Effect Of Angiogenesis Inhibitors, IFN-α And IFN-γ On Lymphangiogenesis

Objective The study was focused on the effect and mechanism of IFN-α and IFN-γ on the proliferation and migration of the lymphatic endothelial cell (LEC), and further discovering the safe, effective and practical inhibitors of lymphangiogenesis.Materials and Methods LECs were taken from the pig thoracic duct. 1. Identification of LEC: LYVE-1 and VEGFR-3 were used to identify LEC. 2. Microscope and electron microscope observation. 3. Inhibitory experiment: Two groups, IFN-α and IFN-γ, were designed in the inhibitory experiment. In each group, we adopted two methods to observe the proliferation and migration of LECs which were affected by IFN-α and IFN-γ. (1) Method of scraping line: In IFN-α group and IFN-γ group, control group and 3 experimental groups of different doses were set respectively. (2) MTT: The control group and six experimental groups of different concentration were set in IFN-α group and IFN-γ group, respectively. 4. Apoptotic assay: (1) Caspase staining. (2) Hoechst staining. 5. IFN experiment in vivo: Lymphatics of rabbit's two posterior limbs were cut and then IFN-α and 0.9% NaCl solution were injected around the lymphatic of each limb respectively to observe healing of the lymphatics.Results 1. Identification of LEC: The cultured cells were identified as typical LECs by LYVE-1 and VEGFR-3. 2. LM, SEM and TEM observations: Under LM, LECs presented the typical character of "cobblestone". Under TEM, ultrastructure of LECs, desmosome-like structures and so on were observed. 3. Method of scraping line: When concentration of IFN-α and IFN-γ reached 1000ng/ml, a distinct inhibitive effect can be found (P<0.01). Both could distinctly inhibit the proliferation and migration of LEC. 4. MTT: When the dose of IFN-α and IFN-γ reached 2000ng/mI, both could significantly inhibit the proliferation and migration of LEC (P<0.01). 5. Apoptotic tests: (1) Caspase staining: The positive cells of apoptosis stained brown can be observed. (2) Hoechst staining: The apoptotic bodies surrounding nucleus of LECs could be observed. 6. IFN experiment in vivo: After 16 days of the operation, the lymphatic which was injected with 0.9% NaCl solution healed (no leak of dye) and the one with IFN-a solution did not heal (serious leak).Conclusion 1. IFN-α and IFN-γ could distinctly inhibit the proliferation and migration of LEC. 2. IFN-α and IFN-γ could induce apoptosis of LEC.