| Objectives1. To study the effect of conditional media from cultured LoVo cells on the proliferation of HUVECs.2. To study the effect of intercellular interaction between LoVo cells and HUVECs on production of nitric oxide(NO), the activity of nitric oxide synthase(NOS), HUVECs morphology, the expression of the proangiogenic factors and the signalling factor in the co-culture system, in order to explore the role of abnormal intercellular interaction in tumor angiogenesis.3. To investigate the role of NO in the intercellular interaction between LoVo cells and HUVECs, and the inhibition of L-NAME from the interaction.4. To establish an experimental model to research tumor angiogenesis in vitro.Methods1. HUVECs were cultured in the conditional media with or without L-NAME for 0h,24h,48h respectively. The proliferation of HUVECs was measured by MTT method .2. HUVECs were co-cultured with LoVo cells in RPMI1640 media with or without L-NAME. After 0h, 12h, 24h, 36h, 48h, the content of NO and the activity of NOS in the supernatant of co-culture system were measured by spectrophotometry assay. The expressions of VEGF, MMP-2, EMMPRIN and IGFBP-7 in HUVECs were detected by immunocytochemistry and the protein level of NF-κBp65 in HUVECs was determined by Western-blotting and immunocytochemistry. The morphologic change of HUVECs was observed under inverted phase contrast microscope.Results1. Compared with control group, the proliferation of HUVECs were markedly increased in conditional media group(P<0.05) and the proliferation was inhibited by L-NAME(P<0.05).There was no distinct difference between control group and L-NAME group(P>0.05).2. When HUVECs were co-cultured with LoVo cells, the content of NO and the activity of NOS in the supernatant were found to be remarkably elevated compared with control group(P<0.05). L-NAME restrained the increased content of NO and the activity of NOS effectively(P<0.05). There was no distinct difference between control group and L-NAME group(P>0.05). |
