再生障碍性贫血患者雄激素和雄激素受体的改变及其意义
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摘要
目的：通过检测慢性再障患者外周血中雄激素的含量、骨髓单个核细胞中的雄激素受体水平以及骨髓中的T细胞亚群和活化T抑制细胞水平，探讨雄激素—雄激素受体途径在慢性再生障碍性贫血(CAA)免疫病理机制中的作用，并分析CAA患者骨髓中雄激素受体表达与其病情以及用雄激素治疗的预后之间的关系。方法：⑴用放射免疫法测定CAA初发组、CAA治疗组和对照组外周血血浆中的睾酮水平；⑵免疫细胞化学SP法检测并半定量分析CAA初发组、CAA治疗组和对照组骨髓单个核细胞(BMMNC)胞浆和胞核内雄激素受体（AR）的阳性表达水平；⑶流式细胞术检测CAA初发组、CAA治疗组及对照组骨髓中T细胞亚群（CD3+CD4+细胞、CD3+CD8+细胞）和活化T抑制细胞（CD8+CD25+细胞、CD8+HLA-DR+细胞）的含量。结果：⑴CAA初发组、CAA治疗组和对照组三组中各组内男性的血浆睾酮水平均显著高于女性[各组男性分别为：(107.69±15.27)、(124.73±30.24)、(123.61±18.46)；女性分别为：(88.21±20.12)、(90.60±24.16)、(93.99±15.40)（ng/dl）]，同时初发组、治疗组和对照组三组间的睾酮水平均无显著差异。⑵CAA初发组BMMNC胞浆和胞核内AR的阳性水平(68.05±6.74)显著低于治疗组(72.89±7.48)和对照组(79.44±6.71) （个/200个MNC），且治疗组AR阳性水平也显著低于对照组。不同性别间AR的含量未见显著差异。⑶CAA初发组患者的血浆睾酮含量与其骨髓中的AR水平明显正相关(p<0.05)。⑷初发组骨髓中的CD3+CD8+细胞、CD8+HLA-DR+细胞含量[CD3+CD8+细胞: (23.97±1.58)； CD8+HLA-DR+细胞：(19.43±4.16)]均显著高于治疗组[CD3+CD8+细胞: (21.68±3.49)； CD8+HLA-DR+细胞：(17.16±2.50)]和对照组[CD3+CD8+细胞: (19.22±4.47)； CD8+HLA-DR+细胞：(15.72±2.26) (%)]，且治疗组的CD3+CD8+细胞含量显著高于对照组。而三组中骨髓内的CD3+CD4+细胞、CD8+CD25+细胞均未见显著差异。⑸CAA患者初发时骨髓中的AR阳性水平与其治疗前骨髓中的CD3+CD8+细胞负相关(p＜
0.01)，外周血中的网织红细胞百分数正相关(p＜0.01)；与其用雄激素制剂治疗三个月时骨髓中的CD3+CD8+细胞负相关(p＜0.01)，外周血中的网织红细胞百分数和骨髓增生程度正相关(均为p＜0.01)；而与治疗前、后的CD8+HLA-DR+、CD3+CD4+、CD8+CD25+细胞含量、WBC数、Hb量、PLT数均未见明显的直线相关关系。结论：CAA患者发病时BMMNC中AR在胞核和/或胞浆中的表达较正常人减少，从而使雄激素刺激骨髓造血的作用减弱。CAA患者发病时骨髓中异常升高的CD3+CD8+细胞、CD8+HLA-DR+细胞在用雄激素治疗后显著降低，且患者初发时骨髓中AR的表达量与其用雄激素治疗前、后骨髓中的CD3+CD8+细胞含量均呈明显负相关，表明雄激素—雄激素受体途径的异常在一定程度上参与了慢性再生障碍性贫血发病机制中的细胞免疫异常。CAA发病时BMMNC中的AR阳性水平与发病时及治疗后骨髓中的CD3+CD8+细胞、外周血中的网织红细胞数等明显相关，提示检测CAA发病时AR的表达量可能在CAA的病情监测和预后估计方面有一定的价值。
Objective: To explore the function of androgen-androgen receptor way in immune pathogenesis in chronic aplastic anemia by measuring the level of androgen in peripheral blood, androgen receptor in nuclear and /or plasma in bone marrow mononuclear cells and T lymphocyte subpopulations in bone marrow ,and to analyze the correlation of androgen receptor expression and state when newly diagnosed as well as prognosis treated with androgen in bone marrow in chronic aplastic anemia patients. Methods:⑴Radioimmunoassay was used to investigate the contents of testosterone in plasma of 20 chronic aplastic anemia patients.⑵Immunocytochemistry was performed to evaluate the positive level of androgen receptor in nuclear and plasma of bone marrow mononuclear cells in 20 newly diagnosed patients、18 patients with treatment of androgen for three months and 25 healthy controls.⑶Flow cytometry was used to evaluate the contents of T- lymphocyte subpopulations（CD3+CD4+ cells、CD3+CD8+ cells） and activated Ts lymphocytes（CD8+CD25+ cells、CD8+HLA-DR+ cells） in bone marrow in newly diagnosed patients、patients treated with androgen and healthy people. Results: The level of testosterone in male is significantly higher than that of female in each of the above three groups, while there was no significant difference among the three groups mutually. The positive contents of andogen receptor in bone marrow in newly diagnosed group are significantly lower than those of the treatment group and the control group, and those of treatment group are also lower than control group. The level of testosterone and the positive contents of androgen receptor were in linear correlation, and the contents of CD3+CD8+ lymphocytes and CD8+HLA-DR+ lymphocytes in newly diagnosed group were significantly higher than those of the treatment group and the control group, and the contents of CD3+CD8+
