腺病毒介导的uPAR反义核酸转移抑制肿瘤细胞的侵袭
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国家自然科学基金资助项目(30170398).


Inhibition of Invasiveness of Human Lung Cancer Cells by Adenovirus-mediated Gene Transfer of Antisense RNA for Urokinase Receptor
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This work was supported by grants from The National Science Foundation of China(30170398).

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    摘要:

    为了观察尿激酶受体(uPAR)反义核酸对肿瘤细胞体外侵袭的抑制作用,用PCR方法扩增uPAR cDNA 5′端-46 bp~+454 bp一段长500 bp的序列,利用DNA重组技术将其克隆到病毒载体pAdeno-X中, 构建出重组腺病毒载体pAdeno-X-uPAR(-)和pAdeno-X-uPAR(+).线性化后的重组腺病毒载体转染HEK293细胞7天后,可以获得滴度分别为1.5×108 pfu/ml和0.5×108 pfu/ml的uPAR反义和正义核酸表达重组腺病毒,分别命名为Ad-uPAR(-)和Ad-uPAR(+).以不同的病毒感染指数(MOI)感染人肺巨细胞癌高转移株95D肿瘤细胞,3天后用RNA印迹法可以检测肿瘤细胞uPAR正义和反义核酸表达水平,随着MOI的升高,Ad-uPAR(-)感染的肿瘤细胞uPAR蛋白水平逐渐下降,肿瘤细胞的体外侵袭能力也明显下降,而感染Ad-uPAR(+)的肿瘤细胞无明显变化.结果表明,重组腺病毒可以表达uPAR正义和反义核酸,uPAR反义核酸可以明显抑制人肺巨细胞癌高转移株95D肿瘤细胞在体外的侵袭能力.

    Abstract:

    The effect of antisense RNA for urokinase receptor(uPAR) on inhibition of invasiveness of human lung giant cancer cell lines 95D was observed. 500bp fragment of uPAR cDNA between -46bp~+454bp was amplified, and recombined into plasmid pAdeno-X. The recombinant vector was named pAdeno-X-uPAR(-) and pAdeno-X-uPAR(+) respectively. 7 days after transfecting HEK293 cell with linearized pAdeno-X-uPAR(-) and pAdeno-X-uPAR(+), the recombinant adenovirus can be obtained, which were named Ad-uPAR(-) and Ad-uPAR(+) respectively. The virus titre(pfu/ml) of Ad-uPAR(-) was 1.5×108, and the virus titre of Ad-uPAR(+) was 0.5×108. 95D cells were infected with Ad-uPAR(-) and Ad-uPAR(+) in different multiplicity of infection (MOI). Norther blot analysis could detected the expression of antisense and sense RNA for 500 bp fragment of uPAR gene. With the increase of MOI, Western blot analysis indicated that with AD-uPAR(-) infection the protein level of uPAR of 95D cells decreased, and modified Boyden's Chamber assay suggested that the invasive ability of 95D cells also decreased obviously. In 95D cells infected with Ad-uPAR(+), both the mRNA and protein level of uPAR did not decrease, and cells still had high invasive ability. The results indicated that adenovirus is an efficient vector for transferring antisense RNA for uPAR into cells, and antisense RNA for uPAR could obviously inhibit the invasive ability of 95D human lung cancer cells.

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孙兴会,谭理,李平,张宇清,王霞,侯敏,宋后燕,朱运松.腺病毒介导的uPAR反义核酸转移抑制肿瘤细胞的侵袭[J].生物化学与生物物理进展,2003,30(5):761-766

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  • 收稿日期:2003-02-24
  • 最后修改日期:2003-04-24
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