重型再生障碍性贫血患者骨髓T细胞克隆的初步研究

A primary study on establishing T-cell clones in vitro from bone marrow of a severe aplastic anemia patient

目的 :建立源于重型再生障碍性贫血 (重型再障 )患者骨髓的T淋巴细胞克隆 ,以便研究该病的发病机理。方法 :用免疫分选法分别从 1名初诊重型再障患者及对照的骨髓中富集CD34+ 和CD3+ 细胞。将CD3+ 细胞作反应细胞、照射过CD34+ 细胞作刺激细胞 ,进行 14d单向自身混合淋巴细胞培养。对活细胞作有限稀释和单个细胞分孔接种 ,与含IL 2和PHA P的饲养细胞体系共培养。观察挑选细胞生长孔 ,计算接种率 ,用泊松 (Poisson)分布初步判断单个细胞分离过程成功与否。而后将阳性孔细胞作转孔培养 ,定期用饲养抚育刺激、常规用IL 2支持增殖以扩大各克隆的数量。用SABC P免疫细胞化学染色法 ,初步鉴定各克隆CD4 CD8抗原表型。结果 :自身混合淋巴细胞培养后的活细胞数 ,病例为 4× 10 3,对照为 6 5。单细胞分离接种培养后 ,对照组的细胞生长孔数为 0 ;而病例组的培养板上 ,共有 11孔发生细胞增殖 ,相应的实际接种率是 2 1% ,未超出Poisson理论接种率值范围 (<2 6 % ) ,判明平均每个增生孔的细胞克隆前体数不大于 1。这 11个克隆中 ,9个呈CD4单阳性表型 ,阳性为 98% ;2个为CD8单阳性表型 ,阳性率为 98%。结论 :从 1例重型再障患者骨髓中分离出了 11株T淋巴细胞克隆。进一步鉴定分析这些克隆的表型和特异性 。

Objective:To isolate and establish long term T cell clones derived from bone marrow(BM) of a patient with severe aplastic anemia(sAA).Methods:CD34 + cells and CD3 +T cells were enriched from BM aspirates,respectively by positive and negative immunomagnetic sorting protocol.BM of exicised rib of a non hematological patient was used as control. Irradiated CD34 + cells together with T cells were subject to one way autologous mixed lymphocyte culture(AMLC).Viable T cells surviving from AMLC were limit diluted and single cell was plated into microtitier wells along with feeder cells.Then all wells were screened for the growing cell wells and they were transferred into new wells to be expanded and maintained.Each clones obtained were primarily assessed CD4/CD8 phenotype and their homogeneity by immunocytochemical analysis.Results:The purity of sorted CD34 + and T cells was more than 90%. On AMLC,a total of 4×10 3 cells were viable with sAA,but 65 cells viable with control.Upon single cell seeding it 11 growing cell wells were found for sAA,and no one growing cells for control.The actual plating rate(2.1%) was within Poisson theoretic value range (<26%),which means a successful cloning procedure A primary analysis of these 11 clones showed 9 clones expressed CD4 homogeneously,but the other 2 expressed CD8 homogeneously.Conclusion:11 T cell clones were established which seems reactive to CD34 +stem/progenitor cells of a sAA patient.Further studies on these clones should facilitate elucidating immune mechanism of sAA.