are also discussed to reveal the pathogenesis of the three diseases.
Methods: 68 chronic benzene poisoning patients(mild 25 ,mediate 15,severe 28)and 20 sex matched healthy controls were enrolled to detect the T lymphocyte subsets and the levels of immunoglobin(Ig) and complement in the peripheral blood cells with FCM and immunological turbidity kit methods.28 chronic severe benzene poisoning (BPAA)patients were divided into two groups by the T lymphocyte subsets' abnormal,normal patients is in group A,abnormal patients is in group B.A group patients didn't receive immunosuppressive therapy(IST),but B group received IST. Expression of Caspase-9 and Bcl-2 gene mRNA were detected in BMMNC from 28 BPAA patients and 20 normal controls by semi-quantitative reverse transcriptional-polymerase chain reaction (RT-PCR) method. The gel images of DNA after semi-quantitative RT-PCR were captured with gel image processing system and analyzed. Volume of Caspase-9 mRNA and Bcl-2 mRNA was figured out and analyzed with SPSS13.0 statistics software package.
Results
(1) The changes of T lymphocyte subsets:Among the three benzene poisoning groups,T lymphocyte subsets analysis showed significantly decreased CD4~+ T lymphocytes and CD4~+/CD8~+ ratio,except CD3~+ T lymphocytes in severe benzene poisoning group(P<0.05); while T lymphocytes subsets analysis showed significantly decreased CD4~+ T lymphocytes and CD4~+/CD8~+ ratio,except CD8~+ T lymphocytes approached normal value in mild and moderate benzene poisoning groups. Ratio of CD4~+/CD8~+.reached normal level in B group patients after they received IST.
(2) The changes of immunoglobin and complement:The levels of immunoglobin and complement in mild and moderate benzene poisoning groups showed no significance(P>0.05).But compared with the control group,the levels of Immunoglobin-G(IgG),IgA,complement-3(C3) and globulin- y showed significantly decreased while IgM rising in BPAA group(P<0.05).
(3)The expression of Caspase-9 mRNA:Compared with the normal control group,the expression value of Caspase-9 gene in BPAA group showed significantly rising(P<0.05);while the expression value of Caspase-9 gene showed no significance in AA and cytopenia with non-hematopoietic groups(P>0.05).
The expression of Bcl-2 mRNA:Compared with the normal control group,the expression value of Bcl-2 gene in BPAA and AA groups showed significantly decreasing(P<0.05);but the expression value of Bcl-2 gene showed significance rising in cytopenia with non-hematopoietic group(P<0.05).
Conclusion
(1) The abnormal levels of T lymphocyte subsets,immunoglobin and complement in the BPAA patients suggested that patients with severe benzene poisoning had partial deficient function of cellular immunity and humoral immunity. The disorder of immuneoregulation machnisan,portion and function of T lymphocyte may play an important role in HSC injury by benzene.
(2) The abnormal levels of T lymphocyte subsets were correlated with the responsiveness to IST in BPAA patients and might be useful for directing treatment.
(3) The abnormal expression of Caspase-9 mRNA increases sharply and Bcl-2 mRNA decreases significantly in BPAA patients may be related to the progression and pathogenesis of benzene induced aplastic anemia,but the mechanism is still unknown.
Significance To explore the importmant effects of immune dysfunction and abnormal gene expression in the pathogenesis of chronic benzene poisoning,further understand the immunomosurppressive treatment of BPAA. It would offer a new thinking molecular pathway for BPAA.