lymphocytes in the treatment group were significantly higher than those of the control group, while there were no significant difference in CD3+CD4+ cells and CD8+CD25+ cells among the above three groups. In addition, the positive contents of androgen receptor in bone marrow had relationship with reticulocyte count in peripheral blood、CD3+CD8+ lymphocytes in bone marrow before treatment, and with reticulocyte count、bone marrow hyperplasia response、CD3+CD8+ lymphocytes in bone marrow after treatment of androgen for three months. Conclusion: The expression of androgen receptor in nuclear and plasma of bone marrow mononuclear cells when newly diagnosed was less than that of healthy people, therefore the function of androgen to stimulate hematopoiesis in bone marrow was reduced. The abnormally increased CD3+CD8+ lymphocytes and CD8+HLA-DR+ lymphocytes in bone marrow of the CAA patients when newly diagnosed were significantly reduced after treatment with androgen , and the amount of AR expression in bone marrow of those patients had relationship with the contents of CD3+CD8+ lymphocytes in bone marrow before and after administration of androgen, which suggested the abnormality of androgen-androgen receptor way may function in the abnormality of cellular immune in pathogenesis of chronic aplastic anemia. The expression of androgen receptor in bone marrow when newly diagnosed had relationship with several parameters such as CD3+CD8+ lymphocytes and reticular cells before and after treatment of androgen, which suggest identifying the expression of androgen receptor when newly diagnosed may be valuable in indicating state and expecting prognosis in chronic aplastic anemia.
0.01)，外周血中的网织红细胞百分数正相关(p＜0.01)；与其用雄激素制剂治疗三个月时骨髓中的CD3+CD8+细胞负相关(p＜0.01)，外周血中的网织红细胞百分数和骨髓增生程度正相关(均为p＜0.01)；而与治疗前、后的CD8+HLA-DR+、CD3+CD4+、CD8+CD25+细胞含量、WBC数、Hb量、PLT数均未见明显的直线相关关系。结论：CAA患者发病时BMMNC中AR在胞核和/或胞浆中的表达较正常人减少，从而使雄激素刺激骨髓造血的作用减弱。CAA患者发病时骨髓中异常升高的CD3+CD8+细胞、CD8+HLA-DR+细胞在用雄激素治疗后显著降低，且患者初发时骨髓中AR的表达量与其用雄激素治疗前、后骨髓中的CD3+CD8+细胞含量均呈明显负相关，表明雄激素—雄激素受体途径的异常在一定程度上参与了慢性再生障碍性贫血发病机制中的细胞免疫异常。CAA发病时BMMNC中的AR阳性水平与发病时及治疗后骨髓中的CD3+CD8+细胞、外周血中的网织红细胞数等明显相关，提示检测CAA发病时AR的表达量可能在CAA的病情监测和预后估计方面有一定的价值。
Objective: To explore the function of androgen-androgen receptor way in immune pathogenesis in chronic aplastic anemia by measuring the level of androgen in peripheral blood, androgen receptor in nuclear and /or plasma in bone marrow mononuclear cells and T lymphocyte subpopulations in bone marrow ,and to analyze the correlation of androgen receptor expression and state when newly diagnosed as well as prognosis treated with androgen in bone marrow in chronic aplastic anemia patients. Methods:⑴Radioimmunoassay was used to investigate the contents of testosterone in plasma of 20 chronic aplastic anemia patients.⑵Immunocytochemistry was performed to evaluate the positive level of androgen receptor in nuclear and plasma of bone marrow mononuclear cells in 20 newly diagnosed patients、18 patients with treatment of androgen for three months and 25 healthy controls.