Screening of Pathogenicity-Related Genes in Bone Marrow CD4⁺ T Cells of Aplastic Anemia Patients.

Recent clinical and experimental data have proved that CD4⁺ T cells play a crucial role in the pathogenesis of aplastic anemia (AA). However, the mechanisms how CD4⁺ T cells in AA over-proliferate, activate, infiltrate bone marrow and damage hematopoietic cells have largely remained unknown. Therefore we adopted the suppressive subtractive hybridization (SSH) method so as to screen differentially expressed genes of bone marrow CD4⁺ T cells between AA patients and normal donors, which may hopefully help clear up the mystery of the pathogenesis of AA. Firstly, we selected a pair of subjects: a typical first-visit AA patient and a healthy donor of the same age and sex. Their bone marrow (BM) mononuclear cells were isolated using lymphocyte separating medium by density gradient centrifugation. After a 24-hour expansion culture, CD4⁺ T cells were separated with magnetic bead sorting technique. Secondly, with CD4⁺ T cells of the patient as “tester” and those of the normal donor as “driver”, a cDNA library was established by the SSH method. 110 clones were detected by means of PCR in the established subtractive library, which contained an inserted fragment with 200∼700bp respectively, and the positive detected rate was up to 88%. Then 10 of the resulting subtracted cDNA clones were randomly selected for a respective DNA sequencing and honologic analysis. Among 10 sequenced clones, there were 8 known genes including 2 repeated genes. Lastly, we collected BM samples of 20 AA patients and 20 healthy donors so as to validate the expression levels of differentially expressed genes from SSH library with semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). We found, compared with normal donors, there were 6 genes among these 8 genes from SSH library up-expressed in BM CD4⁺ T cells of patients with AA. These genes were transducin (beta) -like 1X-linked receptor 1 (TBL1XR1) (NM024665); ATP-binding cassette, sub-family B (MDR/TAP), member 6 (NM005689); ARP2 actin-related protein 2 homolog (ACTR 2) (NM005722); zinc finger protein 561 (ZNF561) (NM152289); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 (NM002493); eukaryotic translation initiation factor 3, subunit 5(NM003754). These results indicate that some genes are found able to regulate functions of BM CD4⁺ T cells in AA, concerned with protein synthesis, biology oxidation, signal transduction and cell migration, etc. Therefore, screening and cloning these genes are helpful to elucidate the possible mechanisms of AA.