VEGFR_2siRNA腺病毒载体的构建及其对肿瘤血管生成的影响
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摘要
目的:构建血管内皮细胞生长因子受体2(VEGFR_2)siRNA的腺病毒载体,在VEGFR_2表达阳性的肿瘤细胞系LOVO和SGC7901中,观察该重组腺病毒对VEGFR_2蛋白表达水平的影响,并检测该重组腺病毒体内的抗肿瘤活性,进而为肿瘤的血管抑制治疗奠定实验基础。
方法:(1) 构建含有VEGFR_2siRNA的鼠U6载体(mU6-VEGFR_2siRNA);(2) 利用HindⅢ与Xba Ⅰ双酶切将鼠U6载体启动子和VEGFR_2siRNA克隆入无启动子穿梭载体promoterless pShuttle vector,得到pShuttle-mU6pro-VEGFR_2siRNA;(3) 将pShuttlemU6pro-VEGFR_2siRNA与腺病毒骨架质粒(pAdeasy-1)在BJ5183细菌中进行同源重组,得到pAdeasy-mU6pro-VEGFR_2siRNA,并进一步在293细胞中包装、扩增得到高滴度的重组腺病毒;(4) 利用得到的重组腺病毒转染LOVO和SGC7901肿瘤细胞系,采用western blot检测感染前后VEGFR_2蛋白表达水平的变化;(5) 利用细胞生长曲线检测重组腺病毒对肿瘤细胞系LOVO、SGC7901生长的影响;(6) 建立SGC7901-裸鼠肿瘤模型,局部注射重组腺病毒后,观察其对肿瘤生长、肿瘤血管生成的抑制作用。
结果:(1)成功构建血管内皮细胞生长因子受体2(VEGFR_2)siRNA的腺病毒载体,该重组腺病毒显著抑制LOVO和SGC7901细胞中VEGFR_2的蛋白表达水平(p<0.01);(2)细胞生长曲线显示重组腺病毒可明显抑制
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 per
well 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 factor
as 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
方法:(1) 构建含有VEGFR_2siRNA的鼠U6载体(mU6-VEGFR_2siRNA);(2) 利用HindⅢ与Xba Ⅰ双酶切将鼠U6载体启动子和VEGFR_2siRNA克隆入无启动子穿梭载体promoterless pShuttle vector,得到pShuttle-mU6pro-VEGFR_2siRNA;(3) 将pShuttlemU6pro-VEGFR_2siRNA与腺病毒骨架质粒(pAdeasy-1)在BJ5183细菌中进行同源重组,得到pAdeasy-mU6pro-VEGFR_2siRNA,并进一步在293细胞中包装、扩增得到高滴度的重组腺病毒;(4) 利用得到的重组腺病毒转染LOVO和SGC7901肿瘤细胞系,采用western blot检测感染前后VEGFR_2蛋白表达水平的变化;(5) 利用细胞生长曲线检测重组腺病毒对肿瘤细胞系LOVO、SGC7901生长的影响;(6) 建立SGC7901-裸鼠肿瘤模型,局部注射重组腺病毒后,观察其对肿瘤生长、肿瘤血管生成的抑制作用。
结果:(1)成功构建血管内皮细胞生长因子受体2(VEGFR_2)siRNA的腺病毒载体,该重组腺病毒显著抑制LOVO和SGC7901细胞中VEGFR_2的蛋白表达水平(p<0.01);(2)细胞生长曲线显示重组腺病毒可明显抑制
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 per
well 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 factor
as 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
引文
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