Zheng Miao, Sun Hanying, Zhou Jianfeng, Xu Huizhen, Huang Lifang, Liu Wenli
Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2010 Feb;30(1):37-41. doi: 10.1007/s11596-010-0107-3. Epub 2010 Feb 14.
Recent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from peripheral blood and bone marrow (BM) in AA patients. In the current study, BM CD4(+) T cells were isolated from AA patients and healthy controls with immunomagnetic beads sorting, and proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells were compared between them. By (3)H-TdR method, CD4(+) T cells in AA group presented more enhanced proliferative activity. The stimulation index in control group and AA group was 1.47+/-0.24, and 2.51+/-0.34 respectively (P<0.01). After BM CD4(+) T cells were induced by high concentration of CD3 monoclonal antibody for 18 h, evident apoptosis cells could be seen under the electron microscope in both control group and AA group. Flow cytometry revealed that apoptosis rates in the early and late stages of AA group were significantly higher than in control group (P<0.01). Early-stage apoptosis rate in control and AA groups was (6.85+/-1.48)% and (16.98+/-4.40)%, and late-stage apoptosis rate in control group and AA group was (2.65+/-1.57)% and (7.74+/-0.83)%, respectively (P<0.01). The CFU-GM count in AA group and control group was (74.50+/-9.50)/10(4) cells and (124.25+/-19.80)/10(4) cells respectively under an inverted microscope (P<0.01), and the expression levels of CyclinD3 mRNA and protein in cord blood CD34(+) cells were both down-regulated induced by BM CD4(+) T cell culture supernatant in AA patients. These results indicate that BM CD4(+) T cells of AA patients are likely in an abnormally proliferative, and activated state which can correlate intimately with AA hematopoiesis damage. BM CD4(+) T cells in AA patients can secret some soluble cytokines that can inhibit proliferation of hematopoietic stem cells by suppressing the expression of Cyclin D3, resulting in hematopoiesis failure.
近期研究表明，免疫相关性再生障碍性贫血（AA）类似于胰岛素依赖型糖尿病和慢性自身免疫性甲状腺炎等自身免疫性疾病，这些疾病属于器官特异性自身免疫性疾病。许多独立的研究小组已成功从AA患者的外周血和骨髓（BM）中分离出具有CD4表型、可导致造血功能衰竭的致病相关T细胞克隆。在本研究中，采用免疫磁珠分选法从AA患者和健康对照者中分离出BM CD4(+) T细胞，并比较了它们的增殖能力、凋亡特征以及它们分泌的细胞因子对造血干/祖细胞的影响。通过³H-TdR法检测，AA组CD4(+) T细胞的增殖活性增强更为明显。对照组和AA组的刺激指数分别为1.47±0.24和2.51±0.34（P<0.01）。用高浓度CD3单克隆抗体诱导BM CD4(+) T细胞18小时后，对照组和AA组在电子显微镜下均可见明显的凋亡细胞。流式细胞术检测显示，AA组早期和晚期凋亡率均显著高于对照组（P<0.01）。对照组和AA组早期凋亡率分别为(6.85±1.48)%和(16.98±4.40)%，晚期凋亡率分别为(2.65±1.57)%和(7.74±0.83)%（P<0.01）。倒置显微镜下观察，AA组和对照组的CFU-GM集落数分别为(74.50±9.50)/10⁴细胞和(124.25±19.80)/10⁴细胞（P<0.01），且AA患者脐血CD34(+)细胞中CyclinD3 mRNA和蛋白的表达水平在BM CD4(+) T细胞培养上清诱导下均下调。这些结果表明，AA患者的BM CD4(+) T细胞可能处于异常增殖和活化状态，这与AA的造血损伤密切相关。AA患者的BM CD4(+) T细胞可分泌一些可溶性细胞因子，通过抑制Cyclin D3的表达来抑制造血干细胞的增殖，从而导致造血功能衰竭。
