[Effects of different stimulatory factors on functions of CIK cells].

This study was aimed to investigate the effects of different stimulatory factors on proliferation and function of cytokine induced killer (CIK) cells. Peripheral blood mononuclear cells (PBMNC) were separated by Ficoll-Hypacue gradient. According to supplement of different stimulatory factors (CD28 mAb, IL-15 and IL-21), the experiment was divided into five groups:control group (CIK), CB28+IL-15+IL-21 group, IL-15+IL-21 group, CD28+IL-15 group and CD28+IL-21 group. Effects of different stimulatory factors on the proliferation of CIK cells were assayed by an automated hematology analyzer. Changes of granzyme B,perforin and CD107a were detected by flow cytometry. IL-10, IL-12, INF-γ and TNF-α were quantified by ELISA. Cytotoxicities on lung cancer cell line A549, breast adenocarcinoma cell line MFC-7 and human melanoma cell line HME1 were examined by lactate dehydrogenase release method. The results showed that there were significant differences among different groups. The highest proliferation index on days 10 was observed in group CD28mAb, IL-15 and IL-21(255.3 ± 6.3), which was higher than control group, IL-21+IL-15 group and CD28 mAb+IL-21 group (166.6 ± 13.5, 199.4 ± 15.0 and 228.8 ± 16.6) (P < 0.05). The expression of perforin in CD28 mAb+IL-15 group was higher than the other groups. The expression of perforin,GranB and CD107a of costimulatory groups was higher than control group. The cytotoxicities of CD28 mAb+IL-15 group on A549, MFC-7 and HME1 cells (82.2%, 59.3% and 70.6%) were much higher than that of control group (60.9%, 49.6% and 48.4%) (P < 0.05). The highest IFN-γsecretion was found in CD28 mAb, IL-15 and IL-21 groups. It is concluded that there are significant difference of proliferative capacity, cytokine secretion and cytotoxicity after being activated by different stimulatory factors. Adding corresponding stimulatory factors into the culture system displays a great value for target cells culture.

[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.

[The effect of aspirin on human's gammadeltaT cells killing digestive system tumor cell lines].

AIM: To explore the effect of aspirin on human's gammadeltaT cells killing digestive system tumor cell lines. METHODS: Use the isopentenyl pyrophosphate method to amplify human peripheral blood gammadeltaT cells in vitro. Various concentrations of aspirin were used to induce gammadeltaT cells and digestive system tumor cells SGC-7901, SW-1990, SW-480, SW-1116, LOVO cells lines, LDH assays was used to measure the cytotoxic activity of gammadeltaT cells, flow cytometry analysis the apoptosis percentage of before and after induced gammadeltaT cells and SGC-7901, SW-1990, SW-480, SW-1116, LOVO cell lines. RESULTS: gammadeltaT cells were cultivated for ten days which proliferation ratio increase from 4.21% to 70.35%. When gammadeltaT cells induced via aspirin concentrations rang from 0.4 mmol/L to 0.8 mmol/L which cytotoxic activity on the five kinds of tumor cell lines was the highest, if aspirin's concentration surpassing 3.2 mmol/L, cytotoxic activity of gammadeltaT cells present decrease tendency. SGC-7901, SW-1990, SW-480, SW-1116, LOVO cells lines were induced by different concentrations of aspirin for 24 hours, just SW-480, SW-1116 and LOVO were enhanced as far as the cytotoxic activity of gammadeltaT cells on these cell lines was concerned, other groups and control group have no notable changed. The apoptosis percentage of gammadeltaT cells(52.71%) were induced by aspirin which concentration was 3.2 mmol/L, which was strikingly higher than SGC-7901, SW-1990, SW-480, SW-1116, LOVO cells lines, (respectively 7.88%, 8.89%, 6.21%, 4.47% and 3.67%). CONCLUSION: When aspirin's concentration for clinical routine used can enhance the effect of gammadeltaT cells killing the tumor cells, if surpassing this concentration can obvious inhibited the proliferation capacity and cytotoxic activity of gammadeltaT cells and augment apoptosis ratio, however, it is not obvious for digestive tract tumor lines.