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目的构建靶向抑制骨硬化蛋白(SOST)基因的小干扰RNA(siRNA)腺病毒,感染MDA-MB-231乳腺癌细胞并与MG-63成骨细胞共培养,检测对成骨相关分子表达的影响。方法针对SOST基因RNA序列设计带3个干扰片段的2对引物,以p B2B为模板,扩增“背靠背”排列的H1和U6启动子。两个PCR产物以Gibson DNA Assembly的方式同时连接腺病毒穿梭质粒p Ad Trace-OK,经同源重组、HEK293细胞包装获得SOST-siRNA腺病毒。感染MDA-MB-231乳腺癌细胞,实时定量PCR检测SOST mRNA的表达水平,ELISA检测SOST蛋白水平。将转染该腺病毒的MDA-MB-231细胞与MG-63成骨细胞间接共培养,实时定量PCR检测护骨因子(OPG)、骨钙蛋白(OCN)、整合素结合涎蛋白(IBSP)、核因子κB受体激活蛋白配体(RANKL)的表达情况。结果成功获得带3个干扰片段的腺病毒穿梭质粒,经同源重组和包装后,获得AdR-si SOST腺病毒。用该病毒感染MDA-MB-231细胞后,明显抑制SOST的表达。在乳腺癌骨转移体外模型中感染Ad-si SOST腺病毒,能导致MG-63细胞的OPG、OCN、IBSP mRNA表达上升,RANKL mRNA降低。结论在乳腺癌骨转移体外模型中感染Ad-si SOST腺病毒,能促进MG-63细胞的成骨分化,提高OPG/RANKL的比例。
Objective To construct a small interfering RNA (siRNA) adenovirus targeting to inhibit the osteopetrosis protein (SOST) gene, infect MDA-MB-231 breast cancer cells and co-culture with MG-63 osteoblasts to detect the expression of osteogenic related molecules. influences. Methods Two pairs of primers with three interfering fragments were designed for the SOST gene RNA sequence. The pB2B template was used to amplify the “back-to-back” arrangement of the H1 and U6 promoters. The two PCR products were simultaneously connected to the adenoviral shuttle plasmid p Ad Trace-OK by means of Gibson DNA Assembly, and the SOST-siRNA adenovirus was obtained by homologous recombination and HEK293 cell packaging. Infection of MDA-MB-231 breast cancer cells, real-time quantitative PCR detection of SOST mRNA expression levels, ELISA detection of SOST protein levels. The MDA-MB-231 cells transfected with the adenovirus were indirectly co-cultured with MG-63 osteoblasts. Real-time quantitative PCR was used to detect OPG, OCN, and IBPs. The expression of nuclear factor kappa B receptor activating protein ligand (RANKL). Results The adenovirus shuttle plasmids with three interfering fragments were successfully obtained. After homologous recombination and packaging, AdR-si SOST adenovirus was obtained. Infection of MDA-MB-231 cells with this virus significantly inhibited the expression of SOST. Infection of Ad-si SOST adenovirus in a bone metastasis model of breast cancer can result in the increase of OPG, OCN and IBSP mRNA expression and decrease of RANKL mRNA in MG-63 cells. Conclusion Infection of Ad-si SOST adenovirus in breast cancer bone metastasis model in vitro can promote osteogenesis of MG-63 cells and increase OPG/RANKL ratio.