[方法]选择有机苯中毒患者68例,其中轻度中毒25例、中度中毒15例、重度中毒(即BPAA)28例,及正常对照20例,采用流式细胞仪(flow cytometry,FCM)方法检测其外周血T淋巴细胞亚群;采用免疫散射比浊法检测免疫球蛋白、补体。将28例BPAA患者根据T细胞亚群有无异常分成两组,CD4~+/CD8~+比值正常患者为A组,CD4~+/CD8~+比值降低患者为B组。A组不给予IST治疗,B组给予IST治疗。采用半定量RT-PCR方法检测28例BPAA组患者及20例正常对照骨髓单个核细胞(bone marrow mononuclear cell,BMMNC),对两者Caspase-9和Bcl-2的基因表达的异同进行比较。产物DNA用凝胶分析系统采集图像并分析Caspase-9,Bcl-2的吸光度值数据以均数±标准差((?)±S)的形式表示,有机苯致轻、中、重度中毒组与正常对照组间比较采用单因素方差分析,A与B治疗组比较以及BPAA等全血细胞减少组与正常对照组比较采用组间t检验,数据用SPSS13.0软件包做统计分析,检验水准α=0.05。
[结果]
1.慢性苯中毒患者T淋巴细胞亚群的变化:有机苯致轻度、中度中毒组患者CD8~+T细胞比值与正常对照相比无明显统计学差异(P＞0.05),而CD4~+T细胞和CD4~+/CD8~+比值与正常对照相比,两者均较正常值明显降低,差异有统计学意义(P＜0.05);BPAA组患者CD3~+T细胞比值与正常对照相比无明显统计学差异(P＞0.05),而CD8~+T细胞的增高、CD4~+T细胞和CD4~+/CD8~+比值的降低与正常对照组相比,差异有统计学意义(P＜0.05)。IST治疗后B组患者6个月后随访CD4~+/CD8~+比值恢复正常。
2.慢性苯中毒患者免疫球蛋白及补体的变化:有机苯致轻度与中度中毒组患者免疫球蛋白及补体水平与正常对照组相比,差异无统计学意义(P＞0.05):BPAA组患者IgG、IgA、C3下降及IgM增高与正常对照相比,差异有统计学意义(P＜0.05);a_1、a_2、β球蛋白及白蛋白水平与正常对照组相比,差异无统计学意义(P＞0.05),而γ球蛋白降低与正常对照组相比,差异有统计学意义(P＜0.05)。
3.BPAA患者Caspase-9、Bcl-2的变化:(1)Caspase-9:BPAA组Caspase-9和内参照β-actin吸光度的比值为1.09±0.12,显著高于正常对照组0.83±0.41,差异有统计学意义(P＜0.05);原发性AA的Caspase-9和内参照β-actin吸光度的比值0.89±0.34与正常对照组0.83±0.41相比差异无统计学意义(P＞0.05):非原发性造血系统疾病引起的全血细胞减少组的Caspase-9和内参照β-actin吸光度的比值0.81±0.32与正常对照组0.83±0.41相比差异无统计学意义(P＞0.05)。(2)Bcl-2:BPAA组Bcl-2和内参照β-actin吸光度的比值为0.81±0.38,显著低于正常对照组1.12±0.46,差异有统计学意义(P＜0.05);原发性AA组Bcl-2和内参照β-actin吸光度的比值0.68±0.11,显著低于正常对照组1.12±0.46,差异有统计学意义(P＜0.05);非造血系统疾病引起的全血细胞减少组Bcl-2和内参照β-actin吸光度的比值1.87±0.26,显著高于正常对照组1.12±0.46,差异有统计学意义(P＜0.05)。
[结论]
1.T淋巴细胞亚群、免疫球蛋白和补体的异常提示BPAA患者存在明显的细胞及体液免疫功能异常。T细胞免疫调节机制紊乱、淋巴细胞功能和比例失调在有机苯对造血干细胞损伤中起重要的作用。
2.BPAA的发病机制可能与上调凋亡通路中的Caspase-9以及下调Bcl-2的表达有关。
3.T细胞亚群有无异常可能成为BPAA患者选择应用IST的一项参考指标。
[意义]探讨免疫功能及凋亡相关基因的异常在有机苯中毒发病机制中的作用,进一步加深对BPAA免疫抑制治疗的认识,为BPAA在分子病因学方面开阔了新思路。
Objective: To study the effect of cellular immunity and humoral immunity about mild,moderate,and severe poisoning patients induced by organic benzene;and further understand the benzene' lymphotoxity. Try to know whether IST therapy can improve the immunity dysfunction and recover the bone marrow hematopoietic injury. To study the variation and significance of Caspase-9 and Bcl-2 gene in the BMMNC apoptosis process of BPAA patients,aplastic anemia(AA) patients,and cytopenia patients with non-hematopoietic diseases. Two genes and their effects on BPAA development are also discussed to reveal the pathogenesis of the three diseases.
Methods: 68 chronic benzene poisoning patients(mild 25 ,mediate 15,severe 28)and 20 sex matched healthy controls were enrolled to detect the T lymphocyte subsets and the levels of immunoglobin(Ig) and complement in the peripheral blood cells with FCM and immunological turbidity kit methods.28 chronic severe benzene poisoning (BPAA)patients were divided into two groups by the T lymphocyte subsets' abnormal,normal patients is in group A,abnormal patients is in group B.A group patients didn't receive immunosuppressive therapy(IST),but B group received IST. Expression of Caspase-9 and Bcl-2 gene mRNA were detected in BMMNC from 28 BPAA patients and 20 normal controls by semi-quantitative reverse transcriptional-polymerase chain reaction (RT-PCR) method. The gel images of DNA after semi-quantitative RT-PCR were captured with gel image processing system and analyzed. Volume of Caspase-9 mRNA and Bcl-2 mRNA was figured out and analyzed with SPSS13.0 statistics software package.
Results
(1) The changes of T lymphocyte subsets:Among the three benzene poisoning groups,T lymphocyte subsets analysis showed significantly decreased CD4~+ T lymphocytes and CD4~+/CD8~+ ratio,except CD3~+ T lymphocytes in severe benzene poisoning group(P<0.05); while T lymphocytes subsets analysis showed significantly decreased CD4~+ T lymphocytes and CD4~+/CD8~+ ratio,except CD8~+ T lymphocytes approached normal value in mild and moderate benzene poisoning groups. Ratio of CD4~+/CD8~+.reached normal level in B group patients after they received IST.
