Influence Of Angiogenesis Inhibitors, Endostatin And PF-4, On The Lymphagiogenesis

Objective Lymphangiogenesis, formation of new lymphatic from preexisting vessels, is a prerequisite for many physiological and pathological processes, such as embryonic development, transplantation of organ, wound healing, regeneration of tissue and organ, growth and metastasis of tumor, etc. The study was designed to discover the safe, effective, practical and economic inhibitors of lymphangiogenesis , so that to inhibit local lymphangiogenesis and to stop spread of tumor through the lymphatic system.Mater i a I and Methods Fresh pig thoracic ducts were obtained at the local abattoir and kept fresh until brought to the laboratory. To visulize the thoracic duct, the lymph node in the far end of it was injected with a solution of 1% Evans in phosphate buffered saline. The thoracic duct was dissected about 10-15cm in length and fat was removed from the surface of it. The far end of it was cannulated with polythene vein tube. The thoracic duct was flushed with D~Hanks to remove residual dye and lymph and 0.1% collagen solution in PBS was injected through the vein tube .The thoracic duct was kept full and placed in a mildly state for 10 minutes at 37 癈 . After incubation , the collagenase solution was collected and then the thoracic duct was flushed with medium fully. The total cell suspension was centrifuged at 1000 rpm for 10 minutes and the suprnatant was removed, then the cells were suspended in medium and seeded on the plate. The cells were maintainted at 37癈 in a humidified incubator with 95% air and 5% C02. The medium was changed every two or three days. When the cells reached confluence and formed a monolayer, subcultures were obtained by digestion of primary cultures with 0. 125% trypsin and 0.01% EDTA. The lymphatic endothelial cells (LEG) were identified by factor Vlll-related antigen through immunofluorescence dye, light microscope and electron microscope. When LEG grew to confluence, the cells were digested and distributed into four plates, then feeded with fresh medium for subculture. When the second passage of LEG made a confluence, theconfluent monolayer was damaged by removing a portion (about 10mm in width) of the cells using a scraper (rubber policeman) at the central part of the plate. The remaining cells were further incubated for 24 hours in the presence of different concentration of Endostatin or PF-4. Migration of the cells was determined by measuring the distance from scraped line to the cells furthest from the scraped line and counting the cell number in the area where was taken the pictures under LM. In this study, the second or third generation of LEG was chosen. The experimental groups were divided into Endostatin groups and PF-4 groups. Endostatin groups included control, 50ng/ml,100ng/ml and 150ng/ml groups. PF-4 groups included control, 40ng/ml, 80ng/ml and 120ng/ml groups. All data were obtained by three independent experiments and the results are shown as mean ?standard error. Statistical significances were calculated using Sigma Stat software.Results (1) Morphological characteristics of LEC: They were flat, elongated or polygonal shape. The cell sizes were nearly the same and the nucleus was oval. The nucleus area was more prominent than the non-nucleus area. Immunofluorencence study to demonstrate the presence of Factor VIII related antigens were carried out on confluent cultures. However, the examination showed an intense staining reaction in the plasm region.(2) The cell number of Endostatin control group was 28.6+1.2 (mean + standard error) . The cell numbers of 3 Endostatin experimental groups (50ng/ml, 100ng/ml and 150ng/ml) respectively were 17.5 + 0.6,10.5 + 0.5 and 4.8 + 0.3 (P<0.05). The migration distance of the cells in Endostatin control group was 381. 698 + 9. 661 u m. The migration distance of the cells in 3 Endostatin experimental groups respectively were 252.886 +5.579; 164. 518?. 085 and 91. 209?. 975 u m (P<0. 05). The cell number in PF4 control group was 28. 3 + 1. 0. After the doses of 40ng/ml, 80ng/ml and 120ng/ml of PF-4 were added, the cell numbers...