Tie2 ShRNA Inhibit The Proliferation Of Human Umbilical Vein Endothelial Cells

One of the most remarkable advances in our understanding of cancer pathogenesis is the notion that the progression of solid tumors depends on tumor angiogenesis.This fundamental principle states that tumor growth beyond two mm~3 in size strictly depends on tumor angiogenesis.The tumor cells may produce one angiogenesis factor that can make endothelial cells disintegrate,and then the new capillaries happen,which can offer the tumors enough oxygen and nutrition.At the same time,it also can remove the metabolites of tumors.Therefore,it is widely recognized that angiogenesis is the base of growth and metastasis of solid tumors. And anti-tumor angiogenesis has been the hot spot,especially gene therapy.Endothelial cells play a crucial role in tumor angiogenesis.Development of the vasculature involves two successive processes:vasculogenesis and angiogenesis. Vasculogenesis is the process that the establishment of a primitive vascular network, including endothelial cell differentiation,proliferation,migration and the formation of new vascular tubes.Angiogenesis is the process of the sprouting of new capillaries from preexisting vessels,including endothelial cells degrading the basement membrane,migrating through to form a sprout,and proliferating to extend the new vessel.Therefore,the therapy of anti-angiogenesis by targeting endothelial cells is a hot spot in the field of tumor therapy.The therapy of anti-tumor angiogenesis by targeting tumor vascular endothelial cells has the following advantages.Firstly,the genes in the vascular endothelial cells are stable.And drug resistance rarely happen. Secondly,endothelial cells in mature tissue are quiescent,while endothelial cells in tumors are active.There are many specific molecules,such as Avb3,E Selectin, VEGF receptor,and Tie.And their expression in tumor is fifty times of the normal tissues.So they are the target point for anti-tumor angiogenesis.Thirdly,theoretically, one endothelial cell can raise about one hundred tumor cells,so it is more effective. Fourthly,tumor angiogenesis is common in all tumors,so it is applicable for various kinds tumor.Umbilical veins are probably the most widely used source for human endothelial cell,since they are more easily available than many other vessels,they are more sensitive to many factors include inflammatory factors,and they are physiologically more relevant than many established cell lines.Tie2 has been recently identified as anther important gene,which has a critical role in promoting vascular homeostasis and vessel maturation,as well as vascular destabilization and remodeling besides vascular endothelial cell growth factor (VEGF).Tie2 is an endothelium-specific receptor tyrosine kinase known to have strong specificity on the process of regulation,which process includes endothelial cell proliferation,degrading the basement membrane,migration and the formation of new vascular tubes.Studies indicated the disruption of Tie2 function in transgenic mice results in embryonic lethality,therefore indicating that Tie2 has an important role in angiogenesis.Studies indicated that some tumors are unaffected by means of interference with the VEGF/VEGF receptor system,and these tumors often express Tie2.Interference with the Tie2 system resulted in inhibited these tumors growth, which suggests that Tie2 and VEGF are two independent mechanisms essential for tumor growth.RNA interference is a mean which proves a powerful role in gene silencing.It is a common phenomenon that endogenous gene expression is inhibited by double-stranded RNA(dsRNA) in eukaryotic organism,known as post transcriptional gene silencing(PTGS).RNAi turns into one of the most efficient tools acting as a retro-genetics research measure due to high degree of specificity,high efficiency, sensitivity,amplifying and herediable ability.As a new kind of reverse genetics on gene function,it overcomes the disadvantage of false-positive conclusions that exist in forward genetics analysis on gene function prediction.The high-throuhput gene silencing technology can inhibit each gene in genome,and can study the gene function and the relationship between genes systematically.Our purpose is study the role of Tie2 on the proliferation in human umbilical vein endothelial cells.The study will supply the theory basis to the further animal research on the inhibition of tumor angiogenesis in vivo and provide the experimental evidence for tumor gene therapy.In our experiment,by RNAi technique,we transfected the shRNA of Tie2 into HUVECs by LIPOFECTAMINE 2000,and detected the change of Tie2 expression by real-time PCR and immunocytochemical stain respectively,and detected the proliferation by MTT,and observed the morphous of apoptotic cells under microscope.The experiment divided into four chapters:Chapter One:Culture and identified the human umbilical vein endothelial