A Research For Angelica Sinensis Polysaccharide Induced CIK Cells’ Biological Characteristics, Killing Activity And Its Related Mechanisms

Objective This study was mainly to investigate the effect of Angelica sinensis polysaccharide(ASP) on the proliferation, immune phenotype, the secretion of cytokines and cytotoxicity to K562 of cytokine induced killer cells(CIK) derived from human peripheral blood mononuclear cells(PBMCs). In addition, discuss the possible mechanisms.Methods PBMCs were obtained from healthy volunteers, density gradient centrifugation was used to isolate peripheral blood mononuclear cells(PBMCs), on day 0 add interferon-γ(1 000 IU/ml),after 24 hour add anti-human CD3(50 ng/ml), then the CIK cells were randomly divided into five groups based on adding different concentration of ASP after every 3 days: A group(no ASP), B~E group(add ASP 12.5、25、50、100 μg/ml respectively). All groups were put in 37 ℃ and 5% CO2 incubator, observe the cell morphology change, every 3 days to add fresh cell cultures containing IL-2 or ASP, according to the cell growth situation timely subculture. Automatic blood cell counter was used to count the absolute number of CIK cells at the defined check point, calculate the proliferation ratio and draw the curve of cell proliferation; Flow cytometry were used to analyze the proportion of CD3+CD4+, CD3+ CD8+ and CD3+ CD56+ cells subsets in all group of CIK cells; The expression of TNF alpha and IFN gamma in the supernatant fluid of CIK cells were tested by enzyme linked immunosorbent assay(ELISA); The killing activity of each group CIK cells to K562 were tested by CCK8 method; All CIK cells were stained by Annexin V-FITC/PI and flow cytometry was used to test CIK cells apoptosis rate on day 13; In the end to test the cell cycle of CIK cells on day 13.Results PBMCs were induced by a variety of cytokines in vitro can obtain a large number of amplification of CIK cells. At different testing point, all groups of CIK cells survival rate remained above 90%; On training 16 days the group E(100 μg ASP group) CIK cells proliferation ratio is(142.46±9.45) times, the control group CIK cells proliferation is(109.53±8.27) times,compared with control group,the proliferation ratio of E group is significantly higher than control group(P < 0.05), there were significant statistic differences between E group and control group; The proportion of CD3+ CD4+ cell and CD3+ CD8+ cells subsets in all Groups at different testing point had no obvious difference(P > 0.05),however, the main effector cells proportion like CD3+ CD56+ cells subsets in group D and group E CIK cells is(26.65±3.71) % and(28.36±4.28) %, while the control group is(20.75±3.56) %(P < 0.05); As the extension of incubation time, the expression of TNF-α, IFN –γ is gradually rising in supernatant of all CIK cells, in terms of the expression of cytokine TNF-α, only high concentration of ASP group(group E) TNF-α content is higher compared with the control group(P < 0.05), the difference was statistically significant on day 16, while the rest group also express TNF-αand the content of TNF-αis higher than control group, but no obvious difference compared with control group on day 16. The medium and high concentration of ASP group, group D and group E CIK cells can secrete more IFN-γ,the content of IFN- γ is significantly higher than the control group(P < 0.05), there are significant difference statistically significant on day 16. With the higher effector-target raito, the each group of CIK cells killing activity are enhanced in turn on day 16, when the effector-target raito is 40:1, 20:1,10:1, the killing activity of group E CIK cells to K562 is gradually increased too, the killing activity ratio is(84.19±0.88) %,(76.69±1.54) %,(72.32 ±2.22) %, while the control group of killer activity is(68.05±1.95) %,(64.55±1.44) %,(61.45±4.22) %, compared with the control group,The killing activity of E group at etch effector-target raito are stronger(P < 0.05); Using 100μg/ml ASP induced CIK cells at different testing time points(7, 10, 13 days) the CD25 expression rate is higher, the proportion of CD25+ cells is(40.56±4.47)%(40.56±4.47)%,(55.34±4.71)%,(20.25±4.79)%, while the control group is(34.17±3.86)%,(42.24%±4.58)%,(14.98%±3.67)%, differences are statistically significant(P < 0.05), and its decline trend is slower than control group. 100μg/ml ASP induced CIK cells apoptosis rate is(8.38±0.89)% on day 13, significantly lower than the control group(14.31±1.76)%, the statistical differences is significant(P < 0.05). Similarly, the percentage of G1 stage cells in 100μg/ml ASP induced CIK cells is(58.42±3.73)%, lower than the control group(70.13±4.56)%,(P < 0.05); And the percentage of cells in S phase is(34.21±5.64)%, compared with the control group(24.83±4.63)% is significantly higher, the differences are statistically significant(P < 0.05).Conclusions With the extension of incubation time, the medium and high concentrations of ASP(50μg, 100μg/ml ASP)have a certain role in promoting CIK cells’ proliferation and cytotoxicity; ASP can promote cytokines like TNF alpha, IL-2, IFN gamma’s expression level in the supernatant fluid of CIK cells; Interestingly the medium and high concentration of ASP can increase the proportion of the main effect cells in CIK cells like CD3 + CD56 + cells subsets, but different concentration of ASP does not have much affect on the percentage of CD3+CD4+ and CD3+ CD8+ cells subsets; The high concentration of ASP induced CIK cells have strong killing effect on K562 cells; The possible mechanism of ASP promoting CIK cells proliferation including ASP can induce expression level of CD25(IL-2R) in CIK cells, and promote CIK cells product more endogenous IL-2, IL-2 can combine with IL-2R, this combination can enhance CIK cells activation and secrete more soluble cytokines(e.g. IFN-γ, IL-2, TNF-α), thus promotes the fast expansion of CIK cells; ASP can protect the activation CIK cells from apoptosis; ASP have benefit in promoting CIK cells from G1 phase to S phase quickly, so as to promote its proliferation.