| Objective: The standard treatment for advanced ovarian cancer is cytoreductive surgery followed by platinum-based chemotherapy,but multidrug resistance(MDR)tend to affect the chemotherapy effects,the DC-CIK treatment is one of the adoptive cellular immunotherapy(ACI).The researchs show that DC-CIK can not only kill hematological and a variety of solid tumors,but can increase the sensitivity of tumor cells resistant to chemotherapy drugs,and down regulate the drug resistance gene MDR1 expression of tumor cells.This study is to observe the effects of dendritic cells(DC)co-cultured with cytokine induced killer cells(CIK)revesing multidrug resistance of the human ovarian cancer drug resistance cell lines SKOV3/DDP and the variation of drug resistance gene MDR1 expression in vitro.Methods:1 Collect peripheral blood mononuclear cells(PBMC)from healthy individuals.The non-adherent cells was induced into CIK cells with rhIFN-?,CD3 McAb,rhIL-2 and rhIL-1?,and the adherent cell was induced into DC with rhIL-4,rhGM-CSF and TNF-? in vitro,then the DC and CIK were co-cultured.Observe the cellmorphology by phase-contrast microscopy,and detect the immunophenotype involved CD83+ and CD86+ of DC,CD3+CD56+of CIK by FCM(Flow Cytometry).2 Detect the inhibition ratio after CIK cells and DC-CIK cells acting on SKOV3/DDP 24 h given the E/T are 2.5:1,5:1,10:1,20:1,40:1,and count the non-cytotoxic concentration IC10 of the CIK and DC-CIK on SKOV3/DDP after 24 h.3 Detect the inhibition effects of cis-platinum on different groups target cells SKOV3/DDP respectively,the concentration of cis-platinum were 1ug/ml,2 ug/ml,4 ug/ml,8 ug/ml,16 ug/ml,32 ug/ml.The target cells involve 3groups: SKOV3/DDP as the control group,SKOV3/DDP with non-cytotoxic concentration CIK cells affected(SKOV3/DDP+CIK)as the first experimental group and the SKOV3/DDP with DC-CIK cells(SKOV3/DDP+DC-CIK)as the second experimental group.Estimate if CIK cells and DC-CIK cells can increase the sensitiviy of SKOV3/DDP to cisplatin or not by counting the IC50 of cisplatin to SKOV3/DDP.4 Detect the expression variation of the drug resistance gene MDR1 in group SKOV3,group SKOV3/DDP,group SKOV3/DDP+CIK and group SKOV3/DDP+DC-CIK by Sybr green Real-time PCR.Results:1 DC and CIK cells were induced from PBMC successfully.Observed by phase-contrast microscopy,CIK cells were in a large number of amplification,showing colony growth.DC cells appeared burr swell which is matured DC's typical form.(91.70±3.09)% DC cells expressed matured DC's specific surface marker CD83+,and(91.70±3.09)% DC expressed CD86+,the CD3+CD56+ double positive rate reachde up to(56.63±6.21)%,which proved that all most cells are matured;2 When the E/T were 2.5:1,5:1,10:1,20:1,40:1,the inhibition rate of CIK on SKOV3/DDP 24 h were(4.33±0.31)%,(8.03±0.57)%,(15.60±0.79)%,(22.14±0.61)%,(38.60±0.40)% respectively,and DC-CIK cells on SKOV3/DDP were(5.30±0.26)%,(9.97±0.31)%,(19.67±0.47)%,(32.20±0.58)%,(49.40±0.28)%.The cells kill ability of CIK and DC-CIK enhanced with the increase of E/T.When the E/T were 2.5:1 and 5:1,there was no statistically significant in the difference of inhibition rate(P>0.05),and when the E/T were 10:1,20:1,40:1,the difference was statistically significant(P<0.05).In the same E/T,the cells kill ability of DC-CIK was stronger than CIK cells,the difference was statistically significant(P<0.05).The IC10 of SKOV3/DDP to CIK and DC-CIK were 7.68:1 and 5.75:1 respectively computed by linear regression.According to the literature,when study methods or drugs adverse resistance effects generally choose slightly less than the dose of IC10,therefore this study selected 5:1 as the E/T;3 To detect killing effects of cis-platinum on different groups target cells SKOV3/DDP by CCK kit,the non-cytotoxic concentration 5:1 of CIK and DC-CIK were combined with cisplatin affected different groups SKOV3/DDP for 24 h,and the IC50 of SKOV3/DDP to cisplatin reduced to 14.31ug/ml and11.39ug/ml respectively from 22.33ug/ml,increasing the sensitiviy of SKOV3/DDP to cisplatin;4 The expression of drug resistance gene MDR1 of each groups SKOV3/DDP was detected by Sybr green Real-time PCR,the MDR1 of control group SKOV3 was 1,the MDR1 expression of human drug resistance cell lines SKOV3/DDP was higher,which was(21.498±3.744).After CIK cells or DC-CIK affecting,the MDR1 expression were reduced to(11.248±3.806),(7.554±1.821)respectively,and the MDR1 of group SKOV3/DDP+DC-CIK reduced more obviously,the difference was statistically significant(P<0.05).Conclusions:1 Mature DC cells and a large number of proliferation of CIK cells can induced with corresponding cytokines from PBMC of healthy individuals.The determination of CIK cells and DC via detecting cell immunophenotype by FCM is easy and reliable.2 Both CIK cells and DC-CIK cells can kill the human ovarian cancer drug resistance cell lines SKOV3/DDP,and with the increase of E/T,the cells kill ability enhanced with limits.In addition,the CIK cells co-contruled with DC perfoms stronger effects,indicating that DC cells can ehnhance the CIK cells kill ability.3 Both CIK cells and DC-CIK cells can increase the sensitivity of ovarian cancer drug resitance cell lines SKOV3/DDP to cisplatin,partly reversing the drug resistance,and down-regulate MDR1 expression,but the effects of DC-CIK cells were stronger. |
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