cells;Chapter Two:Expression of Tie2 in HUVECs;Chapter Three:Construction and identified of recombination plasmid pGenesil 1.1-U6-Tie2-shRNA;Chapter Four:Effect of using RNA interference to alter Tie2 gene expression on the proliferation of human umbilical vein endothelial cellsChapter One:Cultured and identified the human umbilical vein endothelial cells 1.Objective:Study the culture method and identification of human umbilical vein endothelial cells(HUVECs) in vitro.2.Methods:Umbilical cords which must be healthy and fresh are obtained from The First Affiliated Hospital of SUN Yat-sen University.HUVECs were isolated from umbilical veins by enzyme digestion,and were cultivated in plate.The cells were identified byⅧmonoclonal antibody,and we set up negative control also.3.Result:The mixed single cell suspension,most of which are HUVECs,was obtained from human umbilical vein digested by Trypsin and purified by repeating cells plating.HUVECs were characterized by the technique of immunofluorescence,factorⅧexpression.And the result was that there were over 95%HUVECs expression fluorescence,which indicated that the acquired cells were vascular endothelial cells.Chapter Two:Expression of Tie2 in HUVECs1.Objective:Detect the expression of Tie2 in HUVECs,for the purpose of monitoring the role of Tie2 in angiogenesis in vitro.2.Method:The cells were harvested and counted.Total RNA was isolated according to instructions of Trizol kit,and the expression of Tie2 mRNA was detected according to the RT-PCR Kit.The protein level of Tie2 in the HUVECs was detected by immunocytochemical stain:using a primary polyclonal antibody against Tie2 and a SABC-DAB kit used according to the manufacture's instructions.PBS was used for negative control and the positive control was offered by BOSTER bioengineering company and ECV304 which highly expressing Tie2.The result was analyzed according to the methods reported by Birner.3.Result:Tie2 was highly expressed in HUVECs.Chapter Three:Construction and identification of recombination plasmid pGenesil 1.1-U6-Tie2-shRNA1.Objective:Constructed pGenesil 1.1-U6-Tie2-shRNA recombinant plasmid.2.Method:We searched Tie2 mRNA sequences in NCBI database,designed according to the shRNA design principles,and synthesized Tie2 shRNA oligonucleotide chain by Genesil Company.The plasmid pGenesil 1.1-U6 was digested by Eco31I following the structure map.Then we connected the diluted and annealing fragments with the linear plasmid,so the pGenesil 1.1-U6-Tie2-shRNA recombinant plasmid achieved. Finally,we checked the recombinant plasmid.3.Result:The recombinant plasmids pGenesil 1.1-U6-Tie2-shRNA was constructed successfully,and the result of recombinant plasmids sequencing matched that of the design.Chapter Four:Effect of using RNA interference to alter Tie2 gene expression on the proliferation of human umbilical vein endothelial cells1.Objective:Study on the inhibition on Tie2 by RNA interference and explore the effect on proliferation in the HUVECs.The study will supply the theory basis to the further animal research on the inhibition of tumor angiogenesis in vivo and provide the experimental evidence for tumor gene therapy.2.Method:HUVECs were transfected with the plasmid pGenesil-1.1-U6-Tie2-shRNA,or the negative control plasmid pGenesil-HK,and the transfected cells were compared with untransfected cells.The mRNA and protein expression levels of Tie2 in HUVECs transfected by the recombinant plasmids were analyzed by real-time PCR, immunocytochemical stain and western blot in each group respectively.The proliferation of HUVECs was examined by methyl thiazolyl tetrazolium(MTT),and the morphous of apoptotic cells was observed under microscope.3.Result:The expression of Tie2mRNA in pGenesil 1.1- Tie2 group was lower than in the pGenesil-HK and empty control groups.The expression of Tie2 protein in pGenesil 1.1-Tie2 group was significantly lower than in the pGenesil-HK and empty control groups(P＜0.05).The apoptosis rate of HUVECs was significantly higher in the pGenesil-Tie2 group than in the pGenesil-HK and empty control groups(P＜0.05). And the proliferation rate was significantly inhibited in the pGenesil-Tie2 group (P＜0.05).Conclusion:(1)Highly purified endothelial cells were isolated by trypsin.And factorⅧexpression in HUVECs showed that the acquired cells were vascular endothelial cells.(2) Tie2 was highly expressed in HUVECs.(3) The plasmid of pGenesil 1.1-U6-Tie2-shRNA was constructed successfully.(4) pGenesil 1.1-U6-Tie2-shRNA could inhibit the mRNA and protein expression of Tie2 in vitro.The apoptosis rate in the pGenesil-Tie2 group was significantly higher than in the pGenesil-HK and empty control groups.The proliferation rate was significantly inhibited in the pGenesil-Tie2 group.This study will supply the theory basis to the further animal research on the inhibition of tumor angiogenesis in vivo and provide the experimental evidence for tumor gene therapy.