TRANSDUCTION OF CALCITONING GENE-RELA TED PEPTIDE GENE INTO HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS MEDIATED WITH RETROVIRUSTRANSDUCTION OF CALCITONING GENE-RELA TED PEPTIDE GENE INTO HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS MEDIATED WITH RETROVIRUS
摘要(Abstract)：
<正> BACKGROUND:Several lines of evidence indicate that the process of atherosclerosisand lurninal narrowing after PTCA of atherosclerotic lesions is largely due to endothelialinjury and smooth muscle cells hyperplasia Calcitoning gene-related peptide(CGRP)wasreported to stimulate the proliferation of human umbilical vein endothelial cells(HUVECs)and inhibit the growth of vascular smooth muscle cells.This study was to define thepotential for gene transfer to facilitate endothelial cell regeneration and promote recoveryof endothelial dysfunction.METHODS AND RESULTS:Recombinant retrovirus comtaining human α-CGRPcDNA(termed pRCGRP)was constructed and used to infect HUVECs in culture.Thelevel of CGRP gene expression and the mitogenic effects of CGRP on cultured HUVECswere determined by RT-PCR,radioimmunoassay(RIA)and cell proliferative activity.CGRP gere expression was observed only when cultured HUVECs were transduced withvectors containing the CGRP cDNA.No evidence of aimilar gene expression wasobserved in nontransfected or empty vector transfected HUVECs.Twelve days aftertransduction,CGRP level in the supernatant of CGRP vectors tranduced cells wasdetected at 12.4±0.5pM,whereas that of normal and control group were undectableTransduction of HUVECs with vectors bearing CGRP cDNA construct showed a markedincrease in the number of viable cells observed at 24～120 hours after transductioncompared with transduced cultures containing the empty(control)vector CGRP vectorsenhanced cell cycle progression,as determined by the MTT metabolic assay and FlowCytometry.CONCLUSIONS:These primary results represent the first demonstration of theproliferative effects of CGRP retroviral vector on HUVEC.The CGRP gene cam beeffectively transfered into HUVEC and stably expressed with retroviral vector.CGRPvectors containing the leading sequence enabled mature CGRP to be secreted from thecells.Taken together,these data indicate the potential utility of CGRP constructs in thedevelopment of a novel gene therapy spproach to vascular restenosis and atherosclerosisand may lay solid foundations for in vivo gene transfer next.
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