(2) The changes of immunoglobin and complement:The levels of immunoglobin and complement in mild and moderate benzene poisoning groups showed no significance(P>0.05).But compared with the control group,the levels of Immunoglobin-G(IgG),IgA,complement-3(C3) and globulin- y showed significantly decreased while IgM rising in BPAA group(P<0.05).
(3)The expression of Caspase-9 mRNA:Compared with the normal control group,the expression value of Caspase-9 gene in BPAA group showed significantly rising(P<0.05);while the expression value of Caspase-9 gene showed no significance in AA and cytopenia with non-hematopoietic groups(P>0.05).
The expression of Bcl-2 mRNA:Compared with the normal control group,the expression value of Bcl-2 gene in BPAA and AA groups showed significantly decreasing(P<0.05);but the expression value of Bcl-2 gene showed significance rising in cytopenia with non-hematopoietic group(P<0.05).
Conclusion
(1) The abnormal levels of T lymphocyte subsets,immunoglobin and complement in the BPAA patients suggested that patients with severe benzene poisoning had partial deficient function of cellular immunity and humoral immunity. The disorder of immuneoregulation machnisan,portion and function of T lymphocyte may play an important role in HSC injury by benzene.
(2) The abnormal levels of T lymphocyte subsets were correlated with the responsiveness to IST in BPAA patients and might be useful for directing treatment.
(3) The abnormal expression of Caspase-9 mRNA increases sharply and Bcl-2 mRNA decreases significantly in BPAA patients may be related to the progression and pathogenesis of benzene induced aplastic anemia,but the mechanism is still unknown.
Significance To explore the importmant effects of immune dysfunction and abnormal gene expression in the pathogenesis of chronic benzene poisoning,further understand the immunomosurppressive treatment of BPAA. It would offer a new thinking molecular pathway for BPAA.
引文
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37.Viale M,Merli A,Bacigalupo A,et al.Analysis at the clonal level of T cell phenolype and functions in severe aplastic anemia patients[J].Blood,1991,78(5):1268-1274.
38.Posnett DN,Edinger JW,Manavalan JS,et al.Differentiation of human CD8T cells:implications for in vivo persistence of CD8+ CD28- cytotoxic effector clones[J].Int Immunol,1999,11(2):229-241.
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45.傅晋翔,张学光.再生障碍性贫血外周血和骨髓免疫活性分子变化及临床意义.中国免疫学杂志.2002,7(18):499-501
46.陈苏生,华明,王秀芹,等.苯中毒致再生障碍性贫血13例分析[J].中国职业医学,2002,29(6):34-35.
47.张丽,罗文达,郭群依,等.苯中毒再生障碍性贫血患者骨髓细胞免疫功能和Fas、CD34抗原变化[J].中国工业医学杂志,2005,18(2):82-84.
48.王鲁群,迟翠芳,张明珙,等.抗T淋巴细胞单克隆抗体治疗苯中毒致再生障碍性贫血[J].山东医科大学学报,1999,37(1):68-69,73.
49.Bratton SB,Mac-Farlanc M,Cain K,et al.protein complexesactivate distinct caspase cascades in death receptor and stress induced apoptosis [J].Exp Cell Res,2000,256(1):27-33.
50.Ashe P C,Berry M D.Apoptotic signaling cascades.Prog Neuro Psychopharmacol Biol Psychiarty,2003,27(2):199.
51.Denault J B,Salvesen G S.Caspase:key in the ignition of cell death.Chem Rev,2002,102(12):4489.
52.Thomberry X A,Lazebnik Y.Caspases:enemie within cell apoptosis [J].Science,1998,281(5381):1312-1316.
53.Cohen GM.Caspases:the excutioner of apoptosis[J].Bio Chem J,1997,326(1):116-118.
54.Marcus WL.Chemical of current interest-benzene.Toxicol Ind Health,1987,3:205-66
55.Snyder R.Recent developments in the understanding of benzene toxicity and leukemogenesis,Drug Chem Toxicol,2000,23:13-25.
56.TSUMOTOY,COSSMAN J,JAFFE E,et al.Involvement of the bcl-2 gene in human follicular lymphoma[J].Science,1985,228(4706):1440.
57.Farroew S N,White J H,Martinou I,et al.Cloning of a Bcl-2 homologue by interaction with adenovirus EIB19K[J].Nature,1995,374(6524):731-733.
58.Krajewski S,Krajewska M,Shabaik A,et al.Immunohisto-chemical determination of in vivo distribution of Bax,a dominant inhibitor of Bcl-2[J].Am J Pathol,1994,145(6):1323-1336.
59.陈惠新,许岸高,刘集鸿.黄参膏对胃癌癌前病变细胞凋亡及Bcl-2蛋白表达的影响.新医学,2000,31(4):216.
60.Philpott NJ,Scopes J,Marsch J CW,et al,Increased apoptosis in aplastic anemia bone marrow progenitor cells:possible patho physiologic significance[J].Exp Hematol,1995,23:1642-1648.
61.刘红,吴芳颐,姜胜华,等.二例重症再生障碍性贫血患者血清体外加速正常人CD34+细胞凋亡的研究[J].中华血液学杂志,1998,19:514-517.
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