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Objective:To investigate the effect and mechanism of Wogonin on CD3AK cell proliferation and cytotoxicity to SMMC-7721.Methods:CD3AK cells were cultured from peripheral blood mononuclear cells ( PBMC) in vitro by a variety of cytokines for 7 d,and treated with different concentrations of Wogonin for 48 h.CD3AK cells proliferation was measured by CCK-8 assay.SMMC-7721 cell growth was detected by MTT.The expression of perforin (PFP),granzyme B (GrB) and CD107a on CD3AK cells were measured by flow cytometry ( FCM).The cytotoxicity to SMMC-7721 cells was detected by LDH release assay.The expression of ERK1/2 on CD3AK cells was detected by Western blot.The mobility of SMMC-7721 cells was detected with transwell chambers.The merge of SMMC-7721 cells were measured with Wound healing assay.Results:Wogonin could significantly promote CD3AK cells proliferation, especially at 3.2 mg/L (23%higher than that of control group,P<0.05).The highest cytotoxicity to SMMC-7721 was also at the con-centration 3.2 mg/L (60.4%).The expression of PFP,GrB,CD107a were significantly higher than that of control group( P<0.05).The expression of ERK1/2 was obviously improved,especially at 12.5-0.8mg/L.After treated with Wogonin 50,12.5,3.2,0.8,0.2 mg/L for 48 h,the lowest transwell cell was at 12.5 mg/L and lowest merge rate was at 3.2 mg/L.Conclusion:Wogonin could promote CD3AK cell proliferation and enhance the cytotoxicity to SMMC-7721.Wogonin could also inhibit SMMC-7721 cell growth,migration and cell merge.The mechanism may be related to activated ERK1/2 and increase the expression of PFP,GrB,CD107a.
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Objective:To study the effects of ICA on HepG2.2.15 cell proliferation, their sensitivity to the lysis by CD3AK effector cell, to investigate the reversal action of ICA on hepatocarcinoma cells from immune escape through Fas/FasL pathway.To provide the theoretical and experimental bases for ICA development.Methods:MTT assay was used to detect cell proliferation and CD3AK cells cytotoxicity activity;flow cytometry assay was used to examine expression of surface molecules and apoptosis rate of HepG2.2.15 cells.Results:When HepG2.2.15 cells line was treated with 50 ?g/ml ICA,a significant reduction of the rate of cell proliferation was observed. Inhibition rate at 48h was 22.04%,and 29.68% at 72h.Kinetic study showed that inhibition of cell proliferation was time dependent (P0.05).ICA could inhibit apoptosis of Jurkat cells induced by HepG2.2.15 cells. In the co-culture system of HepG2.2.15 cell and Jurkat T cell, apoptosis ratio of Jurkat cell was reduced from 46.66% to 18.20% by ICA (P
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Human CD3AK cells were prepared from peripheral blood mononuclear cells by culturing with recombinant IL-2 and antiCD3AK McAb. The mechanism and regulation of CD3AK cytotoxic activity with cytokines (rhIFN-?, rhIFN-?, TNF) and chernotherapeutic agents (CDDP or ADM) were observed by LDH-release assay, ABC-CELISA and the flow cytometric assay. The results showed: (1) Adhesion molecules ICAM-l/LFA-1 participated in CD3AK-mediated killing of tumor cells, hrlFN-? and TNF enhanced cytotoxicity of CD3AK through this pathway. (2) CD3AK could indirectly kill tumor cells by releasing soluable cytotoxic factors. (3) The membrane-associated TNF may be involved in CD3AK-mediated cytotoxicity. (4) CD3AK cells could induce the apoptosis of tumor cells. (5) Pretreatment of tumor cells with CDDP or ADM resulted in the increased vulnerability of tumor cells to CD3AK-mediated killing, the enhancement of CD3AK-mediated cytotoxicity by CDDP was relative to the increased expression of ICAM-1, HLA-ABC on tumor cell membrane.
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Objective: To study the propagation and phenotypes changes of killer cell (CD3AK cell activated by CD3 mAb in vitro. Methods: Lymph nodes taken from lung cancer patient is dissociated into single cell suspension by mechanical method and cultured in culture medium added CD3 mAb and a little dose IL-2. We analyze cell immunophenotype by flow cytometry and proliferation by trypan blue exclusion test per 2 days. Results: Immunophenotypic analysis showed that CD3AK expressing CD3, CD8, CD56, CD25 increased, and reached a peak value which is 2.33 times than before culturing in the 8 th day. Conclusion: CD3 mAb added to the culture medium can obviously activate CD3AK cell and stimulate proliferation and keep its killer activity.
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The result showed that CD3AK induced and expanded in vitro could kill MHC I class - negative K562 (NK - sensitive) and Daudi (NK - resistant) tumor cells in a MHC - nonrestricted manner. Induction of necrosis and / or apoptosis of target cells were responsible for the tumorlytic effect mediated by CD3AK.
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Objective:To study the role and mechanism of c myc ASODNS on cell expression of surface antigen of the tumor cells and susceptibility of target cells to immune cells attack.Methods:c myc mRNA was examined by RT PCR.MTT assay was usd to explore the effects of c myc ASODNS on PG cell sentisentity to lysis by CD 3AK effector cells.The expression of HLA ABC,ICAM 1,c myc protein was examined by flow cytometry.Results:When PG cells were treated with ASODNS(1 ?mol/L) there was a markably reduction of expression of c myc protein.Expression ratio of HLA ABC and ICAM 1 surface antigen expression ratio of PG cells were enhanced from 68.44%,38.44% to 83.16% and 42.09% respectively( P
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Objective To induce human mononuclear cell of cord blood into CD 3 activating killing (CD 3AK) cells with anti-CD 3 monoclonal antibody (CD 3McAb) and recombinant human interleukin-2 (rhIL-2), so that their proliferative activity, activity of killing action, phenotypes and level of secretory cytokines can be observed dynamically. Methods The increase of the number of cells was counted by Tapan-blue staining. The killing action can be measured by using methyl -thiazolyl-tetrazolium-array. The phenotypes of cells were analysed by using indirect immunofluorescence assay. The levels of IL-6, interferon-? (IFN-?) and tumor necrotic factor-? (TNF-?)in culture supernatants were analysed by using enzyme-linked irnmunosorbent assay(ELISA). Results The increase of the number of CD 3AK cells from cord blood was the highest amounting to 78.56 times in the second week. The killing action reached the peak on day 12, and all target cells (malignant cell lines) could be killed significantly. The heterogeneous phenotypes of CD 3AK cells showed that the number of cells with CD 3+, CD 8+, CD 25+, CD 38+, CD 16+ and CD 56+ increased significantly on day 7,14 compared with those of pre-culture (P