⑶Flow cytometry was used to evaluate the contents of T- lymphocyte subpopulations（CD3+CD4+ cells、CD3+CD8+ cells） and activated Ts lymphocytes（CD8+CD25+ cells、CD8+HLA-DR+ cells） in bone marrow in newly diagnosed patients、patients treated with androgen and healthy people. Results: The level of testosterone in male is significantly higher than that of female in each of the above three groups, while there was no significant difference among the three groups mutually. The positive contents of andogen receptor in bone marrow in newly diagnosed group are significantly lower than those of the treatment group and the control group, and those of treatment group are also lower than control group. The level of testosterone and the positive contents of androgen receptor were in linear correlation, and the contents of CD3+CD8+ lymphocytes and CD8+HLA-DR+ lymphocytes in newly diagnosed group were significantly higher than those of the treatment group and the control group, and the contents of CD3+CD8+
lymphocytes in the treatment group were significantly higher than those of the control group, while there were no significant difference in CD3+CD4+ cells and CD8+CD25+ cells among the above three groups. In addition, the positive contents of androgen receptor in bone marrow had relationship with reticulocyte count in peripheral blood、CD3+CD8+ lymphocytes in bone marrow before treatment, and with reticulocyte count、bone marrow hyperplasia response、CD3+CD8+ lymphocytes in bone marrow after treatment of androgen for three months. Conclusion: The expression of androgen receptor in nuclear and plasma of bone marrow mononuclear cells when newly diagnosed was less than that of healthy people, therefore the function of androgen to stimulate hematopoiesis in bone marrow was reduced. The abnormally increased CD3+CD8+ lymphocytes and CD8+HLA-DR+ lymphocytes in bone marrow of the CAA patients when newly diagnosed were significantly reduced after treatment with androgen , and the amount of AR expression in bone marrow of those patients had relationship with the contents of CD3+CD8+ lymphocytes in bone marrow before and after administration of androgen, which suggested the abnormality of androgen-androgen receptor way may function in the abnormality of cellular immune in pathogenesis of chronic aplastic anemia. The expression of androgen receptor in bone marrow when newly diagnosed had relationship with several parameters such as CD3+CD8+ lymphocytes and reticular cells before and after treatment of androgen, which suggest identifying the expression of androgen receptor when newly diagnosed may be valuable in indicating state and expecting prognosis in chronic aplastic anemia.
引文
杨崇礼主编，再生障碍性贫血（第二版），天津科技翻译出版公司，2000：263 。
Athanasios Mntalans,Nicki Panoskaltsis,et al.Localization of androgen receptor exprssion in human bone marrow.J of Pathology,2001;193: 361-366.
.李殿青，张伟华等，雄激素受体基因在再生障碍性贫血发病中的作用，中华血液学杂志，2001，22（10），548。
王欣，张明拱等，再生障碍性贫血患者细胞免疫功能与造血细胞因子的研究，中华血液学杂志，1998，19（4）：181-183。
曾凤华，沈柏均等，再生障碍性贫血患者骨髓T淋巴细胞的研究，中华血液学杂志，1997，18（10）：541-543。
张之南，沈悌主编，血液病诊断与治疗标准，第2版，北京科学出版社，1998，33-35。
巴德年主编，当代免疫学技术与应用，北京医科大学中国协和医科大学联合出版社，1998：200。
陈修飏，AA中医分型与发TE及秃发区AR相关性的研究，广州中医药大学，2002：38。
Henttu P,Vihko P.Growth factor regulation of gene expression in the human prostatic carcinoma cell line LNCaP.Cancer Res,1993 Mar 1;53(5)1051-8.
Ren F,Zhang S,Mitchell SH,Butler R,Young CY.Tea polyphenols down-regulate the expression of the androgen receptor in LNCaP prostate cancer cells.Oncogene,2000 Apr 6;19(15):1924-32.
Hsieh T,Wu JM.Induction of apoptosis and altered nuclear/cytoplasmic distribution of the androgen receptor and prostate-specific antigen by 1alpha,25-dihydroxyvitamin D3 in androgen-responsive LNCaP cells.Biochem Biophys Res Commun.1997 Jun 27;235(3)539-44.
Jin Y,Penning TM.Steroid 5alpha-reductases and 3alpha-hydroxysteroid dehydrogenases:key enzymes in androgen metabolism.Best Pract Res Clin Endocrinol Metab,2001 Mar;15(1)79-94.
