Electroporation Mediated Kallistatin Gene Expression In Vivo For Prevention And Treatment Of NCI-H446Subcutaneous Xenograft Tumor

Kallistatin(Kal) is an endogenous vascular groth inhibitor, which has a variety ofboilogical activity, such as lowering blood pressure, vasodilating, promotingneointimal formation, anti-inflammatory and anti-oxidation. Recent research haveshown that Kal, which can work in multi-target and multi-signaling pathway that arenecessary as anticancer drugs, has a broad-spectrum anti-tumor activity, so it isworthy to investigate Kallistatin. The technology of Electroporation transgenicexogenous gene impots the exogenous gene into animal target or organ cells byelectric field. Plasmid coding Kal is imported into the organization by the vivoelectroporation so that it reaches to the long-term effect of treament. Currently,however, the reports about vivo electroporation with Kal transfer encoding for thetreatment of cancer are few at home and abroad, an tissue Electroporation withpAM-CAG-AAV-Ka（lpAAV-Kal） for the treatment of H446subcutaneous xenografttumor has been not reported yet.This research does the vitro experiments at first. HUVEC is transfected withplasmid pAAV-Kal. After that, the relative proliferation rate of cell and the migrationrate of cell are significantly reduced. The concentration of Kal protein is up to2.5ng/mL in cell culture medium. Transfected H446, we got that the relativeproliferation rate and morphology were no significant changes, the apoptoticproportion increased markedly after24h and48h transfection, and the concentrationof Kal protein was up to56ng/mL in cell culture medium. Additionally, A549transfection and H460transfection are similar to H446’s, the relative proliferation rateand morphology are no significant changes. The apoptotic proportion of A549increased markedly after24h transfection, but increased notably after48h transfectionin H460cell. However the concentration of Kal protein in A549transfection is up to69ng/mL, and The concentration of Kal protein is up to3.6ng/mL in H460.The optimal optimized Electroporation conditions, including the distance betweenelectrodes is6mm, Plasmid concentration is1.0mg/mL, Electrode voltage is60V, and injection two times, are got through Orthogonal experiment.Employing these Electroporation conditions for transfection pAAV-Kal to preventsubcutaneous xenograft tumor, the results show that it has no significant preventioneffect. And HE staining on the tissue sections, especial the leg muscle, we evaluatedthe damage to animals; at the same time we stained the tumor sections with antibodiesagainst Ki-67, CD34and E-cadherin to test the preventation.The results of treatment for H446subcutaneous xenograft tumor by ElectroporationpAAV-Kal show that tumor volume of Treatment group is smaller than control，and alarge necrotic area is in the tumor tissue of the Treatment. Although this experimentdid not significantly reduce tumor cell proliferation and invasion ability in thetreatment group, CD34immunohistochemical staining has showed that theMicrovessel density decreased significantly.Finally, we used different frequency electric pAAV-Kal to treat H446subcutaneousxenograft tumor. Experimental results show that the only weekly Electroporationcan significantly reduce the size of the tumors,and immunohistochemistry stainingdemonstrates that this weekly Electroporation can significantly reduce microvesseldensity in tumor tissue.