Isolation and amplification of residural normal stem cells from bone maurow of clonal hemopoietic disorders

我们用X染色体失活和磷酸甘油酸激酶(PGK)基因的RFLP或人雄激素受体基因CAG短串联重复序列(HUMARA)的异质性，分析了骨髓再生障碍性综合征(MDS)和再生障碍性贫血等骨髓衰竭患者造血祖细胞的克隆性。CD34阳性的骨髓细胞在添加EPO、GM-CSF和SCF的甲基细胞糖液中培养14天。在倒置显微镜下观察到单个菌落。提取的DNA分为两部分。其中一个被甲基化敏感酶HHA-1消化，另一个仍未被消化。用PGK或Humara引物对扩增出的DNA进行扩增。聚合酶链式反应产物经聚丙烯酰胺凝胶电泳法分析。在本研究中，我们证实了大多数MDS患者骨髓中残留的非克隆性祖细胞。根据MDS中残留的正常祖细胞的结果，我们对患者进行了化疗，证实了病情缓解时非克隆性造血功能的恢复。这些结果有力地表明，分离和扩增残留的正常祖细胞使我们能够开发一种新的MDS治疗策略。

We analyzed the clonality of hemopoietic progenitor cells in bone marrow failure including myelody splastic syndromes (MDS) and aplastic anemia using X-chromosome inactivation and the RFLP of phosphogly cerate kinase (PGK) gene or the heterogeneity of short tandem repeat of CAG sequence in human androgen receptor gene (HUMARA) in female patients. CD34-positive bone marrow cells were cultured in methylcellrose medium supplemented with EPO,GM-CSF and SCF for 14 days. Individual colonies were picked-up under an inverted microscope. Extracted DNA was divided into two parts. One was digested with methylation-sensitive enzyme Hha 1 and the other remained undigested. The resulting DNA were amplified by PCR using PGK or HUMARA primers. PCR products were electrophoresed in polyacrylamide gel. In the present study we demonstrated residual non-clonal progenitor pupulations in bone marrow of most MDS patients. According to the results of residual normal progenitors in MDS we treated the patients with chemotherapy and confirmed the recovery of nonclonal hemopoiesis at remission of the disease. These results strongly suggested that isolation and amplification of residual normal progenitors permit us to develop a new treatment strategy for MDS.