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目的 :利用AdEasyTMvector system构建携带人SMOC1基因的重组腺病毒表达载体,感染人主动脉瓣间质细胞(hAVICs)并检测外源性SMOC1在细胞中的表达和对细胞增殖的影响。方法:用PCR的方法扩增SMOC1 cDNA,将其克隆到pGEM-T Easy载体中并测序鉴定。经NotⅠ和XhoⅠ双酶切后接入pShuttle-CMV穿梭载体,构建重组腺病毒的穿梭质粒pShuttle-CMV-SMOC1。将经PmeⅠ线性化的pShuttle-CMV穿梭载体与pAdEasyTMDNA电穿孔共转化BJ5183重组细菌,获取重组腺病毒质粒pAdEasy-SMOC1,再将经PacⅠ线性化的重组病毒骨架质粒转染HEK293A包装细胞,包装并扩增重组腺病毒(Ad-SMOC1)。用Ad-SMOC1感染hAVICs,以Western blot法和免疫荧光染色法检测重组SMOC1在hAVICs中的表达与定位,用MTS法检测SMOC1对主动脉瓣间质细胞增殖的影响。结果:成功构建并包装表达SMOC1蛋白的重组腺病毒,该重组腺病毒在体外能有效感染hAVICs并高表达SMOC1蛋白,且其介导表达的SMOC1蛋白主要定位于hAVICs的胞浆和胞膜上,MTS分析表明重组SMOC1能够显著促进主动脉瓣间质细胞的增殖。结论:成功地构建了携带人SMOC1基因的重组腺病毒,并证实SMOC1具有促进增殖的作用。本研究为进一步研究SMOC1在瓣膜间质细胞中的作用奠定了实验基础。
OBJECTIVE: To construct a recombinant adenovirus expression vector carrying human SMOC1 gene by AdEasyTM vector system and infect human aortic valve interstitial cells (hAVICs) and to detect the expression of exogenous SMOC1 in cells and its effect on cell proliferation. Methods: SMOC1 cDNA was amplified by PCR and cloned into pGEM-T Easy vector and sequenced. The recombinant plasmid pShuttle-CMV-SMOC1 was constructed by double digestion with NotⅠ and XhoⅠ and ligated into pShuttle-CMV shuttle vector. The PmeI linearized pShuttle-CMV shuttle vector and pAdEasyTM DNA were electroporated into BJ5183 recombinant bacterium to obtain the recombinant adenovirus plasmid pAdEasy-SMOC1. The PacI-linearized recombinant virus backbone plasmid was transfected into HEK293A packaging cells and packaged and expanded Addition of recombinant adenovirus (Ad-SMOC1). HACICs were infected with Ad-SMOC1, the expression and localization of recombinant SMOC1 in hAVICs were detected by Western blot and immunofluorescence staining, and the effect of SMOC1 on the proliferation of aortic valve interstitial cells was detected by MTS. Results: Recombinant adenovirus expressing SMOC1 protein was successfully constructed and packaged. The recombinant adenovirus can effectively infect hAVICs in vitro and express SMOC1 protein highly. SMOC1 protein mediated by SMOC1 mainly locates in the cytoplasm and membrane of hAVICs, MTS analysis showed that recombinant SMOC1 can significantly promote the proliferation of aortic valve interstitial cells. Conclusion: The recombinant adenovirus carrying human SMOC1 gene was successfully constructed and proved that SMOC1 can promote proliferation. This study lays the foundation for the further study of the role of SMOC1 in valvular mesenchymal cells.