Effects Of Indoleamine 2,3 - Dioxygenase On Angiogenesis In

Objective To establish a human hepatoma carcinoma cell line with IDO gene steady transfection.investigate the effect of Indoleamine2,3-dioxygenase (IDO) on tumor angiogenesis and its probable mechanism in hepatocellular careinoma(HCC).Methods:1.By using the lipofectamine TM2000the eukaryotic expression plasmid pcDNA3.1-IDO and empty vector pcDNA3.1were transfected in a SMMC-7721cell line,After screened with G418, the single cell clone was sought out and cultured. The expression of IDO was detected by reverse transcription polymerase chain reaction(RT-PCR) and western blot,then we got the new cell line,which can express IDO steadily.2.Human endothelial cells were isolated from umbilical veins, cultured in20%fetal bovine serum(FBS)RPMI-1640, and morphologic observation and evalution of HUVEC with light microscopy and immunohistochemistry under inverted microscope.3.Cells were divided into the groups of the non-transfection,the empty plasmid transfection,and the IDO transfection.to collect the cell culture medium of these groups,they were then co-cultured with human umbilical vein endothelial cells(HUVEC) by transwell, the co-cultured HUVECs were seeded in96holes plates,that was lined with Matrigel, add the collection of cell culture medium was added in the corresponding HUVECs the Angiogenesis of HUVEC were observed.,the levels of vascular endothelial growth factor(VEGF) in every group were detected by enzyme-linked immunosorbent assay(ELISA)4. SMMC-7721cells transfected with IDO were added to1-D-MT and L-tryptophan respectively, and we got the groups of the IDO transfection,the1-D-MT,the L-tryptophan. the cell culture medium of these groups was collected. the SMMC-7721cell trancfected with pcDNA3.1-IDO were co-cultured with HUVECs,then the co-cultured HUVECs were seeded in96holes plates,which was lined with Matrigethe, the collection of cell culture medium was added in HUVECs respectively,the Angiogenesis of HUVEC and the expression of VEGF were detected.Result:1.By RT-PCR and Westrn blot,we can find IDO expressed in the SMMC-7721cell trancfected with pcDNA3.1-IDO much higher than the SMMC-7721cell and the SMMC-7721cell trancfected with pcDNA3.1.2.The HUVEC had a cobble stone appearance with a strict monolayer growth,and grow to monolayer after one week.And the Ⅷ factor in the HUVEC showed positive reaction by immunohi stochemi stry.3.the angiogenesis number of HUVEC:the SMMC-7721cell trancfected with pcDNA3.1-IDO (24.56±0.16) much more than the SMMC-7721cell (10.38±0.67) and the SMMC-7721cell trancfected with pcDNA3.1(11.02±0.14)(P<0.05);The expression of VEGF in the SMMC-7721cell trancfected with pcDNA3.1-IDO (727.65±0.48) was significantly higher than the SMMC-7721cell (512.05±1.11) and the SMMC-7721cell trancfected with pcDNA3.1(519.35±0.38)(P<0.05).4.the angiogenesis number of HUVEC:the SMMC-7721cell trancfected with pcDNA3.1-IDO (24.84±0.54) more than the tryptophan group (15.14±0.24) and the1-MT group (14.68±0.84) P<0.05; The expression of VEGF:the SMMC-7721cell trancfected with pcDNA3.1-IDO (727.65±0.83) higher than the tryptophan group (576.00±1.34) and the1-D-MT group (574.65±5.83) P<0.05.Conclusion:stablity transfected SSMC-7721of pcDNA3.1-IDO plasmid can effect tumor angiogenesis throught tryptophan metabolism and the expression of VEGF.