Effects Of Transfection Of IL-18 Gene Combined With Diterpenoid Alkaloids In Inducing Proliferation And Apoptosis Of Tongue Squamous Cacinoma Cell Tscca

ObjectiveWe constructed IL-18 eukaryotic expression vector and tranfected it into tongue squamous carcinoma cells Tscca.At the same time,different concentrations of diterpenoid alkaloids were added,and the apoptosis and proliferation was detected by flow cytometry and MTT,finally,we investigated the possible apoptosis mechanism of IL-18 combined with diterpenoid alkaloids on tongue cell Tscca by detecting Akt/p-Akt pathway.Methods1.IL-18 gene-specific primers was designed and synthesized according to IL-18 DNA sequence in Gen Bank.2.Extraction of PBMC from fresh blood and total RNA of PBMC.The c DNA was extracted by RT-PCR and the target fragment was amplified by PCR.Construction of PEGFPN3-IL-18 recombinant plasmid,transfected into Tscca cells after digestion and sequencing were all correctly.The transfection efficiency of the plasmid was observed under fluorescence microscope.3.Dissolved diterpenoid alkaloids by using DMSO,adjust the concentrationof 0.2 mg/ml,0.4 mg/ml,0.6 mg/ml;4.The control group(untreated Tscca cells),IL-18 group(transfected with IL-18 only),diterpenoid alkaloid group(divided into 3 groups as 0.2 mg/ml,0.4mg/ml,0.6 mg/ml)and combined group(divided into 3 groups as IL-18+0.2mg/ml,IL-18+0.4 mg/ml,IL-18+0.6 mg/ml)were used to detect the growth and apoptosis of tongue squamous cell carcinoma Tscca by MTT assay and flow cytometry.5.Western blot was used to detect the protein level of kinase Akt/p-Akt in each group.It was analyzed whether IL-18 combined with diterpenoid alkaloid played a role in this pathway to inhibit the proliferation of tongue cancer cell Tscca.Results1.Successfully constructed PEGFPN3-IL-18 eukaryotic expression plasmid;2.Successful transfection of tongue squamous cell carcinoma,the expression of Tscca cells transfected with PEGFPN3-IL-18 eukaryotic expression plasmid was higher than that of untransfected cells.3.When the concentration of diterpenoid alkaloids was 0.2,0.4 and 0.6mg/ml,the apoptosis of Tscca cells increased with the increase of concentration,and p<0.05,the difference was statistically significant;4.IL-18 combined with diterpenoid alkaloids on Tscca cells for 48 h,compared with the use of diterpenoid alkaloids alone was significantly inhibited,and showed a concentration-dependent,p<0.05,the difference was statisticallysignificant;5.IL-18 combined with diterpenoid alkaloids also dose-dependently reduced p-Akt protein levels,and were p<0.05,the difference was statistically significant.While the total Akt level remained almost unchanged.Conclusions1.Diterpenoid alkaloids have proliferation inhibition and proapoptotic effect on Tscca cells,and showed a concentration-dependent;2.IL-18 combined with diterpenoid alkaloids on the tongue squamous cell carcinoma Tscca synergistic inhibition,and inhibition than the use of IL-18 alone or diterpenoid alkaloids are strong;3.IL-18 combined with diterpenoid alkaloids in the inhibition of p-Akt expression and activation of the signaling pathway to play a role in inhibiting the tongue cancer cell Tscca proliferation and promote its apoptosis.