RNAa Targeted-regulating VEGF Signaling Pathways To The Treatment Of PE

Part one Upregulation of VEGF by small activating RNA(ds VEGF-706)in HTR-8/Svneo cells【 Objective 】 Transfect HTR-8/Svneo cells with ds VEGF-706 sequences,and the ds VEGF-706 was labeled with 5’-carboxyfluorescein,green fluorescent.Test The transfection efficiency and evaluate the transfection effect on VEGF m RNA and protein level.【Methods】 1.experiment was divided into three groups,the control group,mock group and ds VEGF-706 group.After transfected for 72 h,The transfection efficiency of ds VEGF-706 in HTR-8/SVneo cells was detected by a flow cytometer.and Fluorescence images 2.We use Real-Time PCR to detect the expression of VEGF m RNA in HTR-8/SVneo cells after being transfected for 72 h.3.We use Western Blot to detect the expression of VEGF in protein level in HTR-8/SVneo cells after being transfected for 72h【Results】 1.The fluorescence signal in the HTR-8/SVneo cells of ds VEGF-706 group was higher than control group.Additionally,the flow cytometer assay showed that the transfection efficiency was approximately 94.83% 2.The m RNA level of VEGF in ds VEGF-706 group was obviously higher than the control group.(P=0.003)3.the protein level of VEGF in ds VEGF-706 group was markedly higher than the control group(P=0.012)【Conclusion】The small activating RNA could be transfected into HTR-8/SVneo cells with high transfection efficiency,and can elevate VEGF expession both in m RNA and protein level.Part two The effects and mechanism of Upregulated VEGF on migration ability and tube formation function of HTR-8/SVneo cells,【Objective】to investigate the effect of The effects and mechanism of Upregulated VEGF on migration ability and tube formation function of HTR-8/SVneo cells【Methods】 1.The cell wound scratch assay was applied to test the the migration capability of HTR-8/SVneo cells after being transfected with ds VEGF-706 2.Tube formation assay was performed to detect tube formation ability of each group with different treatment.3.The nitrate reductase assay was used to test The NO released from diferent group of EVT cells.Then we use Western Blot to detect the expression of e NOS of each group.【Results】 1.The data indicate that overexpression of VEGF significantly increased cell migration in HTR-8/SVneo cells,relative to the control group(P= 0.024).2.,ds VEGF-706 transfection can significantly promoted tube formation of HTR-8/SVneo cells(P=0.013).And,when there is a presence of TNF-a,the angiogenesis of HTR-8/SVneo cells was inhibited.(P= 0.001).However,there was also a significant increase in the ds VEGF-706 transfected HTR-8/SVneo cells’ tubule genesis in the TNF-a treated group when compared with TNF-a treated non-transfected group(P=0.014)。 3.The nitrate reductase assay showed that,compared with the control group,after being transfected with VEGF-706 for 72 h,the NO secretion of EVT cells significantly increased(P=0.0003).when treat the trophoblast cells with TNF-a for 72 h could inhibit NO secretion by 51%,compared with untreated cells(P=0.001).when incubate of ds VEGF-706 transfected EVT cells with TNFa could attenuate TNF-a mediated inhibition of NO secretion,restoring the secretion of NO to control group level.Western Blot showed that the e NOS expression in transfected EVT cells(transfected with ds VEGF-706 for 72h)was significantly elevated than untransfected group(P=0.032).and TNF-a treatment significantly down regulated e NOS expression of EVT cells(P=0.004),while incubation oftransfected EVT cells with TNF-a restored the secretion to control group levels.【Conclusion】The upregulation of VEGF in EVT cells could significantly enhance HTR-8/Svneo cells,migration ability and tube formation ability,and can restore TNF-a mediated inhibition of angiogenesis.A possible mechanism for ds VEGF-706 in regulating HTR-8/SVneo cells,migration ability and tube formation ability maybe that the overexpress VEGF could activate the e NOS-NO signaling pathways which could enhance HTR-8/SVneo cells,migration ability and tube formation ability.This finding may be a promising method for the treatment of preeclampsia.