| Objectives To construct Adenovirus vector for efficient delivery of siRNA targeted VEGFR2 into two cells including LOVO and SGC7901 which express VEGFR2 , next to observe the influence of VEGFR2siRNA on the expression of KDR in LOVO and SGC7901 and on the growth of SGC7901 in vitro and in vivo.Methods Firstly, the murine U6 vector carrying the VEGFR2siRNA was constructed. Secondly, after the digestion with Hindlll and Xba I , the promoter of the U6 plasmid and VEGFR2siRNA were cloned into the promoterless shuttle vector pshuttle, pShuttle- mU6pro-VEGFR2siRNA was constructed; then the homologous recombination in BJ5183 between the cotransformed adenoviral backbone plasmid pAdeasy-1 and linearized shuttle vector can produce the pAdeasy-mU6pro-VEGFR2siRNA. The LOVO and SGC7901 cancer cell lines were infected with the recombinant adenovirus and observed the influence of it on the level of VEGFR2 expression. At the same time, MTT assay was performed. SGC7901 and LOVO cell lines in logarithmic growth phase were harvested and seeded into 96-well plates, the cell number was diluted to 5000 cells perwell respectively and incubated in 10 % RPMI1640 medium followed by treating with recombinant virus or empty vector 0.1 ml/each well. The cell viability was determined in 4 wells for each drug using MTT assay and observed at 24h, 72h,120h and 168h. Optical density was measured at 490 nm using a microplate reader. The growth curve was made according to the optical density. Statistical analysis was performed by means of Student's t-test, the results demonstrate that Ad-mU6pro-siRNA KDR can significantly inhibit the growth of SGC7901 and LOVO cell.(p<0.05).Mice were challenged subcutaneously in the right rear of the lamb with a single-cell suspension in DMEM solution without serum containing 2×106 viable SGC7901 cells, determined by cell counting to establish the SGC7901 cell-induced tumor model. After the injection 10 days,the tumor size reached approximately from 240mm3 to 280mm3, then, 15 tumor-bearing nude mice were randomly divided into three groups and immediately inoculatedAd-mU6pro-siRNA.KDR(R-AD), Ad-mU6pro(Blank) or DMEM. In order to locally reach higher titer of recombinant adenovirus and avoid the side effect, the intratumoral injection was chosen. Ad-mU6pro-siRNA.KDR(R-AD), Ad-mU6pro(Blank) or DMEM was intratumorally injected into nude mice at the dose of 0.2ml respectively. Thereafter, measurement of the tumor volumes was performed on each day, tumor volumes were calculated according to the formula: ax(b)2x0.5 (a=largest diameter, b=perpendicular diameter). Animals showing severe distress or ulcer and with tumors exceeding 2cm in diameter were killed for ethical reasons and immunohistochemical examination was performed and paraffin-embedded sections were stained with anti-VIII factoras primary antibody to mark the vessels present within the tumor tissue.Results (l)Successful construction of Ad-mU6pro orAd-mU6pro-siRNAKDR The DNA encoding the murine U6 promoter and siRNA targeting the VEGFR-2/KDR were cloned into the promoterless vector pshuttle,obtaining pshuttle-mU6pro-siRNAKDR. The pshuttle-mU6pro -siRNAKDR or pAd-mU6pro was successfully cotransformed with pAdEasy-1 backbone plasmid into BJ5183, the DNA of recombinant adenovirus pAd-mU6pro-siRNA KDR/pAd-mU6pro was digested using pad and confirmed by electrophoresis, the resulting plasmid pAd-mU6pro-siRNAKDR or pAd-mU6pro is assembled and amplified in 293 cells,obtaining Ad-mU6pro-siRNAKDR or Ad-mU6pro.(2)The down-regulation of VEGFR-2 expression in SGC7901 and LOVO and the suppression of LOVO and SGC7901 growth by transfection of recombinant adenovirus Recombinant adenovirus significantly down-regulated VEGFR-2/KDR expression in LOVO and SGC7901 cancer cell lines and inhibited the growth of LOVO and SGC7901(p<0.05).(3)The retardation of growth of SGC7901-induced-tumor and less microvessel density in the tumors treated with Ad-mU6pro-siRNAKDR The siRNA against VEGFR-2/KDR mediated by adenovirus can exert the antitumor effect on SGC7901 cell-induced tumor model, the tumors in experimental group grew significantly more slowly than control groups. A significant difference in tumor size between the experimental group and the control groups was observed on day 30 (0.654±0.06cm3 vs 1.91±0.12 cm3 ,1.98±0.14 cm3... |