Zhou Z,Speiser PW.Regulation of HSD17B1 and SRD5A1 in lymphocytes.Mol Genet Metab.1999 Nov;68(3):410-7.
A. Mizokami, H. Saiga, T. Matsui, T. Mita & A. Sugita: Regulation of androgen receptor by androgen and epidermal growth factor in a human prostatic cancer cell line, LNCaP. Endocrinologia Japonica 1992，39, 235-243 。
唐晓文，邵景章等，再生障碍性贫血患者外周血T细胞亚群检测，中华血液学杂志，1997，18（4）：205-206。
Maciejewski JP. ,Hibbs JR., et al. bone marrow and peripheral blood lymphocyte phenotype in patients with bone marrow failure.Exp Hematol,1994,22:1102-1110.
傅晋翔，於葛华等，再生障碍性贫血患者淋巴细胞表型变化，中华微生物学和免疫学杂志，2002，22（2）：152-155。
Zoumbos NC. ,Gascon P. ,et al. Circulating activated suppressor T lymphocytes in aplastic anemia. N Eng J Med,1985,312:257.
Zhou Z,Shackleton CH,Pahwa S,White PC,Speiser PW.Prominent sex steroid metabolism in human lymphocytes.Mol Cell Endocrinol,1998 Mar 16;138(1-2):61-9.
Sula K,Stroufova A,Presl J.The early androgen syndrome-effect on the hormonal and immune system.Ceska Gynekol,1994 Aug;59(4)200-5.
姚根宏，侯亚义等，睾酮对未成熟雄性大鼠体液和细胞免疫力的影响，免疫学杂志，2001，17（5）：334-337。
Liva SM,Voskuhl RR.Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production.J Immunol,2001 Aug 15;167(4):2060-7.
Samy TS,Schwacha MG,Cioffi WG,Bland KI,Chaudry IH.Androgen and estrogen receptors in splenic T lymphocytes:effects of flutamide and trauma-hemorrhage.Shock,2000 Oct;14(4):465-70.
Zheng R,Samy TS,Schneider CP,et al.Decreased 5alpha- dihydrotestosterone catabolism suppresses T lymphocyte function in males after trauma-hemorrhage.Am J Physiol.2002 Jun;282(6):C1332-8.
夏长青，再生障碍性贫血的免疫病理机制，中华血液学杂志，1996， 17（11）：610-612。
Olsen NJ,Kovacs WJ.Effects of androgens on T and B lymphocyte development.Immunol Res,2001;23(2-3):281-8.
Olsen NJ,Gu X,Kovacs WJ.Bone marrow stromal cells mediate androgenic suppression of B lymphocyte development.J Clin Invest,2001 Dec;108(11)1697-704.
Benten WP,Lieberherr M,Sekeris CE,Wunderlich F. Testosterone induces Ca2+ influx via non-genomic surface receptors in activated T cells.FEBS Lett,1997 Apr 28;407(2)211-4.
Martino G,Moiola L,Bramilla E,Clementi E,Comi G,Grimaldi LM.Interferon-gamma induces T lymphocyte proliferation in multiple sclerosis via a Ca(2+)-dependent mechanism.J Neuroimmunol.1995 Nov;62(2):169-76.
Fanger CM,Neben AL,Cahalan MD.Diffrential Ca2+ influx,Kca channel activity,and Ca2+ clearance distinguish Th1 and Th2 lymphocytes.J Immunol.2000 Feb 1;164(3):1153-60.
Benten WP,Becker A,Schmitt Wrede HP,Wunderlich F.Developmental regulation of intracellular and surface androgen receptors in T cells. Steroid,2002 Oct;67(11):925-31.
Athanasios Mntalans,Nicki Panoskaltsis,et al.Localization of androgen receptor exprssion in human bone marrow.J of Pathology,2001;193: 361-366.
.李殿青，张伟华等，雄激素受体基因在再生障碍性贫血发病中的作用，中华血液学杂志，2001，22（10），548。
王欣，张明拱等，再生障碍性贫血患者细胞免疫功能与造血细胞因子的研究，中华血液学杂志，1998，19（4）：181-183。
曾凤华，沈柏均等，再生障碍性贫血患者骨髓T淋巴细胞的研究，中华血液学杂志，1997，18（10）：541-543。
张之南，沈悌主编，血液病诊断与治疗标准，第2版，北京科学出版社，1998，33-35。
巴德年主编，当代免疫学技术与应用，北京医科大学中国协和医科大学联合出版社，1998：200。
陈修飏，AA中医分型与发TE及秃发区AR相关性的研究，广州中医药大学，2002：38。
Henttu P,Vihko P.Growth factor regulation of gene expression in the human prostatic carcinoma cell line LNCaP.Cancer Res,1993 Mar 1;53(5)1051-8.
