Expression Of VEGF-C And VEGFR-3 In Tissue Of Pancreatic Adenocarcinoma And Depression Of VEGF-C Gene In PANC-1 By VEGF-C Gene Targeted SiRNA

The morbility of pancreatic carcinoma in P.R.China is 5.1/100,000,It is increasing year by year.This disease has very poor prognosis because of early metastasis and invasion through lymphatic system.In a report of U.S.National Institutes of Health,Survival rate in 1 year is 8%and in 5 years is 3%.Metastasis of this neoplasm through lymphatic system is one of the most important factors which affect the prognosis.Preventing the metastasis through lymphatic system may make great progress in treament of pancreatic carcinoma.Discovering of monoclonal antibody of epithelium of lympgatic vessel has improved the study in microlympgatic vessel in tumour tissue since 2000.It is shows that malignancy cell's metastasis and invasion correlates with lymphangiogenesis induced by the neoplasm.Recently studies show that vascular endothelial growth factor receptor 3 (VEGFR-3) is activated by its specific ligand vascular endothelial growth factor C(VEGF-C) and then promotes Lymphangiogenesis.lt may cause metastasis and invasion of the neoplasms.These facts which correlated with Lymphangiogenesis not only promote lymphangiogenesis but also active several chemical factor adhesion,molecule and receptor secreted by epithelium of lympgatic vessel which infact the interaction between the tumor cell and endothelial cell of lympgatic vessel.So these facts can promote cancer progression. The VEGF-C/VEGFR-3 asix affects tumour progress by regulation lymphagiogensis.And it may be the most important regulators of lymphagiogensis.Some studies show inhibiting it can depress lymphagiogensis and metastasis of tumour through lympgatic vessel.RNA interference(RNAi) by double stranded RNA(dsRNAs) molecules of approx--imately 20-25 nucleotides termed short interfering(siRNAs) is a powerful method for pr--eventing the expression of a particular gene.The Mechanism of RNAi can be divided in three steps:the dsRNAs processed into small interfering RNAs(siRNAs) by an RNaseⅢ--like enzyme,RNA-induced silencing complexes(RISCs) packed and RISCs cleave and destroy the cognate RNA.In mammalian cells,introduction of siRNA initiates post-tran--scriptional gene silencing(PTCG).It is going to be a powerful method in study of fun--ctional genomics especially as tumor related gene and gene therapy because of its highly efficacy and specificity.We study expression of VEGF-C and VEGFR-3 in tissues of pancreatic carcinoma, adjacent tissues of pancreatic carcinoma and tissue of normal pancreas by realtime PCR and the relationship With the lympgatic vessel density in these tissues.Then we use VEGF--C gene targeted siRNA to depress the expression of VEGF-C gene in vitro in order to find a new method of gene therpy for pancreatic carcinoma by inhabiting lymphagiogensis and metastasis of neoplasmsPartⅠ:Expression of VEGF-C and VEGFR-3 in pancreatic adenocareinoma, adjacent tissues of pancreatic carcinoma and normal pancreasObject:Analysis the expression of VEGF-C and VEGFR-3 in pancreatic carcinoma, adjacent tissues of pancreatic carcinoma and normal pancreas to find the relationship bet--ween these expressions and the features of clinical pathology.Method:To preserve 20 tissues of pancreatic carcinoma,their adjacent tissues and tissues of their negative margin in liquid nitrogen(LN) at the pancreectomy in Changhai Hospital.Studing the expression of VEGF-C and VEGFR-3 in these tissues by RT-PCR and studying the relationship with the features of clinical pathology by statistical method.Result:The expression of VEGF-C in tissue of pancreatic carcinoma and VEGFR-3 in adjacent tissues of pancreatic carcinoma is higher than it in pancreatic carcinoma and the normal pancreas(p＜0.05).And these expressions has no relationship with the the features of clinical pathology such as age,gender,position of the neoplasm,degree of differentiation(p＞0.05).But it is higher in tissue with positive lymph node than with negative lymph node(p＜0.05).PartⅡ:Relationship of the expression of VEGF-C and VEGFR-3 in tissues of pancreatic carcinoma,adjacent tissues of pancreatic carcinoma and normal pancreas with lympgatic vessel density(LVD)in these tissuesObject:Studing the difference of lympgatic vessel density(LVD) in pancreatic carcinoma, adjacent tissues of pancreatic carcinoma and normal pancreas and the relationship with expression of VEGF-C and VEGFR-3 in these tissues.Method:Immunohistochemistry were used to detect the expression of LYVE-1 to detect LVD in 20 cases of pancreatic carcinoma,adjacent tissues of pancreatic carcinoma and normal pancreas collected in partⅠand studying the relationship with expression of VEGF-C and VEGFR-3 in these tissues by statistical method.Result:The LVD in adjacent tissues of pancreatic carcinoma is higher than it in pancreatic carcinoma and normal pancreas(p＜0.05) And it correlated with the expression of VEGF-C and VEGFR-3(p＜0.05)PartⅢDepression of the expression of VEGF-C by VEGF-C gene targeted siRNA in vitroObject:To observe change in the expression of VEGF-C of human pancreatic cell (PANC-1 ) after introduction of VEGF-C gene targeted siRNA.Method:VEGF-C gene targeted siRNA is designed and composed according to siRNA design software online.VEGF-C gene targeted siRNA is brought by cationic liposome(Lipofectamine^TM 2000 Reagent) to transfect PANC-1 cell.GAPDH-siRNA is introducted as positive control,Missense sequence of these siRNA is also introducted as negative control.24h, 48h,72h after these transfection.The transfected cell is collected.RT-PCR is performed to analysis the expression of VEGF-C in transfected cell and negative control.Result: PANC-1 cell's survival rate has no changed after dealed with siRNA and gationic liposome respectively.The expression of VEGF-C in transfected cell is highly depressed(p＜0.01) in 24h,and has recovered in 72h.