c-ski基因重组腺病毒质粒的构建及其对肿瘤相关成纤维细胞增殖的影响

Construction of recombinant adenoviral expression vector containing c-ski gene and its effect on proliferation of cancer-associated fibroblasts

目的构建携带c-ski基因的重组腺病毒质粒,并检测其对肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)增殖活力的影响。方法以pcDNA3-c-ski质粒为模板,PCR扩增c-ski基因,与腺病毒穿梭载体pAdTrack-CMV连接,构建重组穿梭质粒pAdTrack-c-ski,经PmeⅠ线性化后,转化感受态大肠埃希菌BJ5183,获得重组腺病毒质粒,经PacⅠ线性化后,转染HEK-293细胞,获得重组腺病毒,经扩增及纯化后测定病毒滴度。将纯化的重组腺病毒感染乳腺癌CAFs,采用Western blot法检测感染细胞中c-Ski蛋白的表达水平;MTT法和细胞计数法检测重组腺病毒对CAFs细胞增殖活力的影响。结果重组腺病毒质粒经PacⅠ酶切鉴定证明构建正确;转染HEK-293细胞48 h后可见大量绿色荧光蛋白的表达,纯化的重组腺病毒的滴度为3.11×1010IFU/ml;感染重组腺病毒的乳腺癌CAFs中c-Ski蛋白的表达水平明显高于空病毒组和空白对照组(P<0.001),且增殖活力明显高于空白对照组(P<0.05)。结论成功构建了表达c-ski基因的重组腺病毒质粒,其可明显促进CAFs的增殖,为进一步研究c-Ski对CAFs增殖分化等生物学功能的影响及作用机制提供了实验依据。

Objective To construct recombinant adenoviral expression vector containing c-ski geue, and investigate its effect on the proliferation of cancer-associated fibroblasts （CAFs）. Methods Using plasmid pcDNA3-c-ski as a template, c-ski gene was amplified by PCR and cloned into adenovirus shuttle vector pAdTrack-CMV. The constructed recombinant shuttle plasmid pAdTraek-c-ski was linearized with Pme I and transformed to competent E. coli BJ5183. The obtained recombinant adenoviral plasmids were linearized with Pac I and transfected to HEK-293 cells. The obtained recombinant adenovirus was propagated, purified and determined for titer. CAFs of breast cancer were infected with the purified recombinant adenovirus and determined for expression of c-Ski protein by Western blot. The effect of recombinant adenovirus on proliferative activity of CAFs was determined by MTT assay and cell counting. Results Digestion with Pac I proved that recombinant adenoviral plasmids were constructed correctly. A large quantity of green fluorescent protein （GFP） was expressed in HEK-293 cells 48 h after transfection, and the recombinant adenovirus reached a titer of 2. 11 × 1010 IFU / ml after purification. The expression level of c-Ski protein in CAFs infected with recombinant adenovirns was significantly higher than those infected with empty adenovirus and those uninfected （P 〈 0. 05 ）. However, the proliferation level of CAFs infected with recombinant adenovirus was significantly higher than those uninfected （P 〈 0. 05）. Conclusion The recombinant adenoviral expression containing c-ski gene was successfully constructed and packaged, which promoted the proliferation of CAFs significantly. It provided an experimental basis for further study on effect ofc-Ski on biological function such as proliferation and differentiation of CAFs as well as the relevant mechanism.