Screening of aplastic anaemia-related genes in bone marrow CD4+ T cells by suppressive subtractive hybridization.
BACKGROUND
CD4(+) T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4(+) T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4(+) T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia.


METHODS
The bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as "tester" and donor as "driver", their CD4(+) T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library.


RESULTS
PCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4(+) T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4(+) T cells in aplastic anaemia.


CONCLUSIONS
Screening and cloning genes, which regulate functions of CD4(+) T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4(+) T cells in aplastic anaemia.
Zheng
Miao
M
Department of Haematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Liu
Wen-li
WL
Fu
Jin-rong
JR
Sun
Han-ying
HY
Zhou
Jian-feng
JF
Xu
Hui-zhen
HZ
D000328:Adult; D000741:Anemia, Aplastic / Q000235:genetics*; D001854:Bone Marrow Cells / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D050882:CREB-Binding Protein / Q000235:genetics; D015723:Gene Library; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051822:T Cell Transcription Factor 1 / Q000235:genetics
D051822:T Cell Transcription Factor 1; C497576:TCF7 protein, human; D050882:CREB-Binding Protein; C494363:CREBBP protein, human