[Co-stimulation of multiple activating factors on proliferation and phenotype of T lymphocytes in peripheral blood in vitro].

AIM

To observe the costimulation of multiple activating factors effects on the proliferation and phenotype of T lymphocytes in vitro.

METHODS

Peripheral blood mononuclear cells (PBMCs) were separated by fractionation on Ficoll-Hypaque gradient. According to adding different cytokines (CD3 mAb, CD28 mAb, IFN-γ, IL-1α, IL-2 and IL-15), the experiments were divided into seven groups. Effects of different cytokines on the proliferation of PBMC were counted by automated hematology analyzer five categories. The phenotypes (CD3, CD4, CD8, CD28, CD16, CD56(+);CD16, CD3(+);CD8(+);, CD3(+);CD4(+);, CD3(+); CD56(+);, CD45RO) expressing on the surface of costimulatory cells were detected by flow cytometry, and the cytotoxicity of costimulatory cells on SGC-7901, SW-1990 and SW-116 cell lines was examined by lactate dehydrogenase release method.

RESULTS

The proliferation has significant difference when adding different cytokines into PBMCs culture system, the highestest proliferation multiples group is the one contains cytokines CD3, CD28, IFN-γ, IL-2, IL-1α, IL-15 and IL-21, which proliferation multiple is 255.3±6.3 at the tenth day of cell culture, obviously higher than the other culture systems which only contains CD3, IFN-γ and IL-2 (166.6±5.5) (P<0.05). Part of cells'phenotype changed when adding different activating factors. Without IL-15, the proportion of CD16(+);CD56(+);(NK) cells and CD3(+);CD56(+); cells was higher than the other groups; CD45RO(+); memory cells is most evident when delayed adding IL-15 and IL-21 for three days. The cytotoxicity of PBMCs cultured for ten days with different activating factors had significant difference, the highest was the one which delayed adding IL-15 and IL-21 for three days (76.2%, 60.3% and 70.6%, respectively.), higher than the cell culture groups containing CD3, IFN-γ and IL-2 (54.9%, 44.6% and 50.4%, respectively) (P<0.05). The cultured cells had the strongest cytotoxicity on SGC-7901 gastric adenocarcinoma cells.

CONCLUSION

The PBMCs' proliferation, phenotype and cytotoxicity had significant difference after being activated by different stimulating factors, adding matching stimulating factors into the culture system have great value on cell-directed culture.