Study Of Anti-mouse Hepatocellular Carcinoma Effect On Cytokine-induced Killer Cells Activated By Dendritic Cells

Objective:To investigate anti-mouse hepatocellular carcinoma effect on cytokine-induced killer cells(CIK)activated by dendritic cells(DCs)transfected with mouse hepatocellular carcinoma(HCC)total RNA in vitro.Methods:(1)DCs were generated from the bone marrow of mice.In brief,the bone marrow was flushed from the long bones of the limbs.The DC precursors and DCs harvested from the bone marrow were incubated with recombinant murine granulocyte marcophage-colony stimulating factor(rmGMCSF) and recombinant murine interleukin-4(rmIL-4)in vitro.Then the DCs were identified and immunized.(2)Mice were killed by cervical dislocation. Suspensions of spleen single cells were pooled in serum-free RPMI1640 medium by filtering the suspension through mesh with the aid of a glass homogenizer to exert gentle pressure on the spleen fragments.Erythrocytes were lysed with distilled water.Obtained cells(splenocytes)were resuspended in RPMI1640 medium with 10%calf Serum in vitro.(3)Adherent cells of Suspensions of spleen single cells were removed via adherence to glass surfaces. Nonadherent splenocytes were obtained.Recombinant murine IFN-gamma (IFN-g)was added on d 0.After 24h of incubation,purified anti-mouse CD3 (anti-CD3),recombinant murine L-2(rmIL-2),recombinant murine IL-1b(rmIL-1b) were added.After 48h of incubation,fresh rmIL-2 and fresh medium were added.Then fresh rmIL-2 and fresh medium were added every 3 d for 14d.(4) Total RNA was isolated from H22 cells by standard methods using little amount tissue/cells extracting kit(W6701)in vitro.(5)Transfecting DCs with H22 cells total RNA was performed on the day of Des culture for 7 days.Total RNA were mixed in DCs.The complex was incubated for 2 days.On the day of culture of CIK cells or splenocytes culture for 9 days,DCs transfecting with total RNA or Des without transfecting total RNA were solitudely mixed in CIK cellsor splenocytes.The complex was incubated for 5d.Fresh medium were added every 3d for 3days.(6)Cytotoxicity assay The MTT colorimetric assay was used for testing cytotoxic activity in vitro.At effector cells:target cells ratios of 10:1.Effector cells included the following groups:CIK cells activated DCs transfected with Total RNA,CIK cells activated DCs without transfected total RNA,CIK cells activated total RNA,CIK cells,splenocytes activated DCs transfected with total RNA,splenocytes activated DCs without transfected total RNA,splenocytes activated total RNA,splenocytes.Target cells included H22 cells andS180 cells.Results:(1)(2-3)×10~6 Des was harvested from bone marrow of one mouse,and its purity was more than 70%.(2)In this trial,H22 cell total RNA's OD260/OD280＞1.90,the result of total RNA electrophoresis presented two complete straps.(3)(2-4)×10~7 splenocytes could be harvested from per mouse spleen.It can be proliferated about 3 times after culture for 14 days.(4)On the first day or on the second day of CIK cells culture in vitro,bulk of cells presented dimin,cells colony did not appear.On the third day,plenty of cells died,a few cells colonise appeared.On the day of cells culture for 7 days,bulk of cells presented magnus,shape of cells presented many types.CIK cells proliferated unactively in the 4 before days.After 4 days of CIK cells culture, CIK cells started to growing actively.After 13 days of CIK cells culture,CIK cells proliferated about 35-50 times.(5)In Vitro Cytotoxicity Assay①The cytotoxicity of each effector cells groups included CIK cells on H22 cells was obviously higher than each control effector cells groups included splenocytes on H22 cells respectively,p＜0.05.②CIK cells activated by DCs transfected total RNA of H22 cells achieved the highest cytotoxicity on H22 cells in all groups(p＜0.05).③CIK cells activated by DCs transfected total RNA of H22 cells achieved higher cytotoxicity on H22 cells than on S180 cells(p＜0.05).Conclusion:(1)DCs generated from bone marrow of one mouse proliferated utilitily in vitro.(2)In this trial,total RNA isolated from H22 cells by using little amount tissue/cells extracting kit(W6701)in vitro,presented complete.(3)CIK cells activated by DCs transfected total RNA of H22 cells presented high efficientely and specifitely cytotoxicity on H22 cells in vitro...