[Biological characteristics of human peripheric CD4+ CD25- T cells isolated and cultured in vitro].
OBJECTIVE
To isolate peripheric CD4(+) CD25(-) T cells in vitro, and study their biological characteristics.


METHODS
Human peripheric blood CD4(+) CD25(-) T cells were isolated by discontinuous density gradient centrfugal and dynal immunomagnetic beads, and then divided into three groups: control group(A), LPS group (B) and LPS+TGF-beta1 mAb group (D), After they were prepared 4 h, 3 d and 5 d, the percentage of CD4(+) CD25(+) T cells were tested by FCM, the levels of TGF-beta1 in cultured supernatants were detected by ELISA, and the expression of FOXP3 mRNA was examined by RT-PCR.


RESULTS
(1)LM showed CD4(+) CD25(-) T cells were of spherical cell nucleus, After CD4(+) CD25(-) T cells were cultured by anti-CD3/CD28 in vitro, their cellular volume increased and cytoplasm contained more particles. TEM showed CD4(+) CD25(-) T cells were of oval or kidney-shaped caryon. (2)FCM showed the purity of CD4(+) CD25(-) T cells was 91.5%-96%. The trypan blue experiment showed the energometry before and after detachment had no obvious difference(P>0.05). (3)FCM showed the percentage of CD4(+) CD25(+) T cells in B5d group(55.99+/-1.42)% increased markedly compared with that in A5d group(1.29+/-0.04)%. (4)ELISA showed the levels of TGF-beta1 in B3d(1.60+/-0.09) microg/L, in B5d(1.83+/-0.14) microg/L were significantly increased compared with those in A3d (0.35+/-0.04) microg/L and A5d(0.33+/-0.08) microg/L(P<0.01); but the levels in group D and group A had no significant difference; (5)RT-PCR showed FOXP3 mRNA in B3d group(0.84+/-0.07) and B5d group(1.85+/-0.24) increased strikingly compared with those in A3d(0.05+/-0.02) and A5d(0.04+/-0.02)(P<0.01), but those in group A and group D had no significant difference. (6)There was significantly positive correlation between the expression of TGF-beta1 and FOXP3 mRNA in the induced CD4(+) CD25(-) T cells (r=0.812, P<0.01).


CONCLUSIONS
Human peripheric blood CD4(+) CD25(-) T cells can be isolated in vitro by discontinuous density gradient centrfugal and dynal immunomagnetic beads. The expression of FOXP3 mRNA in E.coli LPS-induced human CD4(+) CD25(-) T cells may correlate with the expression of TGF-beta1.
Su
Miao-shang
MS
Department of Pediatrics, Second Affiliated Hospital of Wenzhou Medical College, Wenzhou 325027, China. sumish@163.com
Xu
Man-huan
MH
Chen
Fu-jiang
FJ
Li
Chang-chong
CC
Zhang
Wei-xi
WX
Chen
Xiao-fang
XF
Chen
Hui
H
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004797:Enzyme-Linked Immunosorbent Assay; D051858:Forkhead Transcription Factors / Q000235:genetics; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D046529:Microscopy, Electron, Transmission; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
D000911:Antibodies, Monoclonal; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D008070:Lipopolysaccharides; D053773:Transforming Growth Factor beta1; D017493:Leukocyte Common Antigens