| Objective: Use zoledronic phosphonic acid(Zol)and interleukin-2(IL-2)to induced and proliferated ??T cells.To investigate the cytotoxicity and other potential effect of ??T cells on ovarian cancer cell lines SKOV3.Methods: 1.Collected the peripheral blood from healthy volunteers,use the density gradient centrifugation method to isolated Peripheral blood mononuclear cells(PBMC)then use Zol and IL-2 to induce and proliferated ??T cells until day 14.2.Flow cytometry(FCM)measured the purity of ??T cells on day 0 and day 14.3.Collected ??T cell which cultured 14 days,use magnetic activated cell sorting(MACS)to separate the ??T cell,then flow cytometry(FCM)measured the purity of ??T cells.4.MTT assay was used to determine the cytotoxicity of ??T cells on SKOV3 cells and the cytotoxicity when ??T cells combined with cisplatin on SKOV3 cells.5.Flow cytometry(FCM)measured the apoptosis effect of ??T cells on SKOV3 cells.6.enzyme-linked immunosorbent assay(ELISA)was used to measure the concentration of INF-??TNF-? in culture supernatant which collected from Co-culture system.Results:1.PBMC can be effectively proliferated by used Zol and IL-2,until day 14 could obtain high-purity ??T cells.The purity increased from 7.67±0.41% to 76.95±4.08%.2.Use MACS can obtainultra high purity??T cells,the purityafter separated was 98.7%±0.35%.3.The ??T cells were co-cultured with SKOV3 cells at different effector-target ratios(5:1,10:1,20:1,40:1)for 24 hours,the k i l l r a t e s w e r e 5 : 1(1.7 0 ± 0.3 6 %),1 0 : 1(2.4 0 ± 0.2 %),2 0 : 1(3.6 3 ± 0.3 5 %),40:1(8.00±0.56%).Kill rate increased follow effector-target ratios((both P<0.05).4.??T cells combined with cisplatin had synergistic effects on SKOV3 cells,use effector-target ratios(20:1)combined with cisplatin(20ug/ml)which kill rate i s(8 3.2 7 ± 1.7 4 %)?? T c e l l s a l o n e g r o u p 4 3.8 0 ± 2.0 0 %,c i s p l a t i n a l o n e group53.53±0.67%(both P<0.05).5.??T cells have apoptosis effect on SKOV3 cells,the apoptosis rate at different effector-target ratios were 5:1(1.70±0.36%),10:1(2.40±0.2%),20:1(3.63±0.35%),40:1(8.00±0.56%)respectively.6.ELISA was used to measure the concentration of TNF-?,INF-? in culture supernatant which collected from Co-culture system.Concentration of TNF-? at different effector-target ratios5:1(34.20±8.64pg/ml),10:1(53.30±5.23pg/ml),20:1(53.30±5.23pg/ml),40:1(107.70±14.93pg/ml),concentration of INF-? at different effector-target ratios 5:1(250.03±76.7pg/ml),10:1(452.45±160.37pg/ml),20:1(841.35±279.53pg/ml),40:1(1342.36±231.23pg/ml).7.Used ELISA measure the concentration of TNF-?,INF-? in culture supernatant which collected from joint destruction system,the concentration of TNF-? severally,??T cells combined with cisplatin group(75.87±4.84pg/ml),??T cells only(75.88±2.40pg/ml),two group compared(P ? 0.05),the concentration of INF-? severally,??T cells combined with cisplatin group(826.93±87.06pg/ml),??T cells only(826.07±93.88pg/ml),two group compared(P?0.05).Conclusions:Use use Zol and IL-2 to induce PBMCs could obtain high-purity ??T cells,the ??T cells have obviously cytotoxicity on SKOV3 cells,and the kill rate increase follow effector-target ratios.Its cytotoxicity mechanism was related to induced apoptosis and secretion of anti-tumor cytokines like TNF-?,INF-?,and the concentration of TNF-? and INF-? increase follow effector-target ratios.??T cells combined with cisplatin have synergistic effect on SKOV3 cells,Its synergistic effect mechanism may related to ??T cells secretion of anti-tumor cytokines TNF-?,INF-? it can reduce MDR gene expression which can increase chemotherapy drugs sensitivity of SKOV3 cells... |
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