Ren F,Zhang S,Mitchell SH,Butler R,Young CY.Tea polyphenols down-regulate the expression of the androgen receptor in LNCaP prostate cancer cells.Oncogene,2000 Apr 6;19(15):1924-32.
Hsieh T,Wu JM.Induction of apoptosis and altered nuclear/cytoplasmic distribution of the androgen receptor and prostate-specific antigen by 1alpha,25-dihydroxyvitamin D3 in androgen-responsive LNCaP cells.Biochem Biophys Res Commun.1997 Jun 27;235(3)539-44.
Jin Y,Penning TM.Steroid 5alpha-reductases and 3alpha-hydroxysteroid dehydrogenases:key enzymes in androgen metabolism.Best Pract Res Clin Endocrinol Metab,2001 Mar;15(1)79-94.
Zhou Z,Speiser PW.Regulation of HSD17B1 and SRD5A1 in lymphocytes.Mol Genet Metab.1999 Nov;68(3):410-7.
A. Mizokami, H. Saiga, T. Matsui, T. Mita & A. Sugita: Regulation of androgen receptor by androgen and epidermal growth factor in a human prostatic cancer cell line, LNCaP. Endocrinologia Japonica 1992，39, 235-243 。
唐晓文，邵景章等，再生障碍性贫血患者外周血T细胞亚群检测，中华血液学杂志，1997，18（4）：205-206。
Maciejewski JP. ,Hibbs JR., et al. bone marrow and peripheral blood lymphocyte phenotype in patients with bone marrow failure.Exp Hematol,1994,22:1102-1110.
傅晋翔，於葛华等，再生障碍性贫血患者淋巴细胞表型变化，中华微生物学和免疫学杂志，2002，22（2）：152-155。
Zoumbos NC. ,Gascon P. ,et al. Circulating activated suppressor T lymphocytes in aplastic anemia. N Eng J Med,1985,312:257.
Zhou Z,Shackleton CH,Pahwa S,White PC,Speiser PW.Prominent sex steroid metabolism in human lymphocytes.Mol Cell Endocrinol,1998 Mar 16;138(1-2):61-9.
Sula K,Stroufova A,Presl J.The early androgen syndrome-effect on the hormonal and immune system.Ceska Gynekol,1994 Aug;59(4)200-5.
姚根宏，侯亚义等，睾酮对未成熟雄性大鼠体液和细胞免疫力的影响，免疫学杂志，2001，17（5）：334-337。
Liva SM,Voskuhl RR.Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production.J Immunol,2001 Aug 15;167(4):2060-7.
Samy TS,Schwacha MG,Cioffi WG,Bland KI,Chaudry IH.Androgen and estrogen receptors in splenic T lymphocytes:effects of flutamide and trauma-hemorrhage.Shock,2000 Oct;14(4):465-70.
Zheng R,Samy TS,Schneider CP,et al.Decreased 5alpha- dihydrotestosterone catabolism suppresses T lymphocyte function in males after trauma-hemorrhage.Am J Physiol.2002 Jun;282(6):C1332-8.
夏长青，再生障碍性贫血的免疫病理机制，中华血液学杂志，1996， 17（11）：610-612。
Olsen NJ,Kovacs WJ.Effects of androgens on T and B lymphocyte development.Immunol Res,2001;23(2-3):281-8.
Olsen NJ,Gu X,Kovacs WJ.Bone marrow stromal cells mediate androgenic suppression of B lymphocyte development.J Clin Invest,2001 Dec;108(11)1697-704.
Benten WP,Lieberherr M,Sekeris CE,Wunderlich F. Testosterone induces Ca2+ influx via non-genomic surface receptors in activated T cells.FEBS Lett,1997 Apr 28;407(2)211-4.
Martino G,Moiola L,Bramilla E,Clementi E,Comi G,Grimaldi LM.Interferon-gamma induces T lymphocyte proliferation in multiple sclerosis via a Ca(2+)-dependent mechanism.J Neuroimmunol.1995 Nov;62(2):169-76.
Fanger CM,Neben AL,Cahalan MD.Diffrential Ca2+ influx,Kca channel activity,and Ca2+ clearance distinguish Th1 and Th2 lymphocytes.J Immunol.2000 Feb 1;164(3):1153-60.
Benten WP,Becker A,Schmitt Wrede HP,Wunderlich F.Developmental regulation of intracellular and surface androgen receptors in T cells. Steroid,2002 Oct;67(11):925-31.
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