TNF-α Promotes Micro Lymphatic Vessel Formation Of Gallbladder Carcinoma Through Upregulating Expression Of VEGF-d

【Objective】Primary gallbladder cancer is a common malignant tumor in the biliary tract, with a high degree of malignancy, early metastasis happen easily at early stage, Its early lymphatic metastasis is an important prognostic factor. The study found that VEGF-C and VEGF-D is closely associated with lymphangiogenesis and lymph node metastasis, and therefore both known as lymphangiogenesis factor. Our previous study found that, TNF-α can promote micro lymphatic vessel formation of gallbladder in vitro and in vivo, and partly rely on promoting secretion VEGF-C of gallbladder cancer cells, but But whether it can increase the secretion of VEGF-D, thus contribute to lymphangiogenesis is still unclear.This study was to investigate whether TNF alpha can promote the secretion of VEGF-D in gallbladder cancer so as to promote micro-lymphangiogenesis in vitro. In order to provide laboratory evidence to the study of lymph node metastasis and provide targets for the study of new drugs.【Method】three gallbladder cancer cells(NOZ, GBC-SD and SGC-996) were cultured in vitro and were stimulated with different concentration gradients of TNF-α 12 h and 24 h.Real-time PCR and ELSIA method were used to detect the level of transcription and translation of VEGF-D expression in three lines of gallbladder cancer cell lines(NOZ, GBC-SD and SGC-996). Lentiviral with VEGF-Dsi RNA infected with NOZ cells.Transfection rate was observed under a fluorescence microscope. were detected interference effect on the expression of VEGF-D from the m RNA and protein levels by RT-PCR and Western-blot. Establish gallbladder NOZ cells and human dermal lymphatic endothelial cell 3D co-culture system to observe the effect of TNF-α on lymphatic endothelial cells into tubes.The number of lymph tube was calculated to analyze whether TNF-α up-regulate the secretion of VEGF-D to promote gallbladder cancer lymphangiogenesis.【Result】Real-time PCR and ELISA results showed that TNF-α in the concentration of 10-50 ng / ml can promote VEGF-D m RNA and protein in both NOZ and GBC-SD cells in a concentration-dependent manner. which was significant when the cell were stimulated 24 hour. compared with the control group,Each treatment group VEGF-D m RNA and protein expression levels were increased significant in 24h(p <0.05),as well as among the treatment groups.(p <0.05). When TNF-α stimulation at a concentration of 50 ng / ml, VEGF-D expression level was highest, but when the stimulus concentration rose to 100 ng / ml, the expression level of VEGF-D declined. But the study also found that, TNF-α expression had no effect(p> 0.05) on VEGF-D m RNA and protein in SGC-996 cells.NOZ cells were infectide with lentiviral with VEGF-Dsi RNA 72 h and were observed under fluorescence microscopy The result showed the transfection rate was up to 90%.RT-PCR and Western-blot detected that the expression of VEGF-D in interference group was significantly inhibited(P <0.05). NOZ cells and HDLEC 3D co-culture cells showed that the number of lymph tubules generated in VEGF-D interference group(NOZ / sh VEGF-D group) was significantly lower than control group(NOZ) and the negative control group(NOZ / sh CTRL group),there were significant differences(P <0.05). Stimulated with TNF-α,the numbers of tubes in the control group, negative control group and the interference group were increased in different degrees, but the magnitude of the interference group increased was significantly lower than the control group and negative control group, statistically significant difference(P <0.05).【Conclusion】1, Low dose of TNF-α can increase VEGF-D nucleic acid and protein expression in a time- and concentration-dependent manner in NOZ and GBC-SD gallbladder cancer cells,but SGC-996 cells. 2, TNF-α can promote micro lymphangiogenesis in gallbladder cancer through upregulating expression of VEGF-D.